US20120184007A1 - Engineered microorganisms with enhanced fermentation activity - Google Patents

Engineered microorganisms with enhanced fermentation activity Download PDF

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US20120184007A1
US20120184007A1 US13/382,903 US201013382903A US2012184007A1 US 20120184007 A1 US20120184007 A1 US 20120184007A1 US 201013382903 A US201013382903 A US 201013382903A US 2012184007 A1 US2012184007 A1 US 2012184007A1
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activity
nucleic acid
yeast
sequence
ruminococcus
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Stephen Picataggio
Kirsty Anne Lily Salmon
Jose Miguel Laplaza
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Verdezyne Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the entire contents of the foregoing patent applications are incorporated herein by reference, including, without limitation, all text, tables and drawings.
  • the technology relates in part to genetically modified microorganisms that have enhanced fermentation activity, and methods for making and using such microorganisms.
  • Microorganisms employ various enzyme-driven biological pathways to support their own metabolism and growth.
  • a cell synthesizes native proteins, including enzymes, in vivo from deoxyribonucleic acid (DNA).
  • DNA first is transcribed into a complementary ribonucleic acid (RNA) that comprises a ribonucleotide sequence encoding the protein.
  • RNA then directs translation of the encoded protein by interaction with various cellular components, such as ribosomes.
  • the resulting enzymes participate as biological catalysts in pathways involved in production of molecules utilized or secreted by the organism.
  • pathways can be exploited for the harvesting of the naturally produced products.
  • the pathways also can be altered to increase production or to produce different products that may be commercially valuable.
  • Advances in recombinant molecular biology methodology allow researchers to isolate DNA from one organism and insert it into another organism, thus altering the cellular synthesis of enzymes or other proteins.
  • Such genetic engineering can change the biological pathways within the host organism, causing it to produce a desired product.
  • Microorganic industrial production can minimize the use of caustic chemicals and production of toxic byproducts, thus providing a “clean” source for certain products.
  • microorganisms having enhanced fermentation activity.
  • such microorganisms are capable of generating a target product with enhanced fermentation efficiency by, for example, (i) preferentially utilizing a particular glycolysis pathway, which increases yield of a target product, upon a change in fermentation conditions; (ii) reducing cell division rates upon a change in fermentation conditions, thereby diverting nutrients towards production of a target product; (iii) having the ability to readily metabolize five-carbon sugars; and/or (iv) having the ability to readily metabolize carbon dioxide; and combinations of the foregoing.
  • a target product is ethanol or succinic acid.
  • engineered microorganisms that comprise: (a) a functional Embden-Meyerhoff glycolysis pathway that metabolizes six-carbon sugars under aerobic fermentation conditions, and (b) a genetic modification that reduces an Embden-Meyerhoff glycolysis pathway member activity upon exposure of the engineered microorganism to anaerobic fermentation conditions, whereby the engineered microorganisms preferentially metabolize six-carbon sugars by the Enter-Doudoroff pathway under the anaerobic fermentation conditions.
  • the genetic modification is insertion of a promoter into genomic DNA in operable linkage with a polynucleotide that encodes the Embden-Meyerhoff glycolysis pathway member activity.
  • the genetic modification is provision of a heterologous promoter polynucleotide in operable linkage with a polynucleotide that encodes the Embden-Meyerhoff glycolysis pathway member activity.
  • the genetic modification is a deletion or disruption of a polynucleotide that encodes, or regulates production of, the Embden-Meyerhoff glycolysis pathway member, and the microorganism comprises a heterologous nucleic acid that includes a polynucleotide encoding the Embden-Meyerhoff glycolysis pathway member operably linked to a polynucleotide that down-regulates production of the member under anaerobic fermentation conditions.
  • the Embden-Meyerhoff glycolysis pathway member activity is a phosphofructokinase activity.
  • the activity of one or more (e.g., 2, 3, 4, 5 or more) pathway members in an EM pathway is reduced or removed to undetectable levels.
  • engineered microorganisms that comprise a genetic modification that inhibits cell division upon exposure to a change in fermentation conditions, where: the genetic modification comprises introduction of a heterologous promoter operably linked to a polynucleotide encoding a polypeptide that regulates the cell cycle of the microorganism; and the promoter activity is altered by the change in fermentation conditions.
  • engineered microorganisms that comprise a genetic modification that inhibits cell division and/or cell proliferation upon exposure of the microorganisms to a change in fermentation conditions.
  • the genetic modification inhibits cell division, inhibits cell proliferation, inhibits the cell cycle and/or induces cell cycle arrest.
  • the change in fermentation conditions is a switch to anaerobic fermentation conditions, and in certain embodiments, the change in fermentation conditions is a switch to an elevated temperature.
  • the polypeptide that regulates the cell cycle has thymidylate synthase activity.
  • the promoter activity is reduced by the change in fermentation conditions.
  • the genetic modification is a temperature sensitive mutation.
  • a target product produced by an engineered microorganism which comprise: (a) culturing an engineered microorganism described herein under aerobic conditions; and (b) culturing the engineered microorganism after (a) under anaerobic conditions, whereby the engineered microorganism produces the target product.
  • Also provided in some embodiments are methods for producing a target product by an engineered microorganism which comprise: (a) culturing an engineered microorganism described herein under a first set of fermentation conditions; and (b) culturing the engineered microorganism after (a) under a second set of fermentation conditions different than the first set of fermentation conditions, whereby the second set of fermentation conditions inhibits cell division and/or cell proliferation of the engineered microorganism.
  • the target product is ethanol or succinic acid.
  • the host microorganism from which the engineered microorganism is produced does not produce a detectable amount of the target product.
  • the culture conditions comprise fermentation conditions, comprise introduction of biomass, comprise introduction of a six-carbon sugar (e.g., glucose), and/or comprise introduction of a five-carbon sugar (e.g., xylulose, xylose); or combinations of the foregoing.
  • the target product is produced with a yield of greater than about 0.3 grams per gram of glucose added, and in certain embodiments, a method comprises purifying the target product from the cultured microorganisms. In some embodiments, a method comprises modifying the target product, thereby producing modified target product.
  • a method comprises placing the cultured microorganisms, the target product or the modified target product in a container, and in certain embodiments, a method comprises shipping the container.
  • the second set of fermentation conditions comprises an elevated temperature as compared to the temperature in the first set of fermentation conditions.
  • the genetic modification inhibits the cell cycle of the engineered microorganism upon exposure to the second set of fermentation conditions.
  • the genetic modification inhibits cell proliferation, inhibits cell division, inhibits the cell cycle and/or induces cell cycle arrest upon exposure to the second set of fermentation conditions.
  • the genetic modification inhibits thymidylate synthase activity upon exposure to the change in fermentation conditions, and sometimes the genetic modification comprises a temperature sensitive mutation.
  • Also provided in certain embodiments are methods for manufacturing an engineered microorganism which comprise: (a) introducing a genetic modification to a host microorganism that reduces an Embden-Meyerhoff glycolysis pathway member activity upon exposure of the engineered microorganism to anaerobic conditions; and (b) selecting for engineered microorganisms that (i) metabolize six-carbon sugars by the Embden-Meyerhoff glycolysis pathway under aerobic fermentation conditions, and (ii) preferentially metabolize six-carbon sugars by the Enter-Doudoroff pathway under the anaerobic fermentation conditions.
  • the genetic modification is insertion of a promoter into genomic DNA in operable linkage with a polynucleotide that encodes the Embden-Meyerhoff glycolysis pathway member activity.
  • the genetic modification sometimes is provision of a heterologous promoter polynucleotide in operable linkage with a polynucleotide that encodes the Embden-Meyerhoff glycolysis pathway member activity.
  • the genetic modification is a deletion or disruption of a polynucleotide that encodes, or regulates production of, the Embden-Meyerhoff glycolysis pathway member
  • the microorganism comprises a heterologous nucleic acid that includes a polynucleotide encoding the Embden-Meyerhoff glycolysis pathway member operably linked to a polynucleotide that down-regulates production of the member under anaerobic fermentation conditions.
  • the Embden-Meyerhoff glycolysis pathway member activity is a phosphofructokinase activity.
  • the activity of one or more (e.g., 2, 3, 4, 5 or more) pathway members in an EM pathway is reduced or removed to undetectable levels.
  • methods for manufacturing an engineered microorganism which comprise: (a) introducing a genetic modification to a host microorganism that inhibits cell division upon exposure to a change in fermentation conditions, thereby producing engineered microorganisms; and (b) selecting for engineered microorganisms with inhibited cell division upon exposure of the engineered microorganisms to the change in fermentation conditions.
  • the change in fermentation conditions comprises a change to anaerobic fermentation conditions.
  • the change in fermentation conditions sometimes comprises a change to an elevated temperature.
  • the genetic modification inhibits the cell cycle of the engineered microorganism upon exposure to the change in fermentation conditions.
  • the genetic modification sometimes inhibits cell division, inhibits the cell cycle, inhibits cell proliferation and/or induces cell cycle arrest upon exposure to the change in fermentation conditions.
  • the genetic modification inhibits thymidylate synthase activity upon exposure to the change in fermentation conditions, and in certain embodiments, the genetic modification comprises a temperature sensitive mutation.
  • the microorganism comprises a genetic modification that adds or alters a five-carbon sugar metabolic activity.
  • the microorganism comprises a genetic alteration that adds or alters xylose isomerase activity.
  • the microorganism comprises a genetic alteration that adds or alters five-carbon sugar transporter activity, and sometimes the transporter activity is a transporter facilitator activity or an active transporter activity.
  • the microorganism comprises a genetic alteration that adds or alters carbon dioxide fixation activity, and sometimes the genetic alteration that adds or alters phosphoenolpyruvate (PEP) carboxylase activity.
  • PEP phosphoenolpyruvate
  • FIG. 1 depicts a metabolic pathway that produces ethanol as by product of cellular respiration.
  • the solid lines represent activities present in the Embden-Meyerhoff pathway (e.g., aerobic respiration). Dashed lines represent activities associated with the Entner-Doudoroff pathway (e.g., anaerobic respiration).
  • One or both pathways often can be operational in a microorganism.
  • the level of activity of each pathway can vary from organism to organism.
  • the arrow from FBP e.g., Fructose-1,6-bisphosphate, also referred to as F-1,6-BP
  • G3P e.g., glcyeraldehyde-3-phosphate
  • FIGS. 2 , 3 and 5 a smaller arrow from FBP to G3P is illustrated, indicating reduced or no conversion of FBP to G3P.
  • the reduction in conversion of FBP to G3P illustrated in FIGS. 2 , 3 and 5 is a result of the reduction or elimination of the previous activity that converts fructose-6-phosphate (F6P) to FBP (e.g., the activity of PFK).
  • FIG. 2 depicts an engineered metabolic pathway that can be used to produce ethanol more efficiently in a host microorganism in which the pathway has been engineered.
  • the solid lines in FIGS. 2-5 represent the metabolic pathway naturally found in a host organism (e.g., Saccharomyces cerevisiae , for example).
  • the dashed lines in FIGS. 2-5 represent a novel activity or pathway engineered into a microorganism to allow increased ethanol production efficiency.
  • the activity of an enzyme in the Embden-Meyerhoff pathway, phosphofructokinase (e.g., PFK) is permanently or temporarily reduced or eliminated.
  • the inactivation is shown as the “X” in FIG. 2 .
  • FIG. 4 depicts an engineered metabolic pathway that can be used to increase the efficiency of ethanol production (and other products) by introducing the ability to fix atmospheric carbon dioxide into a microorganism.
  • the engineered microorganism can incorporate or fix atmospheric carbon dioxide into organic molecules using the introduced phosphoenolpyruvate carboxylase activity. Carbon dioxide incorporated in this manner can be used as an additional carbon source that can increase production of many organic molecules, including ethanol.
  • Non-limiting examples of other products whose production can benefit from carbon fixation include; pyruvate, oxaloacetate, glyceraldehyde-3-phosphate and the like.
  • FIG. 4 illustrates the introduction of the novel carbon dioxide fixation activity in the background of a fully functional EM pathway, and an introduced ED pathway. It is understood the introduction of the carbon fixation activity can benefit microorganisms that have no other modifications to any metabolic pathways. It also is understood that microorganism modified in one, or multiple, other metabolic pathways can benefit from the introduction of a carbon fixation activity.
  • FIG. 5 shows a combination of some engineered metabolic pathways described herein.
  • the combination of engineered metabolic pathways shown in FIG. 5 can provide significant increases in the production of ethanol (or other products) when compared to the wild type organism or organisms lacking one, two, three or more of the modifications.
  • Other combinations of engineered metabolic pathways not shown in FIG. 5 are possible, including but not limited to, combinations including increased alcohol tolerance, modified alcohol dehydrogenase 2 activity and/or modified thymidylate synthase activity, as described herein. Therefore, FIG.
  • FIG. 7 shows a representative western blot used to detect the presence of an enzyme associated with an activity described herein.
  • FIGS. 8A and 8B show representative Western blots used to detect levels of various exogenous EDD and EDA gene combinations expressed in a host organism. Experimental conditions and results are described in Example 9.
  • FIG. 9 graphically displays the relative activities of the various EDD/EDA combinations generated as described in Example 10.
  • FIGS. 12A and 12B graphically illustrate fermentation data for engineered yeast strains described herein.
  • FIG. 12A illustrates the fermentation data for engineered strain BF738 (BY4742 tal1 with vector controls p426GPD and p425GPD).
  • FIG. 12B illustrates the fermentation data for engineered strain BF741 (BY4742 tal1 with plasmids pBF290 (EDD-PAO1) and pBF292 (EDA-PAO1). Experimental conditions and results are described in Example 13.
  • microorganisms used in industrial fermentation process also are incapable of significant carbon fixation.
  • the ability to incorporate atmospheric carbon dioxide, or carbon dioxide waste from respiration in fermentation processes, can increase the amount of industrial chemical product produced per gram of feedstock, in certain embodiments.
  • the microorganisms described herein also can be modified to add or increase the ability to incorporate carbon from carbon dioxide into industrial chemical products, in some embodiments.
  • the microorganisms described herein are engineered to express enzymes such as phosphoenolpyruvate carboxylase (“PEP” carboxylase) and/or ribulose 1,5-bis-phosphate carboxylase (“Rubisco”), thus allowing the use of carbon dioxide as an additional source of carbon.
  • PEP phosphoenolpyruvate carboxylase
  • Rubisco ribulose 1,5-bis-phosphate carboxylase
  • a particularly useful industrial chemical product produced by fermentation is ethanol.
  • Ethanol is an end product of cellular respiration and is produced from acetaldehyde by an alcohol dehydrogenase activity (e.g., by an enzyme like alcohol dehydrogenase 1 or ADH1, for example).
  • alcohol dehydrogenase 2 e.g., ADH2
  • microorganisms described herein are modified to reduce or eliminate the activity of ADH2, to allow increased yields of ethanol.
  • the engineered microorganisms described herein also are modified to have a higher tolerance to alcohol, thus enabling even higher yields of alcohol as a fermentation product without inhibition of cellular processes due to increased levels of alcohol in the growth medium.
  • a microorganism selected often is suitable for genetic manipulation and often can be cultured at cell densities useful for industrial production of a target product.
  • a microorganism selected often can be maintained in a fermentation device.
  • engineered microorganism refers to a modified microorganism that includes one or more activities distinct from an activity present in a microorganism utilized as a starting point (hereafter a “host microorganism”).
  • An engineered microorganism includes a heterologous polynucleotide in some embodiments, and in certain embodiments, an engineered organism has been subjected to selective conditions that alter an activity, or introduce an activity, relative to the host microorganism.
  • an engineered microorganism has been altered directly or indirectly by a human being.
  • a host microorganism sometimes is a native microorganism, and at times is a microorganism that has been engineered to a certain point.
  • an engineered microorganism is a single cell organism, often capable of dividing and proliferating.
  • a microorganism can include one or more of the following features: aerobe, anaerobe, filamentous, non-filamentous, monoploid, dipoid, auxotrophic and/or non-auxotrophic.
  • an engineered microorganism is a prokaryotic microorganism (e.g., bacterium), and in certain embodiments, an engineered microorganism is a non-prokaryotic microorganism.
  • an engineered microorganism is a eukaryotic microorganism (e.g., yeast, fungi, amoeba).
  • Yeast include, but are not limited to, Yarrowia yeast (e.g., Y. lipolytica (formerly classified as Candida lipolytica )), Candida yeast (e.g., C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis ), Rhodotorula yeast (e.g., R. glutinus, R. graminis ), Rhodosporidium yeast (e.g., R. toruloides ), Saccharomyces yeast (e.g., S. cerevisiae, S. bayanus, S.
  • Yarrowia yeast e.g., Y. lipolytica (formerly classified as Candida lipolytica )
  • Candida yeast e.g., C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis
  • Rhodotorula yeast e.g., R. glutinus, R. graminis
  • Rhodosporidium yeast
  • a yeast is a S. cerevisiae strain including, but not limited to, YGR240CBY4742 (ATCC accession number 4015893) and BY4742 (ATCC accession number 201389). In some embodiments, a yeast is a Y.
  • a yeast is a C. tropicalis strain that includes, but is not limited to, ATCC20336, ATCC20913, SU-2 (ura3 ⁇ /ura3 ⁇ ), ATCC20962, H5343 (beta oxidation blocked; U.S. Pat. No. 5,648,247) strains.
  • Any suitable fungus may be selected as a host microorganism, engineered microorganism or source for a heterologous polynucleotide.
  • fungi include, but are not limited to, Aspergillus fungi (e.g., A. parasiticus, A. nidulans ), Thraustochytrium fungi, Schizochytrium fungi and Rhizopus fungi (e.g., R. arrhizus, R. oryzae, R. nigricans ), Orpinomyces or Piromyces .
  • a fungus is an A. parasiticus strain that includes, but is not limited to, strain ATCC24690, and in certain embodiments, a fungus is an A. nidulans strain that includes, but is not limited to, strain ATCC38163.
  • Any suitable prokaryote may be selected as a host microorganism, engineered microorganism or source for a heterologous polynucleotide.
  • a Gram negative or Gram positive bacteria may be selected. Examples of bacteria include, but are not limited to, Bacillus bacteria (e.g., B. subtilis, B. megaterium, B. stearothermophilus ), Bacteroides bacteria (e.g., Bacteroides uniformis, Bacteroides thetaiotaomicron ), Clostridium bacteria (e.g., C. phytofermentans, C. thermohydrosulfuricum, C.
  • H10 cellulyticum
  • Acinetobacter bacteria Norcardia baceteria
  • Lactobacillus bacterial e.g., Lactobacillus pentosus
  • Xanthobacter bacteria Escherichia bacteria
  • Escherichia bacteria e.g., E. coli (e.g., strains DH10B, Stb12, DH5-alpha, DB3, DB3.1), DB4, DB5, JDP682 and ccdA-over (e.g., U.S. application Ser. No.
  • Streptomyces bacteria e.g., Streptomyces rubiginosus, Streptomyces murinus
  • Erwinia bacteria Klebsiella bacteria
  • Serratia bacteria e.g., S. marcessans
  • Pseudomonas bacteria e.g., P. aeruginosa
  • Salmonella bacteria e.g., S. typhimurium, S.
  • Thermus bacteria e.g., Thermus thermophilus
  • Thermotoga bacteria e.g., Thermotoga maritiima, Thermotoga neopolitana
  • Ruminococcus e.g., Ruminococcus environmental samples, Ruminococcus albus, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus flavefaciens, Ruminococcus gaenteauii, Ruminococcus gnavus, Ruminococcus lactaris, Ruminococcus obeum, Ruminococcus sp., Ruminococcus sp.
  • Ruminococcus sp. 15975 Ruminococcus sp. 16442, Ruminococcus sp. 18P13, Ruminococcus sp. 25F6, Ruminococcus sp. 25F7, Ruminococcus sp. 25F8, Ruminococcus sp. 4 — 1 — 47FAA, Ruminococcus sp. 5, Ruminococcus sp. 5 — 1 — 39BFAA, Ruminococcus sp. 7L75, Ruminococcus sp. 8 — 1 — 37FAA, Ruminococcus sp. 9SE51, Ruminococcus sp.
  • Ruminococcus sp. CB10 Ruminococcus sp. CB3, Ruminococcus sp. CCUG 37327 A, Ruminococcus sp. CE2, Ruminococcus sp. CJ60, Ruminococcus sp. CJ63, Ruminococcus sp. CO1, Ruminococcus sp. CO12, Ruminococcus sp. CO22, Ruminococcus sp. CO27, Ruminococcus sp. CO28, Ruminococcus sp. CO34, Ruminococcus sp. CO41, Ruminococcus sp. CO47, Ruminococcus sp.
  • Ruminococcus sp. CS1 Ruminococcus sp. CS6, Ruminococcus sp. DJF_VR52, Ruminococcus sp. DJF_VR66, Ruminococcus sp. DJF_VR67, Ruminococcus sp. DJF_VR70k1, Ruminococcus sp. DJF_VR87, Ruminococcus sp. Eg2, Ruminococcus sp. Egf, Ruminococcus sp. END-1, Ruminococcus sp. FD1, Ruminococcus sp. GM2/1, Ruminococcus sp. ID1, Ruminococcus sp.
  • Ruminococcus sp. K-1 Ruminococcus sp. KKA Seq234, Ruminococcus sp. M-1, Ruminococcus sp. M10, Ruminococcus sp. M22, Ruminococcus sp. M23, Ruminococcus sp. M6, Ruminococcus sp. M73, Ruminococcus sp. M76, Ruminococcus sp. MLG080-3, Ruminococcus sp. NML 00-0124, Ruminococcus sp. Pei041, Ruminococcus sp. SC101, Ruminococcus sp. SC103, Ruminococcus sp.
  • Bacteria also include, but are not limited to, photosynthetic bacteria (e.g., green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g., C. aurantiacus ), Chloronema bacteria (e.g., C. gigateum )), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C.
  • photosynthetic bacteria e.g., green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g., C. aurantiacus ), Chloronema bacteria (e.g., C. gigateum )), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C.
  • Pelodictyon bacteria e.g., P. luteolum
  • purple sulfur bacteria e.g., Chromatium bacteria (e.g., C. okenii )
  • purple non-sulfur bacteria e.g., Rhodospirillum bacteria (e.g., R. rubrum )
  • Rhodobacter bacteria e.g., R. sphaeroides, R. capsulatus
  • Rhodomicrobium bacteria e.g., R. vanellii
  • Cells from non-microbial organisms can be utilized as a host microorganism, engineered microorganism or source for a heterologous polynucleotide.
  • Examples of such cells include, but are not limited to, insect cells (e.g., Drosophila (e.g., D. melanogaster ), Spodoptera (e.g., S. frugiperda Sf9 or Sf21 cells) and Trichoplusa (e.g., High-Five cells); nematode cells (e.g., C.
  • elegans cells e.g., elegans cells
  • avian cells e.g., amphibian cells (e.g., Xenopus laevis cells); reptilian cells; and mammalian cells (e.g., NIH3T3, 293, CHO, COS, VERO, C127, BHK, Per-C6, Bowes melanoma and HeLa cells).
  • amphibian cells e.g., Xenopus laevis cells
  • reptilian cells e.g., NIH3T3, 293, CHO, COS, VERO, C127, BHK, Per-C6, Bowes melanoma and HeLa cells.
  • mammalian cells e.g., NIH3T3, 293, CHO, COS, VERO, C127, BHK, Per-C6, Bowes melanoma and HeLa cells.
  • Microorganisms or cells used as host organisms or source for a heterologous polynucleotide are commercially available. Microorganisms and cells described herein, and other suitable microorganisms and cells are available, for example, from Invitrogen Corporation, (Carlsbad, Calif.), American Type Culture Collection (Manassas, Va.), and Agricultural Research Culture Collection (NRRL; Peoria, Ill.).
  • Host microorganisms and engineered microorganisms may be provided in any suitable form.
  • such microorganisms may be provided in liquid culture or solid culture (e.g., agar-based medium), which may be a primary culture or may have been passaged (e.g., diluted and cultured) one or more times.
  • Microorganisms also may be provided in frozen form or dry form (e.g., lyophilized). Microorganisms may be provided at any suitable concentration.
  • Embden-Meyerhoff pathway operates primarily under aerobic (e.g., oxygen rich) conditions.
  • the other pathway operates primarily under anaerobic (e.g., oxygen poor) conditions, producing pyruvate that can be converted to lactic acid. Lactic acid can be further metabolized upon a return to appropriate conditions.
  • the EM pathway produces two ATP for each six-carbon sugar metabolized, as compared to one ATP produced for each six-carbon sugar metabolized in the ED pathway.
  • the ED pathway yields ethanol more efficiently than the EM pathway with respect to a given amount of input carbon, as seen by the lower net energy yield.
  • yeast preferentially use the EM pathway for metabolism of six-carbon sugars, thereby preferentially using the pathway that yields more energy and less desired product.
  • the following steps and enzymatic activities metabolize six-carbon sugars via the EM pathway.
  • Six-carbon sugars (glucose, sucrose, fructose, hexose and the like) are converted to glucose-6-phosphate by hexokinase or glucokinase (e.g., HXK or GLK, respectively).
  • Glucose-6-phosphate can be converted to fructose-6-phosphate by phosphoglucoisomerase (e.g., PGI).
  • Fructose-6-phosphate can be converted to fructose-1,6-bisphosphate by phosphofructokinase (e.g., PFK).
  • Fructose-1,6-bisphosphate represents a key intermediate in the metabolism of six-carbon sugars, as the next enzymatic reaction converts the six-carbon sugar into two 3 carbon sugars.
  • the reaction is catalyzed by fructose bisphosphate aldolase and yields a mixture of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G-3-P).
  • DHAP dihydroxyacetone phosphate
  • G-3-P glyceraldehyde-3-phosphate
  • the mixture of the two 3 carbon sugars is preferentially converted to glyceraldehyde-3-phosphate by the action of triosephosphate isomerase.
  • G-3-P is converted is converted to 1,3-diphosphoglycerate (1,3-DPG) by glyceraldehyde-3-phosphate dehydrogenase (GLD).
  • 1,3-DPG is converted to 3-phosphoglycerate (3-P-G by phosphoglycerate kinase (PGK).
  • 3-P-G is converted to 2-phosphoglycerate (2-P-G) by phophoglycero mutase (GPM).
  • 2-P-G is converted to phosphoenolpyruvate (PEP) by enolase (ENO).
  • PEP is converted to pyruvate (PYR) by pyruvate kinase (PYK).
  • PYR is converted to acetaldehyde by pyruvate dicarboxylase (PDC).
  • Acetaldehyde is converted to ethanol by alcohol dehydrogenase 1 (ADH1).
  • enzymes in the EM pathway are reversible.
  • the enzymes in the EM pathway that are not reversible, and provide a useful activity with which to control six-carbon sugar metabolism, via the EM pathway include, but are not limited to phosphofructokinase and alcohol dehydrogenase.
  • reducing or eliminating the activity of phosphofructokinase may inactivate the EM pathway.
  • Engineering microorganisms with modified activities in PFK and/or ADH may yield increased product output as compared to organisms with the wild type activities, in certain embodiments.
  • modifying a reverse activity may also yield an increase in product yield by reducing or eliminating the back conversion of products by the backwards reaction.
  • the activity which catalyzes the conversion of ethanol to acetaldehyde is alcohol dehydrogenase 2 (ADH2). Reducing or eliminating the activity of ADH2 can increase the yield of ethanol per unit of carbon input due to the inactivation of the conversion of ethanol to acetaldehyde, in certain embodiments.
  • certain reversible activities also can be used to control six-carbon sugar metabolism via the EM pathway, in some embodiments.
  • a non-limiting example of a reversible enzymatic activity that can be utilized to control six-carbon sugar metabolism includes phosphoglucose isomerase (PGI).
  • a microorganism may be engineered to include or regulate one or more activities in the Embden-Meyerhoff pathway, for example. In some embodiments, one or more of these activities may be altered such that the activity or activities can be increased or decreased according to a change in environmental conditions. In certain embodiments, one or more of the activities (e.g., PGI, PFK or ADH2) can be altered to allow regulated control and an alternative pathway for more efficient carbon metabolism can be provided (e.g., one or more activities from the ED pathway, for example).
  • An engineered organism with the EM pathway under regulatable control and a novel or enhanced ED pathway would be useful for producing significantly more ethanol or other end product from a given amount of input feedstock.
  • Ethanol (or other product) producing activity can be provided by any non-mammalian source in certain embodiments. Such sources include, without limitation, eukaryotes such as yeast and fungi and prokaryotes such as bacteria. In some embodiments, the activity of one or more (e.g., 2, 3, 4, 5 or more) pathway members in an EM pathway is reduced or removed to undetectable levels.
  • An engineered microorganism may, in some embodiments, preferentially metabolize six-carbon sugars via the ED pathway as opposed to the EM pathway under certain conditions.
  • Such engineered microorganisms may metabolize about 60% or more of the available six-carbon sugars via the ED pathway (e.g., about 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than any one of the foregoing), and such fraction of the available six-carbon sugars are not metabolized by the EM pathway, under certain conditions.
  • a microorganism may metabolize six-carbon sugars substantially via the ED pathway, and not the EM pathway, in certain embodiments (e.g., 99% or greater, or 100%, of the available six-carbon sugars are metabolized via the ED pathway).
  • a six-carbon sugar is deemed as being metabolized via a particular pathway when the sugar is converted to end metabolites of the pathway, and not intermediate metabolites only, of the particular pathway.
  • a microorganism may preferentially metabolize certain sugars under the ED pathway after a certain time after the microorganism is exposed to a certain set of conditions (e.g., there may be a time delay after a microorganism is exposed to a certain set of conditions before the microorganism preferentially metabolizes sugars by the ED pathway).
  • Certain novel activities involved in the metabolism of six-carbon sugars by the ED pathway can be engineered into a desired yeast strain to increase the efficiency of ethanol (or other products) production.
  • Yeast do not have an activity that converts 6-phophogluconate to 2-keto-3-deoxy-6-p-gluconate or an activity that converts 2-keto-3-deoxy-6-p-gluconate to pyruvate.
  • Addition of these activities to engineered yeast can allow the engineered microorganisms to increase fermentation efficiency by allowing yeast to ferment ethanol under anaerobic condition without having to use the EM pathway and expend additional energy.
  • the engineered microorganism can benefit by producing ethanol more efficiently, with respect to a given amount of input carbon, than by using the native EM pathway.
  • Bacteria often have enzymatic activities that confer the ability to anaerobically metabolize six-carbon sugars to ethanol. These activities are associated with the ED pathway and include, but are not limited to, phosphogluconate dehydratase (e.g., the EDD gene, for example), and 2-keto-3-deoxygluconate-6-phosphate aldolase (e.g., the EDA gene, for example).
  • Phosphogluconate dehydratase converts 6-phophogluconate to 2-keto-3-deoxy-6-p-gluconate.
  • 2-keto-3-deoxygluconate-6-phosphate aldolase converts 2-keto-3-deoxy-6-p-gluconate to pyruvate.
  • these activities can be introduced into a host organism to generate an engineered microorganism which gains the ability to use the ED pathway to produce ethanol more efficiently than the non-engineered starting organism, by virtue of the lower net energy yield by the ED pathway.
  • a microorganism may be engineered to include or regulate one or more activities in the Entner-Doudoroff pathway. In some embodiments, one or more of these activities may be altered such that the activity or activities can be increased or decreased according to a change in environmental conditions.
  • Nucleic acid sequences encoding Embden-Meyerhoff pathway and Entner-Doudoroff pathway activities can be obtained from any suitable organism (e.g., plants, bacteria, and other microorganisms, for example) and any of these activities can be used herein with the proviso that the nucleic acid sequence is naturally active in the chosen microorganism when expressed, or can be altered or modified to be active.
  • Yeast also can have endogenous or heterologous enzymatic activities that enable the organism to anaerobically metabolize six carbon sugars.
  • Saccharomyces cerevisiae used in fermentation often convert glucose-6-phospate (G-6-P) to fructose-6-phosphate (F-6-P) via phosphoglucose isomerase (EC 5.3.1.9), up to 95% of G-6-P is converted to F-6-P in this manner for example. Only a minor proportion of G-6-P is converted to 6-phophoglucono-lactone (6-PGL) by an alternative enzyme, glucose-6-phosphate dehydrogenase (EC 1.1.1.49).
  • Yeast engineered to carry both Entner-Doudoroff (ED) and Embden-Meyerhoff (EM) pathways often covert sugars to ethanol using the EM pathway preferentially. Inactivation of one or more activities in the EM pathway can result in conversion of sugars to ethanol using the ED pathway preferentially, in some embodiments.
  • ED Entner-Doudoroff
  • EM Embden-Meyerhoff
  • Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the reversible interconversion of glucose-6-phosphate and fructose-6-phosphate.
  • Phosphoglucose isomerase is encoded by the PGI1 gene in S. cerevisiae .
  • the proposed mechanism for sugar isomerization involves several steps and is thought to occur via general acid/base catalysis. Since glucose 6-phosphate and fructose 6-phosphate exist predominantly in their cyclic forms, PGI is believed to catalyze first the opening of the hexose ring to yield the straight chain form of the substrates.
  • Glucose 6-phosphate and fructose 6-phosphate then undergo isomerization via formation of a cis-enediol intermediate with the double bond located between C-1 and C-2.
  • Phosphoglucose isomerase sometimes also is referred to as glucose-6-phosphate isomerase or phosphohexose isomerase.
  • PGI is involved in different pathways in different organisms. In some higher organisms PGI is involved in glycolysis, and in mammals PGI also is involved in gluconeogenesis. In plants PGI is involved in carbohydrate biosynthesis, and in some bacteria PGI provides a gateway for fructose into the Entner-Doudoroff pathway. PGI also is known as neuroleukin (a neurotrophic factor that mediates the differentiation of neurons), autocrine motility factor (a tumor-secreted cytokine that regulates cell motility), differentiation and maturation mediator and myofibril-bound serine proteinase inhibitor, and has different roles inside and outside the cell.
  • neuroleukin a neurotrophic factor that mediates the differentiation of neurons
  • autocrine motility factor a tumor-secreted cytokine that regulates cell motility
  • differentiation and maturation mediator and myofibril-bound serine proteinase inhibitor
  • PGI In the cytoplasm, PGI catalyses the second step in glycolysis, while outside the cell it serves as a nerve growth factor and cytokine. PGI activity is involved in cell cycle progression and completion of the gluconeogenic events of sporulation in S. cerevisiae.
  • phosphoglucose isomerase activity is altered in an engineered microorganism. In some embodiments phosphoglucose isomerase activity is decreased or disrupted in an engineered microorganism. In certain embodiments, decreasing or disrupting phosphoglucose isomerase activity may be desirable to decrease or eliminate the isomerization of glucose-6-phosphate to fructose-6-phosphate, thereby increasing the proportion of glucose-6-phosphate converted to gluconolactone-6-phosphate by the activity encoded by ZWF1 (e.g., glucose-6-phosphate dehydrogenase).
  • ZWF1 e.g., glucose-6-phosphate dehydrogenase
  • Increased levels of gluconolactone-6-phosphate can be further metabolized and thereby improve fermentation of sugar to ethanol via activities in the Entner-Doudoroff pathway, even in the presence of the enzymes comprising the Embden-Meyerhoff pathway.
  • Decreased or disrupted phosphoglucose isomerase (EC 5.3.1.9) activity in yeast may be achieved by any suitable method, or as described herein.
  • Non-limiting examples of methods suitable for decreasing or disrupting the activity of phosphoglucose isomerase include use of a regulated promoter, use of a weak constitutive promoter, disruption of one of the two copies of the gene in a diploid yeast, disruption of both copies of the gene in a diploid yeast, expression of an anti-sense nucleic acid, expression of an siRNA, over expression of a negative regulator of the endogenous promoter, alteration of the activity of an endogenous or heterologous gene, use of a heterologus gene with lower specific activity, the like or combinations thereof.
  • a gene used to knockout one activity can also introduce or increase another activity.
  • PGI1 genes may be native to S. cerevisiae , or may be obtained from a heterologous source.
  • Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) catalyzes the first step of the pentose phosphate pathway, and is encoded by the S. cerevisiae gene, zwf1.
  • the reaction for the first step in the PPP pathway is;
  • D-glucose 6-phosphate+NADP + D-glucono-1,5-lactone 6-phosphate+NADPH+H +
  • the enzyme regenerates NADPH from NADP+ and is important both for maintaining cytosolic levels of NADPH and protecting yeast against oxidative stress.
  • Zwf1p expression in yeast is constitutive, and the activity is inhibited by NADPH such that processes that decrease the cytosolic levels of NADPH stimulate the oxidative branch of the pentose phosphate pathway.
  • Amplification of glucose-6-phosphate dehydrogenase activity in yeast may be desirable to increase the proportion of glucose-6-phosphate converted to 6-phosphoglucono-lactone and thereby improve fermentation of sugar to ethanol via the Entner-Doudoroff pathway, even in the presence of the enzymes comprising the Embden-Meyerhoff pathway.
  • Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity in yeast may be amplified by over-expression of the zwf1 gene by any suitable method.
  • methods suitable to amplify or over express zwf1 include amplifying the number of ZWF1 genes in yeast following transformation with a high-copy number plasmid (e.g., such as one containing a 2 uM origin of replication), integration of multiple copies of ZWF1 into the yeast genome, over-expression of the ZWF1 gene directed by a strong promoter, the like or combinations thereof.
  • the ZWF1 gene may be native to S. cerevisiae , or it may be obtained from a heterologous source.
  • 6-phosphogluconolactonase (EC 3.1.1.31) catalyzes the second step of the ED (e.g., pentose phosphate pathway), and is encoded by S. cerevisiae genes SOL3 and SOL4.
  • the reaction for the second step of the pentose phosphate pathway is;
  • Amplification of 6-phosphogluconolactonase activity in yeast may be desirable to increase the proportion of 6-phospho-D-glucono-1,5-lactone converted to 6-phospho-D-gluconate and thereby improve fermentation of sugar to ethanol via the Entner-Doudoroff pathway, even in the presence of the enzymes comprising the Embden-Meyerhoff pathway.
  • over expression of SOL3 is known to increase the rate of carbon source utilization to result in faster growth on xylose than wild type.
  • the Saccharomyces cerevisiae SOL protein family includes Sol3p and Sol4p. Both localize predominantly in the cytosol, exhibit 6-phosphogluconolactonase activity and function in the pentose phosphate pathway. 6-phosphogluconolactonase (EC 3.1.1.31) activity in yeast may be amplified by over-expression of the SOL3 and/or SOL4 gene(s) by any suitable method.
  • Non-limiting examples of methods to amplify or over express SOL3 and SOL4 include increasing the number of SOL3 and/or SOL4 genes in yeast by transformation with a high-copy number plasmid, integration of multiple copies of SOL3 and/or SOL4 gene(s) into the yeast genome, over-expression of the SOL3 and/or SOL4 gene(s) directed by a strong promoter, the like or combinations thereof.
  • the SOL3 and/or SOL4 gene(s) may be native to S. cerevisiae , or may be obtained from a heterologous source.
  • Sol3p and Sol4p have similarity to each other, and to Candida albicans Sol1p, Schizosaccharomyces pombe Sol1p, human PGLS which is associated with 6-phosphogluconolactonase deficiency, and human H6PD which is associated with cortisone reductase deficiency.
  • Sol3p and Sol4p are also similar to the 6-phosphogluconolactonases in bacteria ( Pseudomonas aeruginosa ) and eukaryotes ( Drosophila melanogaster, Arabidopsis thaliana , and Trypanosoma brucei ), to the glucose-6-phosphate dehydrogenase enzymes from bacteria ( Mycobacterium leprae ) and eukaryotes ( Plasmodium falciparum and rabbit liver microsomes), and have regions of similarity to proteins of the Nag family, including human GNPI and Escherichia coli NagB.
  • Phosphogluconate dehydrogenase (EC:1.1.1.44) catalyzes the second oxidative reduction of NADP+ to NADPH in the cytosolic oxidative branch of the pentose phosphate pathway, and is encoded by the S. cerevisiae genes GND1 and GND2.
  • GND1 encodes the major isoform of the enzyme accounting for up to 80% of phosphogluconate dehydrogenase activity
  • GND2 encodes the minor isoform of the enzyme.
  • Phosphogluconate dehydrogenase sometimes also is referred to as phosphogluconic acid dehydrogenase, 6-phosphogluconic dehydrogenase, 6-phosphogluconic carboxylase, 6-phosphogluconate dehydrogenase (decarboxylating), and 6-phospho-D-gluconate dehydrogenase.
  • Phosphogluconate dehydrogenase belongs to the family of oxidoreductases, specifically those acting on the CH—OH group of donor with NAD + or NADP + as the acceptor. The reaction for the second oxidative reduction of NADP+ to NADPH in the cytosolic oxidative branch of the pentose phosphate pathway is;
  • Decreasing the level of 6-phosphogluconolactonase activity in yeast may be desirable to decrease the proportion of 6-phospho-D-gluconate converted to D-ribulose 5-phosphate thereby increasing the levels of the intermediate gluconate-6-phosphate available for conversion to 6-dehydro-3-deoxy-gluconate-6-phosphate, in some embodiments involving engineered microorganisms including increased EDA and EDD activities, thereby improving fermentation of sugar to ethanol via the Entner-Doudoroff pathway, even in the presence of the enzymes comprising the Embden-Meyerhoff pathway.
  • Decreasing or disrupting 6-phosphogluconolactonase activity in yeast may be achieved by any suitable method, or as described herein.
  • methods suitable for decreasing the activity of 6-phosphogluconate dehydrogenase include use of a regulated promoter, use of a weak constitutive promoter, disruption of one of the two copies of the gene in a diploid yeast (e.g., partial gene knockout), disrupting both copies of the gene in a diploid yeast (e.g., complete gene knockout) expression of an anti-sense nucleic acid, expression of an siRNA, over expression of a negative regulator of the endogenous promoter, alteration of the activity of an endogenous or heterologous gene, use of a heterologus gene with lower specific activity, the like or combinations thereof.
  • a gene used to knockout one activity can also introduce or increase another activity.
  • GND1 and/or GND2 gene(s) may be native to S. cerevisiae , or may be obtained from a heterologous source.
  • S. cerevisiae GND1 and GND2 have similarity to each other, and to the phosphogluconate dehydrogenase nucleotide sequences of Candida parapsilosis, Cryptococcus neoformans and humans.
  • xylose is the second most abundant carbohydrate in nature.
  • energy e.g., ethanol, for example
  • Biomass and waste biomass contain both cellulose and hemicellulose.
  • Many industrially applicable organisms can metabolize five-carbon sugars (e.g., xylose, pentose and the like), but may do so at low efficiency, or may not begin metabolizing five-carbon sugars until all six-carbon sugars have been depleted from the growth medium.
  • yeast and fungus grow slowly on xylose and other five-carbon sugars. Some yeast, such as S. cerevisiae do not naturally use xylose, or do so only if there are no other carbon sources.
  • An engineered microorganism e.g., yeast, for example
  • yeast that could grow rapidly on xylose and provide ethanol and/or other products as a result of fermentation of xylose can be useful due to the ability to use a feedstock source that is currently underutilized while also reducing the need for petrochemicals.
  • the pentose phosphate pathway (PPP), which is a biochemical route for xylose metabolism, is found in virtually all cellular organisms where it provides D-ribose for nucleic acid biosynthesis, D-erythrose 4-phosphate for the synthesis of aromatic amino acids and NADPH for anabolic reactions.
  • the PPP is thought of as having two phases. The oxidative phase converts the hexose, D-glucose 6P, into the pentose, D-ribulose 5P, plus CO2 and NADPH.
  • the non-oxidative phase converts D-ribulose 5P into D-ribose 5P, D-xylulose 5P, D-sedoheptulose 7P, D-erythrose 4P, D-fructose 6P and D-glyceraldehyde 3P.
  • D-Xylose and L-arabinose enter the PPP through D-xylulose.
  • Certain organisms require two or more activities to convert xylose to a usable from that can be metabolized in the pentose phosphate pathway.
  • the activities are a reduction and an oxidation carried out by xylose reductase (XYL1) and xylitol dehydrogenase (XYL2), respectively.
  • XYL1 xylose reductase
  • XYL2 xylitol dehydrogenase
  • Xylose reductase converts D-xylose to xylitol.
  • Xylitol dehydrogenase converts xylitol to D-xylulose.
  • the use of these activities sometimes can inhibit cellular function due to cofactor and metabolite imbalances.
  • xylose isomerase converts xylose directly to xylulose. Xylulose can then be converted to xylulose-5-phosphate by xylulose kinase. Phosphorylation of xylulose then allows the five-carbon sugar to be further converted by transketolase (e.g., TKL1/TKL2) to enter the EM pathway for further metabolism at either fructose-6-phosphate or glyceraldehyde-3-phosphate.
  • transketolase e.g., TKL1/TKL2
  • a microorganism with xylose isomerase activity may allow rapid growth on xylose when compared to the non-engineered microorganism, while avoiding cofactor and metabolite imbalances.
  • a microorganism may be engineered to include or regulate one or more activities in a five-carbon sugar metabolism pathway (e.g., pentose phosphate pathway, for example).
  • an engineered microorganism can comprise a xylose isomerase activity.
  • the xylose isomerase activity may be altered such that the activity can be increased or decreased according to a change in environmental conditions.
  • Nucleic acid sequences encoding xylose isomerase activities can be obtained from any suitable bacteria (e.g., Piromyces, Orpinomyces, Bacteroides thetaiotaomicron, Clostridium phytofermentans, Thermus thermophilus and Ruminococcus (e.g., R. flavefaciens ) and any of these activities can be used herein with the proviso that the nucleic acid sequence is naturally active in the chosen microorganism when expressed, or can be altered or modified to be active.
  • Microorganisms grown in fermentors often are grown under anaerobic conditions, with limited or no gas exchange. Therefore the atmosphere inside fermentors sometimes is carbon dioxide rich. Unlike photosynthetic organisms, many microorganisms suitable for use in industrial fermentation processes do not incorporate atmospheric carbon (e.g., CO 2 ) to any significant degree, or at all. Thus, to ensure that increasing levels of carbon dioxide do not inhibit cell growth and the fermentation process, methods to remove carbon dioxide from the interior of fermentors can be useful.
  • atmospheric carbon e.g., CO 2
  • Photosynthetic organisms make use of atmospheric carbon by incorporating the carbon available in carbon dioxide into organic carbon compounds by a process known as carbon fixation.
  • the activities responsible for a photosynthetic organism's ability to fix carbon dioxide include phosphoenolpyruvate carboxylase (e.g., PEP carboxylase) or ribulose 1,5-bis-phosphate carboxylase (e.g., Rubisco).
  • PEP carboxylase catalyzes the addition of carbon dioxide to phosphoenolpyruvate to generate the four-carbon compound oxaloacetate.
  • Oxaloacetate can be used in other cellular processes or be further converted to yield several industrially useful products (e.g., malate, succinate, citrate and the like).
  • Rubisco catalyzes the addition of carbon dioxide and ribulose-1,5-bisphosphate to generate 2 molecules of 3-phosphoglycerate. 3-phosphoglycerate can be further converted to ethanol via cellular fermentation or used to produce other commercially useful products.
  • Nucleic acid sequences encoding PEP carboxylase and Rubisco activities can be obtained from any suitable organism (e.g., plants, bacteria, and other microorganisms, for example) and any of these activities can be used herein with the proviso that the nucleic acid sequence is either naturally active in the chosen microorganism when expressed, or can be altered or modified to be active.
  • engineered microorganisms can include modifications to one or more (e.g., 1, 2, 3, 4, 5, 6 or all) of the following activities: phosphofructokinase activity (PFK1 A subunit, PFK2 B subunit), phosphogluconate dehydratase activity (EDD), 2-keto-3-deoxygluconate-6-phosphate aldolase activity (EDA), xylose isomerase activity (xylA), phosphoenolpyruvate carboxylase activity (PEP carboxylase), alcohol dehydrogenase 2 activity (ADH2), thymidylate synthase activity, phosphoglucose isomerase activity (PGI1), transaldolase activity (TAL1), transketolase activity (TKL1, TKL2), 6-phosphogluconolactonase activity (SOL3, SOL4), Glucose-6-phosphate dehydrogenase activity (ZWF1), 6-phosphogluconate dehydrogenase
  • Phosphofructokinase activity refers to conversion of fructose-6-phosphate to fructose-1,6-bisphosphate. Phosphofructokinase activity may be provided by an enzyme that includes one or two subunits (referred to hereafter as “subunit A” and/or “subunit B”).
  • activating the Embden-Meyerhoff pathway refers to reducing or eliminating the activity of one or more activities in the Embden-Meyerhoff pathway, including but not limited to phosphofructokinase activity.
  • the phosphofructokinase activity can be reduced or eliminated by introduction of an untranslated RNA molecule (e.g., antisense RNA, RNAi, and the like, for example).
  • the untranslated RNA is encoded by a heterologous nucleotide sequence introduced to a host microorganism.
  • the phosphofructokinase activity can be temporarily or permanently reduced or eliminated by genetic modification, as described below.
  • the genetic modification renders the activity responsive to changes in the environment.
  • the genetic modification disrupts synthesis of a functional nucleic acid encoding the activity or produces a nonfunctional polypeptide or protein.
  • Nucleic acid sequences that can be used to reduce or eliminate the activity of phosphofructokinase activity can have sequences partially or substantially complementary to sequences described herein. Presence or absence of the amount of phosphofructokinase activity can be detected by any suitable method known in the art, including requiring a five-carbon sugar carbon source or a functional Entner-Doudoroff pathway for growth.
  • substantially complementary with respect to sequences refers to nucleotide sequences that will hybridize with each other. The stringency of the hybridization conditions can be altered to tolerate varying amounts of sequence mismatch.
  • regions of counterpart, target and capture nucleotide sequences 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more complementary to each other.
  • phosphogluconate dehydratase activity refers to conversion of 6-phophogluconate to 2-keto-3-deoxy-6-p-gluconate.
  • the phosphogluconate dehydratase activity can be provided by a polypeptide.
  • the polypeptide is encoded by a heterologous nucleotide sequence introduced to a host microorganism. Nucleic acid sequences conferring phosphogluconate dehydratase activity can be obtained from a number of sources, including Zymomonas mobilis and Escherichia coli .
  • 2-keto-3-deoxygluconate-6-phosphate aldolase activity refers to conversion of 2-keto-3-deoxy-6-p-gluconate to pyruvate.
  • the 2-keto-3-deoxygluconate-6-phosphate aldolase activity can be provided by a polypeptide.
  • the polypeptide is encoded by a heterologous nucleotide sequence introduced to a host microorganism. Nucleic acid sequences conferring 2-keto-3-deoxygluconate-6-phosphate aldolase activity can be obtained from a number of sources, including Zymomonas mobilis and Escherichia coli .
  • Examples of an amino acid sequence of a polypeptide having 2-keto-3-deoxygluconate-6-phosphate aldolase activity, and a nucleotide sequence of a polynucleotide that encodes the polypeptide, are presented below in tables. Presence, absence or amount of 2-keto-3-deoxygluconate-6-phosphate aldolase activity can be detected by any suitable method known in the art, including western blot analysis.
  • xylose isomerase activity refers to conversion of xylose to xylulose.
  • the xylose isomerase activity can be provided by a polypeptide.
  • the polypeptide is encoded by a heterologous nucleotide sequence introduced to a host microorganism.
  • Nucleic acid sequences conferring xylose isomerase activity can be obtained from a number of sources, including Piromyces, Orpinomyces, Bacteroides (e.g., B. thetaiotaomicron, B. uniformis, B. stercoris ), Clostrialies (e.g., Clostrialies BVAB3), Clostridium (e.g., C.
  • phytofermentans C. thermohydrosulfuricum, C. cellulyticum
  • Thermus thermophilus Eschericia coli
  • Streptomyces e.g., S. rubiginosus, S. murinus
  • Bacillus stearothermophilus Lactobacillus pentosus
  • Thermotoga e.g., T. maritime, T.
  • Ruminococcus e.g., Ruminococcus environmental samples, Ruminococcus albus, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus flavefaciens, Ruminococcus gaenteauii, Ruminococcus gnavus, Ruminococcus lactaris, Ruminococcus obeum, Ruminococcus sp., Ruminococcus sp. 14531, Ruminococcus sp. 15975, Ruminococcus sp. 16442, Ruminococcus sp. 18P13, Ruminococcus sp. 25F6, Ruminococcus sp.
  • Ruminococcus sp. 25F8 Ruminococcus sp. 4 — 1 — 47FAA, Ruminococcus sp. 5, Ruminococcus sp. 5 — 1 — 39BFAA, Ruminococcus sp. 7L75, Ruminococcus sp. 8 — 1 — 37FAA, Ruminococcus sp. 9SE51, Ruminococcus sp. C36, Ruminococcus sp. CB10, Ruminococcus sp. CB3, Ruminococcus sp. CCUG 37327 A, Ruminococcus sp. CE2, Ruminococcus sp.
  • DJF_VR67 Ruminococcus sp. DJF_VR70k1, Ruminococcus sp. DJF_VR87, Ruminococcus sp. Eg2, Ruminococcus sp. Egf, Ruminococcus sp. END-1, Ruminococcus sp. FD1, Ruminococcus sp. GM2/1, Ruminococcus sp. ID1, Ruminococcus sp. ID8, Ruminococcus sp. K-1, Ruminococcus sp. KKA Seq234, Ruminococcus sp. M-1, Ruminococcus sp. M10, Ruminococcus sp.
  • YE71 Ruminococcus sp. ZS2-15, Ruminococcus torques.
  • phosphoenolpyruvate carboxylase activity refers to the addition of carbon dioxide to phosphoenolpyruvate to generate the four-carbon compound oxaloacetate.
  • the phosphoenolpyruvate carboxylase activity can be provided by a polypeptide.
  • the polypeptide is encoded by a heterologous nucleotide sequence introduced to a host microorganism. Nucleic acid sequences conferring phosphoenolpyruvate carboxylase activity can be obtained from a number of sources, including Zymomonas mobilis .
  • alcohol dehydrogenase 2 activity refers to conversion of ethanol to acetaldehyde, which is the reverse of the forward action catalyzed by alcohol dehydrogenase 1.
  • activation of the conversion of ethanol to acetaldehyde refers to a reduction or elimination in the activity of alcohol dehydrogenase 2. Reducing or eliminating the activity of alcohol dehydrogenase 2 activity can lead to an increase in ethanol production.
  • the alcohol dehydrogenase 2 activity can be reduced or eliminated by introduction of an untranslated RNA molecule (e.g., antisense RNA, RNAi, and the like, for example).
  • the untranslated RNA is encoded by a heterologous nucleotide sequence introduced to a host microorganism.
  • the alcohol dehydrogenase 2 activity can be temporarily or permanently reduced or eliminated by genetic modification, as described below.
  • the genetic modification renders the activity responsive to changes in the environment.
  • the genetic modification disrupts synthesis of a functional nucleic acid encoding the activity or produces a nonfunctional polypeptide or protein.
  • Nucleic acid sequences that can be used to reduce or eliminate the activity of alcohol dehydrogenase 2 can have sequences partially or substantially complementary to nucleic acid sequences that encode alcohol dehydrogenase 2 activity. Presence or absence of the amount of alcohol dehydrogenase 2 activity can be detected by any suitable method known in the art, including inability to grown in media with ethanol as the sole carbon source.
  • thymidylate synthase activity refers to a reductive methylation, where deoxyuridine monophosphate (dUMP) and N5,N10-methylene tetrahydrofolate are together used to generate thymidine monophosphate (dTMP), yielding dihydrofolate as a secondary product.
  • dUMP deoxyuridine monophosphate
  • dTMP thymidine monophosphate
  • temporary inactivate thymidylate synthase activity refers to a temporary reduction or elimination in the activity of thymidylate synthase when the modified organism is shifted to a non-permissive temperature. The activity can return to normal upon return to a permissive temperature. Temporarily inactivating thymidylate synthase uncouples cell growth from cell division while under the non permissive temperature. This inactivation in turn allows the cells to continue fermentation without producing biomass and dividing, thus increasing the yield of product produced during fermentation.
  • the thymidylate synthase activity can be temporarily reduced or eliminated by genetic modification, as described below.
  • the genetic modification renders the activity responsive to changes in the environment.
  • Nucleic acid sequences conferring temperature sensitive thymidylate synthase activity can be obtained from S. cerevisiae strain 172066 (accession number 208583).
  • the cdc21 mutation in S. cerevisiae strain 172066 has a point mutation at position G139S relative to the initiating methionine. Examples of nucleotide sequences used to PCR amplify the polynucleotide encoding the temperature sensitive polypeptide, are presented below in tables. Presence, absence or amount of thymidylate synthase activity can be detected by any suitable method known in the art, including growth arrest at the non-permissive temperature.
  • Thymidylate synthase is one of many polypeptides that regulate the cell cycle.
  • the cell cycle may be inhibited in engineered microorganisms under certain conditions (e.g., temperature shift, dissolved oxygen shift), which can result in inhibited or reduced cell proliferation, inhibited or reduced cell division, and sometimes cell cycle arrest (collectively “cell cycle inhibition”).
  • a microorganism may display cell cycle inhibition after a certain time after the microorganism is exposed to the triggering conditions (e.g., there may be a time delay after a microorganism is exposed to a certain set of conditions before the microorganism displays cell cycle inhibition).
  • cell proliferation rates may be reduced by about 50% or greater, for example (e.g., reduced by about 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than any one of the foregoing).
  • the rate of cell division may be reduced by about 50% or greater, for example (e.g., the number of cells undergoing division is reduced by about 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than any one of the foregoing).
  • cells may be arrested at any stage of the cell cycle (e.g., resting G 0 phase, interphase (e.g., G 1 , S, G 2 phases), mitosis (e.g., prophase, prometaphase, metaphase, anaphase, telophase)) and different percentages of cells in a population can be arrested at different stages of the cell cycle.
  • stage of the cell cycle e.g., resting G 0 phase, interphase (e.g., G 1 , S, G 2 phases), mitosis (e.g., prophase, prometaphase, metaphase, anaphase, telophase)
  • mitosis e.g., prophase, prometaphase, metaphase, anaphase, telophase
  • phosphoglucose isomerase activity refers to the conversion of glucose-6-phosphate to fructose-6-phosphate.
  • activation of the conversion of glucose-6-phosphate to fructose-6-phosphate refers to a reduction or elimination in the activity of phosphoglucose isomerase. Reducing or eliminating the activity of phosphoglucose isomerase activity can lead to an increase in ethanol production.
  • the phosphoglucose isomerase activity can be reduced or eliminated by introduction of an untranslated RNA molecule (e.g., antisense RNA, RNAi, and the like, for example).
  • the untranslated RNA is encoded by a heterologous nucleotide sequence introduced to a host microorganism.
  • the phosphoglucose isomerase activity can be temporarily or permanently reduced or eliminated by genetic modification, as described below.
  • the genetic modification renders the activity responsive to changes in the environment.
  • the genetic modification disrupts synthesis of a functional nucleic acid encoding the activity or produces a nonfunctional polypeptide or protein.
  • Nucleic acid sequences that can be used to reduce or eliminate the activity of phosphoglucose isomerase can have sequences partially or substantially complementary to nucleic acid sequences that encode phosphoglucose isomerase activity. Presence or absence of the amount of phosphoglucose isomerase activity can be detected by any suitable method known in the art, including nucleic acid based analysis and western blot analysis.
  • glucose-6-phosphate dehydrogenase activity refers to conversion of glucose-6-phosphate to gluconolactone-6-phosphate coupled with the generation of NADPH.
  • the glucose-6-phosphate dehydrogenase aldolase activity can be provided by a polypeptide.
  • the polypeptide is encoded by a heterologous nucleotide sequence introduced to a host microorganism. Nucleic acid sequences conferring glucose-6-phosphate dehydrogenase activity can be obtained from a number of sources, including, but not limited to S. cerevisiae Examples of a nucleotide sequence of a polynucleotide that encodes the polypeptide, are presented below in tables. Presence, absence or amount of glucose-6-phosphate dehydrogenase activity can be detected by any suitable method known in the art, including western blot analysis.
  • 6-phosphogluconolactonase activity refers to conversion of gluconolactone-6-phosphate to gluconate-6-phosphate.
  • the 6-phosphogluconolactonase activity can be provided by a polypeptide.
  • the polypeptide is encoded by a heterologous nucleotide sequence introduced to a host microorganism. Nucleic acid sequences conferring 6-phosphogluconolactonase activity can be obtained from a number of sources, including, but not limited to S. cerevisiae .
  • an amino acid sequence of a polypeptide having 6-phosphogluconolactonase activity and a nucleotide sequence of a polynucleotide that encodes the polypeptide, are presented below in tables. Presence, absence or amount of 6-phosphogluconolactonase activity can be detected by any suitable method known in the art, including nucleic acid based analysis and western blot analysis.
  • 6-phosphogluconate dehydrogenase (decarboxylating) activity refers to the conversion of gluconate-6-phosphate to ribulose-5-phosphate.
  • activation of the conversion of gluconate-6-phosphate to ribulose-5-phosphate refers to a reduction or elimination in the activity of 6-phosphogluconate dehydrogenase. Reducing or eliminating the activity of 6-phosphogluconate dehydrogenase (decarboxylating) activity can lead to an increase in ethanol production.
  • the 6-phosphogluconate dehydrogenase (decarboxylating) activity can be reduced or eliminated by introduction of an untranslated RNA molecule (e.g., antisense RNA, RNAi, and the like, for example).
  • an untranslated RNA molecule e.g., antisense RNA, RNAi, and the like, for example.
  • the untranslated RNA is encoded by a heterologous nucleotide sequence introduced to a host microorganism.
  • the 6-phosphogluconate dehydrogenase (decarboxylating) activity can be temporarily or permanently reduced or eliminated by genetic modification, as described below.
  • the genetic modification renders the activity responsive to changes in the environment.
  • the genetic modification disrupts synthesis of a functional nucleic acid encoding the activity or produces a nonfunctional polypeptide or protein.
  • Nucleic acid sequences that can be used to reduce or eliminate the activity of 6-phosphogluconate dehydrogenase (decarboxylating) can have sequences partially or substantially complementary to nucleic acid sequences that encode 6-phosphogluconate dehydrogenase (decarboxylating) activity. Presence or absence of the amount of 6-phosphogluconate dehydrogenase (decarboxylating) activity can be detected by any suitable method known in the art, including nucleic acid based analysis and western blot analysis.
  • transketolase activity refers to conversion of xylulose-5-phosphate and ribose-5-phosphate to sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate.
  • the transketolase activity can be provided by a polypeptide.
  • the polypeptide is encoded by a heterologous nucleotide sequence introduced to a host microorganism. Nucleic acid sequences conferring transketolase activity can be obtained from a number of sources, including, but not limited to S.
  • the transketolase activity can be reduced or eliminated by introduction of an untranslated RNA molecule (e.g., antisense RNA, RNAi, and the like, for example).
  • the untranslated RNA is encoded by a heterologous nucleotide sequence introduced to a host microorganism.
  • transaldolase activity refers to conversion of sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate to erythrose 4-phosphate and fructose 6-phosphate.
  • the transaldolase activity can be provided by a polypeptide.
  • the polypeptide is encoded by a heterologous nucleotide sequence introduced to a host microorganism.
  • Nucleic acid sequences conferring transaldolase activity can be obtained from a number of sources, including, but not limited to S. cerevisiae, Kluyveromyces, Pichia, Escherichia, Bacillus, Ruminococcus, Schizosaccharomyces , and Candida . Examples of an amino acid sequence of a polypeptide having transaldolase activity, and a nucleotide sequence of a polynucleotide that encodes the polypeptide, are presented below in the examples.
  • the transaldolase activity can be temporarily or permanently reduced or eliminated by genetic modification, as described below.
  • the genetic modification renders the activity responsive to changes in the environment.
  • the genetic modification disrupts synthesis of a functional nucleic acid encoding the activity or produces a nonfunctional polypeptide or protein.
  • Nucleic acid sequences that can be used to reduce or eliminate the activity of transaldolase can have sequences partially or substantially complementary to nucleic acid sequences that encode transaldolase activity. Presence, absence or amount of transaldolase activity can be detected by any suitable method known in the art, including nucleic acid based analysis and western blot analysis.
  • glucose/xylose transport activity refers to the import of glucose and/or xylose into a cell or organism by an activity that transports glucose and/or xylose across cell membranes.
  • the glucose/xylose transport activity can be provided by a polypeptide.
  • the polypeptide is encoded by a heterologous nucleotide sequence introduced to a host microorganism.
  • Nucleic acid sequences conferring glucose/xylose transport activity can be obtained from a number of sources, including, but not limited to Pichia yeast, Saccharomyces cerevisiae, Candida albicans, Debaryomyces hansenii, Schizosaccaromyces pombe .
  • Presence, absence or amount of glucose/xylose transport activity can be detected by any suitable method known in the art, including nucleic acid based analysis and western blot analysis.
  • Activities described herein can be modified to generate microorganisms engineered to allow a method of independently regulating or controlling (e.g., ability to independently turn on or off, or increase or decrease, for example) six-carbon sugar metabolism, five-carbon sugar metabolism, atmospheric carbon metabolism (e.g., carbon dioxide fixation) or combinations thereof.
  • regulated control of a desired activity can be the result of a genetic modification.
  • the genetic modification can be modification of a promoter sequence.
  • the modification can increase of decrease an activity encoded by a gene operably linked to the promoter element.
  • the modification to the promoter element can add or remove a regulatory sequence.
  • the regulatory sequence can respond to a change in environmental or culture conditions. Non-limiting examples of culture conditions that could be used to regulate an activity in this manner include, temperature, light, oxygen, salt, metals and the like. Additional methods for altering an activity by modification of a promoter element are given below.
  • the genetic modification can be to an ORF.
  • the modification of the ORF can increase or decrease expression of the ORF.
  • modification of the ORF can alter the efficiency of translation of the ORF.
  • modification of the ORF can alter the activity of the polypeptide or protein encoded by the ORF. Additional methods for altering an activity by modification of an ORF are given below.
  • the genetic modification can be to an activity associated with cell division (e.g., cell division cycle or CDC activity, for example).
  • the cell division cycle activity can be thymidylate synthase activity.
  • regulated control of cell division can be the result of a genetic modification.
  • the genetic modification can be to a nucleic acid sequence that encodes thymidylate synthase.
  • the genetic modification can temporarily inactivate thymidylate synthase activity by rendering the activity temperature sensitive (e.g., heat resistant, heat sensitive, cold resistant, cold sensitive and the like).
  • the genetic modification can modify a promoter sequence operably linked to a gene encoding an activity involved in control of cell division. In some embodiments the modification can increase of decrease an activity encoded by a gene operably linked to the promoter element. In certain embodiments, the modification to the promoter element can add or remove a regulatory sequence. In some embodiments the regulatory sequence can respond to a change in environmental or culture conditions. Non-limiting examples of culture conditions that could be used to regulate an activity in this manner include, temperature, light, oxygen, salt, metals and the like.
  • an engineered microorganism comprising one or more activities described above or below can be used in to produce ethanol by inhibiting cell growth and cell division by use of a temperature sensitive cell division control activity while allowing cellular fermentation to proceed, thereby producing a significant increase in ethanol yield when compared to the native organism.
  • a nucleic acid (e.g., also referred to herein as nucleic acid reagent, target nucleic acid, target nucleotide sequence, nucleic acid sequence of interest or nucleic acid region of interest) can be from any source or composition, such as DNA, cDNA, gDNA (genomic DNA), RNA, siRNA (short inhibitory RNA), RNAi, tRNA or mRNA, for example, and can be in any form (e.g., linear, circular, supercoiled, single-stranded, double-stranded, and the like).
  • a nucleic acid can also comprise DNA or RNA analogs (e.g., containing base analogs, sugar analogs and/or a non-native backbone and the like).
  • nucleic acid does not refer to or infer a specific length of the polynucleotide chain, thus polynucleotides and oligonucleotides are also included in the definition.
  • Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine.
  • the uracil base is uridine.
  • a nucleic acid sometimes is a plasmid, phage, autonomously replicating sequence (ARS), centromere, artificial chromosome, yeast artificial chromosome (e.g., YAC) or other nucleic acid able to replicate or be replicated in a host cell.
  • a nucleic acid can be from a library or can be obtained from enzymatically digested, sheared or sonicated genomic DNA (e.g., fragmented) from an organism of interest.
  • nucleic acid subjected to fragmentation or cleavage may have a nominal, average or mean length of about 5 to about 10,000 base pairs, about 100 to about 1,000 base pairs, about 100 to about 500 base pairs, or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 base pairs.
  • Fragments can be generated by any suitable method in the art, and the average, mean or nominal length of nucleic acid fragments can be controlled by selecting an appropriate fragment-generating procedure by the person of ordinary skill.
  • the fragmented DNA can be size selected to obtain nucleic acid fragments of a particular size range.
  • Nucleic acid can be fragmented by various methods known to the person of ordinary skill, which include without limitation, physical, chemical and enzymic processes. Examples of such processes are described in U.S. Patent Application Publication No. 20050112590 (published on May 26, 2005, entitled “Fragmentation-based methods and systems for sequence variation detection and discovery,” naming Van Den Boom et al.). Certain processes can be selected by the person of ordinary skill to generate non-specifically cleaved fragments or specifically cleaved fragments.
  • Examples of processes that can generate non-specifically cleaved fragment sample nucleic acid include, without limitation, contacting sample nucleic acid with apparatus that expose nucleic acid to shearing force (e.g., passing nucleic acid through a syringe needle; use of a French press); exposing sample nucleic acid to irradiation (e.g., gamma, x-ray, UV irradiation; fragment sizes can be controlled by irradiation intensity); boiling nucleic acid in water (e.g., yields about 500 base pair fragments) and exposing nucleic acid to an acid and base hydrolysis process.
  • shearing force e.g., passing nucleic acid through a syringe needle; use of a French press
  • irradiation e.g., gamma, x-ray, UV irradiation; fragment sizes can be controlled by irradiation intensity
  • boiling nucleic acid in water e.g., yields about
  • Nucleic acid may be specifically cleaved by contacting the nucleic acid with one or more specific cleavage agents.
  • specific cleavage agent refers to an agent, sometimes a chemical or an enzyme that can cleave a nucleic acid at one or more specific sites. Specific cleavage agents often will cleave specifically according to a particular nucleotide sequence at a particular site. Examples of enzymic specific cleavage agents include without limitation endonucleases (e.g., DNase (e.g., DNase I, II); RNase (e.g., RNase E, F, H, P); CleavaseTM enzyme; Taq DNA polymerase; E.
  • endonucleases e.g., DNase (e.g., DNase I, II); RNase (e.g., RNase E, F, H, P); CleavaseTM enzyme; Taq DNA polymerase; E.
  • coli DNA polymerase I and eukaryotic structure-specific endonucleases murine FEN-1 endonucleases; type I, II or III restriction endonucleases such as Acc I, Afl III, Alu I, Alw44 I, Apa I, Asn I, Ava I, Ava II, BamH I, Ban II, Bcl I, Bgl I.
  • Sample nucleic acid may be treated with a chemical agent, or synthesized using modified nucleotides, and the modified nucleic acid may be cleaved.
  • sample nucleic acid may be treated with (i) alkylating agents such as methylnitrosourea that generate several alkylated bases, including N3-methyladenine and N3-methylguanine, which are recognized and cleaved by alkyl purine DNA-glycosylase; (ii) sodium bisulfite, which causes deamination of cytosine residues in DNA to form uracil residues that can be cleaved by uracil N-glycosylase; and (iii) a chemical agent that converts guanine to its oxidized form, 8-hydroxyguanine, which can be cleaved by formamidopyrimidine DNA N-glycosylase.
  • alkylating agents such as methylnitrosourea that generate several alkylated bases, including N3-methyla
  • Examples of chemical cleavage processes include without limitation alkylation, (e.g., alkylation of phosphorothioate-modified nucleic acid); cleavage of acid lability of P3′-N5′-phosphoroamidate-containing nucleic acid; and osmium tetroxide and piperidine treatment of nucleic acid.
  • alkylation e.g., alkylation of phosphorothioate-modified nucleic acid
  • cleavage of acid lability of P3′-N5′-phosphoroamidate-containing nucleic acid e.g., osmium tetroxide and piperidine treatment of nucleic acid.
  • nucleic acids of interest may be treated with one or more specific cleavage agents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more specific cleavage agents) in one or more reaction vessels (e.g., nucleic acid of interest is treated with each specific cleavage agent in a separate vessel).
  • specific cleavage agents e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more specific cleavage agents
  • a nucleic acid suitable for use in the embodiments described herein sometimes is amplified by any amplification process known in the art (e.g., PCR, RT-PCR and the like). Nucleic acid amplification may be particularly beneficial when using organisms that are typically difficult to culture (e.g., slow growing, require specialize culture conditions and the like).
  • the terms “amplify”, “amplification”, “amplification reaction”, or “amplifying” as used herein, refer to any in vitro processes for multiplying the copies of a target sequence of nucleic acid. Amplification sometimes refers to an “exponential” increase in target nucleic acid.
  • amplifying can also refer to linear increases in the numbers of a select target sequence of nucleic acid, but is different than a one-time, single primer extension step.
  • a limited amplification reaction also known as pre-amplification
  • Pre-amplification is a method in which a limited amount of amplification occurs due to a small number of cycles, for example 10 cycles, being performed.
  • Pre-amplification can allow some amplification, but stops amplification prior to the exponential phase, and typically produces about 500 copies of the desired nucleotide sequence(s).
  • Use of pre-amplification may also limit inaccuracies associated with depleted reactants in standard PCR reactions.
  • a nucleic acid reagent sometimes is stably integrated into the chromosome of the host organism, or a nucleic acid reagent can be a deletion of a portion of the host chromosome, in certain embodiments (e.g., genetically modified organisms, where alteration of the host genome confers the ability to selectively or preferentially maintain the desired organism carrying the genetic modification).
  • nucleic acid reagents e.g., nucleic acids or genetically modified organisms whose altered genome confers a selectable trait to the organism
  • the nucleic acid reagent can be altered such that codons encode for (i) the same amino acid, using a different tRNA than that specified in the native sequence, or (ii) a different amino acid than is normal, including unconventional or unnatural amino acids (including detectably labeled amino acids).
  • native sequence refers to an unmodified nucleotide sequence as found in its natural setting (e.g., a nucleotide sequence as found in an organism).
  • a nucleic acid or nucleic acid reagent can comprise certain elements often selected according to the intended use of the nucleic acid. Any of the following elements can be included in or excluded from a nucleic acid reagent.
  • a nucleic acid reagent may include one or more or all of the following nucleotide elements: one or more promoter elements, one or more 5′ untranslated regions (5′UTRs), one or more regions into which a target nucleotide sequence may be inserted (an “insertion element”), one or more target nucleotide sequences, one or more 3′ untranslated regions (3′UTRs), and one or more selection elements.
  • a nucleic acid reagent can be provided with one or more of such elements and other elements may be inserted into the nucleic acid before the nucleic acid is introduced into the desired organism.
  • a provided nucleic acid reagent comprises a promoter, 5′UTR, optional 3′UTR and insertion element(s) by which a target nucleotide sequence is inserted (i.e., cloned) into the nucleotide acid reagent.
  • a provided nucleic acid reagent comprises a promoter, insertion element(s) and optional 3′UTR, and a 5′ UTR/target nucleotide sequence is inserted with an optional 3′UTR.
  • a nucleic acid reagent comprises the following elements in the 5′ to 3′ direction: (1) promoter element, 5′UTR, and insertion element(s); (2) promoter element, 5′UTR, and target nucleotide sequence; (3) promoter element, 5′UTR, insertion element(s) and 3′UTR; and (4) promoter element, 5′UTR, target nucleotide sequence and 3′UTR.
  • a promoter element typically is required for DNA synthesis and/or RNA synthesis.
  • a promoter element often comprises a region of DNA that can facilitate the transcription of a particular gene, by providing a start site for the synthesis of RNA corresponding to a gene. Promoters generally are located near the genes they regulate, are located upstream of the gene (e.g., 5′ of the gene), and are on the same strand of DNA as the sense strand of the gene, in some embodiments.
  • a promoter often interacts with a RNA polymerase.
  • a polymerase is an enzyme that catalyses synthesis of nucleic acids using a preexisting nucleic acid reagent.
  • the template is a DNA template
  • an RNA molecule is transcribed before protein is synthesized.
  • Enzymes having polymerase activity suitable for use in the present methods include any polymerase that is active in the chosen system with the chosen template to synthesize protein.
  • a promoter e.g., a heterologous promoter
  • a promoter element can be operably linked to a nucleotide sequence or an open reading frame (ORF).
  • RNA corresponding to the nucleotide sequence or ORF sequence operably linked to the promoter can catalyze the synthesis of an RNA corresponding to the nucleotide sequence or ORF sequence operably linked to the promoter, which in turn leads to synthesis of a desired peptide, polypeptide or protein.
  • operably linked refers to a nucleic acid sequence (e.g., a coding sequence) present on the same nucleic acid molecule as a promoter element and whose expression is under the control of said promoter element.
  • Promoter elements sometimes exhibit responsiveness to regulatory control.
  • Promoter elements also sometimes can be regulated by a selective agent. That is, transcription from promoter elements sometimes can be turned on, turned off, up-regulated or down-regulated, in response to a change in environmental, nutritional or internal conditions or signals (e.g., heat inducible promoters, light regulated promoters, feedback regulated promoters, hormone influenced promoters, tissue specific promoters, oxygen and pH influenced promoters, promoters that are responsive to selective agents (e.g., kanamycin) and the like, for example).
  • Promoters influenced by environmental, nutritional or internal signals frequently are influenced by a signal (direct or indirect) that binds at or near the promoter and increases or decreases expression of the target sequence under certain conditions.
  • Non-limiting examples of selective or regulatory agents that can influence transcription from a promoter element used in embodiments described herein include, without limitation, (1) nucleic acid segments that encode products that provide resistance against otherwise toxic compounds (e.g., antibiotics); (2) nucleic acid segments that encode products that are otherwise lacking in the recipient cell (e.g., essential products, tRNA genes, auxotrophic markers); (3) nucleic acid segments that encode products that suppress the activity of a gene product; (4) nucleic acid segments that encode products that can be readily identified (e.g., phenotypic markers such as antibiotics (e.g., ⁇ -lactamase), ⁇ -galactosidase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), and cell surface proteins); (5) nucleic acid segments that bind products that are otherwise detrimental to cell survival and/or function; (6) nucleic acid segments that otherwise inhibit the activity of any of the nucleic acid segments described in Nos.
  • nucleic acid segments that bind products that modify a substrate e.g., restriction endonucleases
  • nucleic acid segments that can be used to isolate or identify a desired molecule e.g., specific protein binding sites
  • nucleic acid segments that encode a specific nucleotide sequence that can be otherwise non-functional e.g., for PCR amplification of subpopulations of molecules
  • nucleic acid segments that, when absent, directly or indirectly confer resistance or sensitivity to particular compounds (11) nucleic acid segments that encode products that either are toxic or convert a relatively non-toxic compound to a toxic compound (e.g., Herpes simplex thymidine kinase, cytosine deaminase) in recipient cells; (12) nucleic acid segments that inhibit replication, partition or heritability of nucleic acid molecules that contain them; and/or (13) nucleic acid segments that encode condition
  • regulation of a promoter element can be used to alter (e.g., increase, add, decrease or substantially eliminate) the activity of a peptide, polypeptide or protein (e.g., enzyme activity for example).
  • a microorganism can be engineered by genetic modification to express a nucleic acid reagent that can add a novel activity (e.g., an activity not normally found in the host organism) or increase the expression of an existing activity by increasing transcription from a homologous or heterologous promoter operably linked to a nucleotide sequence of interest (e.g., homologous or heterologous nucleotide sequence of interest), in certain embodiments.
  • a microorganism can be engineered by genetic modification to express a nucleic acid reagent that can decrease expression of an activity by decreasing or substantially eliminating transcription from a homologous or heterologous promoter operably linked to a nucleotide sequence of interest, in certain embodiments.
  • the activity can be altered using recombinant DNA and genetic techniques known to the artisan. Methods for engineering microorganisms are further described herein. Tables herein provide non-limiting lists of yeast promoters that are up-regulated by oxygen, yeast promoters that are down-regulated by oxygen, yeast transcriptional repressors and their associated genes, DNA binding motifs as determined using the MEME sequence analysis software. Potential regulator binding motifs can be identified using the program MEME to search intergenic regions bound by regulators for overrepresented sequences. For each regulator, the sequences of intergenic regions bound with p-values less than 0.001 were extracted to use as input for motif discovery.
  • the MEME software was run using the following settings: a motif width ranging from 6 to 18 bases, the “zoops” distribution model, a 6th order Markov background model and a discovery limit of 20 motifs.
  • the discovered sequence motifs were scored for significance by two criteria: an E-value calculated by MEME and a specificity score. The motif with the best score using each metric is shown for each regulator. All motifs presented are derived from datasets generated in rich growth conditions with the exception of a previously published dataset for epitope-tagged Gal4 grown in galactose
  • the altered activity can be found by screening the organism under conditions that select for the desired change in activity.
  • certain microorganisms can be adapted to increase or decrease an activity by selecting or screening the organism in question on a media containing substances that are poorly metabolized or even toxic.
  • An increase in the ability of an organism to grow a substance that is normally poorly metabolized would result in an increase in the growth rate on that substance, for example.
  • a decrease in the sensitivity to a toxic substance might be manifested by growth on higher concentrations of the toxic substance, for example.
  • Genetic modifications that are identified in this manner sometimes are referred to as naturally occurring mutations or the organisms that carry them can sometimes be referred to as naturally occurring mutants. Modifications obtained in this manner are not limited to alterations in promoter sequences.
  • screening microorganisms by selective pressure can yield genetic alterations that can occur in non-promoter sequences, and sometimes also can occur in sequences that are not in the nucleotide sequence of interest, but in a related nucleotide sequences (e.g., a gene involved in a different step of the same pathway, a transport gene, and the like).
  • Naturally occurring mutants sometimes can be found by isolating naturally occurring variants from unique environments, in some embodiments.
  • a nucleic acid reagent may include a polynucleotide sequence 70% or more identical to the foregoing (or to the complementary sequences).
  • nucleotide sequence that is at least 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to a nucleotide sequence described herein can be utilized.
  • nucleotide sequences having substantially the same nucleotide sequence when compared to each other.
  • One test for determining whether two nucleotide sequences or amino acids sequences are substantially identical is to determine the percent of identical nucleotide sequences or amino acid sequences shared.
  • sequence identity can be performed as follows. Sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is sometimes 30% or more, 40% or more, 50% or more, often 60% or more, and more often 70% or more, 80% or more, 90% or more, or 100% of the length of the reference sequence.
  • the nucleotides or amino acids at corresponding nucleotide or polypeptide positions, respectively, are then compared among the two sequences.
  • the nucleotides or amino acids are deemed to be identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, introduced for optimal alignment of the two sequences.
  • Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers & Miller, CABIOS 4: 11-17 (1989), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Also, percent identity between two amino acid sequences can be determined using the Needleman & Wunsch, J. Mol. Biol.
  • Sequence identity can also be determined by hybridization assays conducted under stringent conditions.
  • stringent conditions refers to conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6 (1989). Aqueous and non-aqueous methods are described in that reference and either can be used.
  • An example of stringent hybridization conditions is hybridization in 6 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2 ⁇ SSC, 0.1% SDS at 50° C.
  • SSC sodium chloride/sodium citrate
  • stringent hybridization conditions are hybridization in 6 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2 ⁇ SSC, 0.1% SDS at 55° C.
  • a further example of stringent hybridization conditions is hybridization in 6 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2 ⁇ SSC, 0.1% SDS at 60° C.
  • stringent hybridization conditions are hybridization in 6 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2 ⁇ SSC, 0.1% SDS at 65° C. More often, stringency conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2 ⁇ SSC, 1% SDS at 65° C.
  • nucleic acid reagents may also comprise one or more 5′ UTR's, and one or more 3′UTR's.
  • a 5′ UTR may comprise one or more elements endogenous to the nucleotide sequence from which it originates, and sometimes includes one or more exogenous elements.
  • a 5′ UTR can originate from any suitable nucleic acid, such as genomic DNA, plasmid DNA, RNA or mRNA, for example, from any suitable organism (e.g., virus, bacterium, yeast, fungi, plant, insect or mammal). The artisan may select appropriate elements for the 5′ UTR based upon the chosen expression system (e.g., expression in a chosen organism, or expression in a cell free system, for example).
  • a 5′ UTR sometimes comprises one or more of the following elements known to the artisan: enhancer sequences (e.g., transcriptional or translational), transcription initiation site, transcription factor binding site, translation regulation site, translation initiation site, translation factor binding site, accessory protein binding site, feedback regulation agent binding sites, Pribnow box, TATA box, ⁇ 35 element, E-box (helix-loop-helix binding element), ribosome binding site, replicon, internal ribosome entry site (IRES), silencer element and the like.
  • a promoter element may be isolated such that all 5′ UTR elements necessary for proper conditional regulation are contained in the promoter element fragment, or within a functional subsequence of a promoter element fragment.
  • a 5′UTR in the nucleic acid reagent can comprise a translational enhancer nucleotide sequence.
  • a translational enhancer nucleotide sequence often is located between the promoter and the target nucleotide sequence in a nucleic acid reagent.
  • a translational enhancer sequence often binds to a ribosome, sometimes is an 18S rRNA-binding ribonucleotide sequence (i.e., a 40S ribosome binding sequence) and sometimes is an internal ribosome entry sequence (IRES).
  • An IRES generally forms an RNA scaffold with precisely placed RNA tertiary structures that contact a 40S ribosomal subunit via a number of specific intermolecular interactions.
  • ribosomal enhancer sequences are known and can be identified by the artisan (e.g., Mumblee et al., Nucleic Acids Research 33: D141-D146 (2005); Paulous et al., Nucleic Acids Research 31: 722-733 (2003); Akbergenov et al., Nucleic Acids Research 32: 239-247 (2004); Mignone et al., Genome Biology 3(3): reviews0004.1-0001.10 (2002); Gallie, Nucleic Acids Research 30: 3401-3411 (2002); Shaloiko et al., http address www.interscience.wiley.com, DOI: 10.1002/bit.20267; and Gallie et al., Nucleic Acids Research 15: 3257-3273 (1987)).
  • a translational enhancer sequence sometimes is a eukaryotic sequence, such as a Kozak consensus sequence or other sequence (e.g., hydroid polyp sequence, GenBank accession no. U07128).
  • a translational enhancer sequence sometimes is a prokaryotic sequence, such as a Shine-Dalgarno consensus sequence.
  • the translational enhancer sequence is a viral nucleotide sequence.
  • a translational enhancer sequence sometimes is from a 5′ UTR of a plant virus, such as Tobacco Mosaic Virus (TMV), Alfalfa Mosaic Virus (AMV); Tobacco Etch Virus (ETV); Potato Virus Y (PVY); Turnip Mosaic (poty) Virus and Pea Seed Borne Mosaic Virus, for example.
  • TMV Tobacco Mosaic Virus
  • AMV Alfalfa Mosaic Virus
  • ETV Tobacco Etch Virus
  • PVY Potato Virus Y
  • Turnip Mosaic (poty) Virus and Pea Seed Borne Mosaic Virus for example.
  • an omega sequence about 67 bases in length from TMV is included in the nucleic acid reagent as a translational enhancer sequence (e.g., devoid of guanosine nucleotides and includes a 25 nucleotide long poly (CAA) central region).
  • CAA nucleotide long poly
  • a 3′ UTR may comprise one or more elements endogenous to the nucleotide sequence from which it originates and sometimes includes one or more exogenous elements.
  • a 3′ UTR may originate from any suitable nucleic acid, such as genomic DNA, plasmid DNA, RNA or mRNA, for example, from any suitable organism (e.g., a virus, bacterium, yeast, fungi, plant, insect or mammal). The artisan can select appropriate elements for the 3′ UTR based upon the chosen expression system (e.g., expression in a chosen organism, for example).
  • a 3′ UTR sometimes comprises one or more of the following elements known to the artisan: transcription regulation site, transcription initiation site, transcription termination site, transcription factor binding site, translation regulation site, translation termination site, translation initiation site, translation factor binding site, ribosome binding site, replicon, enhancer element, silencer element and polyadenosine tail.
  • a 3′ UTR often includes a polyadenosine tail and sometimes does not, and if a polyadenosine tail is present, one or more adenosine moieties may be added or deleted from it (e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50 adenosine moieties may be added or subtracted).
  • modification of a 5′ UTR and/or a 3′ UTR can be used to alter (e.g., increase, add, decrease or substantially eliminate) the activity of a promoter.
  • Alteration of the promoter activity can in turn alter the activity of a peptide, polypeptide or protein (e.g., enzyme activity for example), by a change in transcription of the nucleotide sequence(s) of interest from an operably linked promoter element comprising the modified 5′ or 3′ UTR.
  • a microorganism can be engineered by genetic modification to express a nucleic acid reagent comprising a modified 5′ or 3′ UTR that can add a novel activity (e.g., an activity not normally found in the host organism) or increase the expression of an existing activity by increasing transcription from a homologous or heterologous promoter operably linked to a nucleotide sequence of interest (e.g., homologous or heterologous nucleotide sequence of interest), in certain embodiments.
  • a novel activity e.g., an activity not normally found in the host organism
  • a nucleotide sequence of interest e.g., homologous or heterologous nucleotide sequence of interest
  • a microorganism can be engineered by genetic modification to express a nucleic acid reagent comprising a modified 5′ or 3′ UTR that can decrease the expression of an activity by decreasing or substantially eliminating transcription from a homologous or heterologous promoter operably linked to a nucleotide sequence of interest, in certain embodiments.
  • a nucleotide reagent sometimes can comprise a target nucleotide sequence.
  • a “target nucleotide sequence” as used herein encodes a nucleic acid, peptide, polypeptide or protein of interest, and may be a ribonucleotide sequence or a deoxyribonucleotide sequence.
  • a target nucleic acid sometimes can comprise a chimeric nucleic acid (or chimeric nucleotide sequence), which can encode a chimeric protein (or chimeric amino acid sequence).
  • chimeric refers to a nucleic acid or nucleotide sequence, or encoded product thereof, containing sequences from two or more different sources.
  • Any suitable source can be selected, including, but not limited to, a sequence from a nucleic acid, nucleotide sequence, ribosomal nucleic acid, RNA, DNA, regulatory nucleotide sequence (e.g., promoter, URL, enhancer, repressor and the like), coding nucleic acid, gene, nucleic acid linker, nucleic acid tag, amino acid sequence, peptide, polypeptide, protein, chromosome, and organism.
  • regulatory nucleotide sequence e.g., promoter, URL, enhancer, repressor and the like
  • a chimeric molecule can include a sequence of contiguous nucleotides or amino acids from a source including, but not limited to, a virus, prokaryote, eukaryote, genus, species, homolog, ortholog, paralog and isozyme, nucleic acid linkers, nucleic acid tags, the like and combinations thereof).
  • a chimeric molecule can be generated by placing in juxtaposition fragments of related or unrelated nucleic acids, nucleotide sequences or DNA segments, in some embodiments.
  • the nucleic acids, nucleotide sequences or DNA segments can be native or wild type sequences, mutant sequences or engineered sequences (completely engineered or engineered to a point, for example).
  • a chimera includes about 1, 2, 3, 4 or 5 sequences (e.g., contiguous nucleotides, contiguous amino acids) from one organism and 1, 2, 3, 4 or 5 sequences (e.g., contiguous nucleotides, contiguous amino acids) from another organism.
  • the organisms sometimes are a microbe, such as a bacterium (e.g., gram positive, gram negative), yeast or fungus (e.g., aerobic fungus, anaerobic fungus), for example.
  • the organisms are bacteria, the organisms are yeast or the organisms are fungi (e.g., different species), and sometimes one organism is a bacterium or yeast and another is a fungus.
  • a chimeric molecule may contain up to about 99% of sequences from one organism (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99%) and the balance percentage from one or more other organisms.
  • a chimeric molecule includes altered codons (in the case of a chimeric nucleic acid) and one or more mutations (e.g., point mutations, nucleotide substitutions, amino acid substitutions).
  • a chimera sometimes is the result of recombination between two or more nucleic acids, nucleotide sequences or genes, and sometimes is the result of genetic manipulation (e.g., designed and/or generated by the hand of a human being).
  • Any suitable nucleic acid or nucleotide sequence and method for combining nucleic acids or nucleotide sequences can be used to generate a chimeric nucleic acid or nucleotide sequence.
  • Non-limiting examples of nucleic acid and nucleotide sequence sources and methods for generating chimeric nucleic acids and nucleotide sequences are presented herein.
  • fragments used to generate a chimera can be juxtaposed as units (e.g., nucleic acid from the sources are combined end to end and not interspersed.
  • nucleotide sequence combinations can be noted as DNA source 1 DNA source 2 or DNA source 1/DNA source 2/DNA source 3, the like and combinations thereof, for example.
  • fragments used to generate a chimera can be juxtaposed such that one or more fragments from one or more sources can be interspersed with other fragments used to generate the chimera (e.g., DNA source 1/DNA source 2/DNA source 1/DNA source 3/DNA source 2/DNA source 1).
  • the nucleotide sequence length of the fragments used to generate a chimera can be in the range from about 5 base pairs to about 5,000 base pairs (e.g., about 5 base pairs (bp), about 10 bp, about 15 bp, about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 125 bp, about 150 bp, about 175 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, about 500 bp, about 550 bp, about 600 bp, about 650 b
  • a chimeric nucleic acid or nucleotide sequence encodes the same activity as the activity encoded by the source nucleic acids or nucleotide sequences. In some embodiments, a chimeric nucleic acid or nucleotide sequence has a similar or the same activity, but the amount of the activity, or kinetics of the activity, are altered (e.g., increased, decreased). In certain embodiments, a chimeric nucleic acid or nucleotide sequence encodes a different activity, and in some embodiments a chimeric nucleic acid or nucleotide sequences encodes a chimeric activity (e.g., a combination of two or more activities).
  • a target nucleic acid sometimes is an untranslated ribonucleic acid and sometimes is a translated ribonucleic acid.
  • An untranslated ribonucleic acid may include, but is not limited to, a small interfering ribonucleic acid (siRNA), a short hairpin ribonucleic acid (shRNA), other ribonucleic acid capable of RNA interference (RNAi), an antisense ribonucleic acid, or a ribozyme.
  • a translatable target nucleotide sequence (e.g., a target ribonucleotide sequence) sometimes encodes a peptide, polypeptide or protein, which are sometimes referred to herein as “target peptides,” “target polypeptides” or “target proteins.”
  • Any peptides, polypeptides or proteins, or an activity catalyzed by one or more peptides, polypeptides or proteins may be encoded by a target nucleotide sequence and may be selected by a person of ordinary skill in the art.
  • Representative proteins include enzymes (e.g., phosphofructokinase activity, phosphogluconate dehydratase activity, 2-keto-3-deoxygluconate-6-phosphate aldolase activity, xylose isomerase activity, phosphoenolpyruvate carboxylase activity, alcohol dehydrogenase 2 activity and thymidylate synthase activity and the like, for example), antibodies, serum proteins (e.g., albumin), membrane bound proteins, hormones (e.g., growth hormone, erythropoietin, insulin, etc.), cytokines, etc., and include both naturally occurring and exogenously expressed polypeptides.
  • enzymes e.g., phosphofructokinase activity, phosphogluconate dehydratase activity, 2-keto-3-deoxygluconate-6-phosphate aldolase activity, xylose isomerase activity, phosphoenolpyruvate carboxylase activity, alcohol dehydrogenase
  • Representative activities include phosphofructokinase activity, phosphogluconate dehydratase activity, 2-keto-3-deoxygluconate-6-phosphate aldolase activity, xylose isomerase activity, phosphoenolpyruvate carboxylase activity, alcohol dehydrogenase 2 activity and thymidylate synthase activity and the like for example.
  • enzyme refers to a protein which can act as a catalyst to induce a chemical change in other compounds, thereby producing one or more products from one or more substrates.
  • polypeptides e.g., enzymes
  • protein refers to a molecule having a sequence of amino acids linked by peptide bonds. This term includes fusion proteins, oligopeptides, peptides, cyclic peptides, polypeptides and polypeptide derivatives, whether native or recombinant, and also includes fragments, derivatives, homologs, and variants thereof.
  • a protein or polypeptide sometimes is of intracellular origin (e.g., located in the nucleus, cytosol, or interstitial space of host cells in vivo) and sometimes is a cell membrane protein in vivo.
  • a genetic modification can result in a modification (e.g., increase, substantially increase, decrease or substantially decrease) of a target activity.
  • a translatable nucleotide sequence generally is located between a start codon (AUG in ribonucleic acids and ATG in deoxyribonucleic acids) and a stop codon (e.g., UAA (ochre), UAG (amber) or UGA (opal) in ribonucleic acids and TAA, TAG or TGA in deoxyribonucleic acids), and sometimes is referred to herein as an “open reading frame” (ORF).
  • a nucleic acid reagent sometimes comprises one or more ORFs.
  • An ORF may be from any suitable source, sometimes from genomic DNA, mRNA, reverse transcribed RNA or complementary DNA (cDNA) or a nucleic acid library comprising one or more of the foregoing, and is from any organism species that contains a nucleic acid sequence of interest, protein of interest, or activity of interest.
  • organisms from which an ORF can be obtained include bacteria, yeast, fungi, human, insect, nematode, bovine, equine, canine, feline, rat or mouse, for example.
  • a nucleic acid reagent sometimes comprises a nucleotide sequence adjacent to an ORF that is translated in conjunction with the ORF and encodes an amino acid tag.
  • the tag-encoding nucleotide sequence is located 3′ and/or 5′ of an ORF in the nucleic acid reagent, thereby encoding a tag at the C-terminus or N-terminus of the protein or peptide encoded by the ORF. Any tag that does not abrogate in vitro transcription and/or translation may be utilized and may be appropriately selected by the artisan. Tags may facilitate isolation and/or purification of the desired ORF product from culture or fermentation media.
  • a tag sometimes specifically binds a molecule or moiety of a solid phase or a detectable label, for example, thereby having utility for isolating, purifying and/or detecting a protein or peptide encoded by the ORF.
  • a tag comprises one or more of the following elements: FLAG (e.g., DYKDDDDKG (SEQ ID NO: 29)), V5 (e.g., GKPIPNPLLGLDST (SEQ ID NO: 30)), c-MYC (e.g., EQKLISEEDL (SEQ ID NO: 31)), HSV (e.g., QPELAPEDPED (SEQ ID NO: 32)), influenza hemaglutinin, HA (e.g., YPYDVPDYA (SEQ ID NO: 33)), VSV-G (e.g., YTDIEMNRLGK (SEQ ID NO: 34)), bacterial glutathione-S-transferase, maltose binding protein
  • a cysteine-rich tag comprises the amino acid sequence CC-Xn-CC (SEQ ID NO: 36), wherein X is any amino acid and n is 1 to 3, and the cysteine-rich sequence sometimes is CCPGCC (SEQ ID NO: 37).
  • the tag comprises a cysteine-rich element and a polyhistidine element (e.g., CCPGCC (SEQ ID NO: 37) and His6 (SEQ ID NO: 35)).
  • a tag often conveniently binds to a binding partner.
  • some tags bind to an antibody (e.g., FLAG) and sometimes specifically bind to a small molecule.
  • a polyhistidine tag specifically chelates a bivalent metal, such as copper, zinc and cobalt;
  • a polylysine or polyarginine tag specifically binds to a zinc finger;
  • a glutathione S-transferase tag binds to glutathione;
  • a cysteine-rich tag specifically binds to an arsenic-containing molecule.
  • Arsenic-containing molecules include LUMIOTM agents (Invitrogen, California), such as FlAsHTM (EDT2[4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2]) and ReAsH reagents (e.g., U.S. Pat. No. 5,932,474 to Tsien et al., entitled “Target Sequences for Synthetic Molecules;” U.S. Pat. No. 6,054,271 to Tsien et al., entitled “Methods of Using Synthetic Molecules and Target Sequences;” U.S. Pat. Nos.
  • a tag sometimes comprises a sequence that localizes a translated protein or peptide to a component in a system, which is referred to as a “signal sequence” or “localization signal sequence” herein.
  • a signal sequence often is incorporated at the N-terminus of a target protein or target peptide, and sometimes is incorporated at the C-terminus. Examples of signal sequences are known to the artisan, are readily incorporated into a nucleic acid reagent, and often are selected according to the organism in which expression of the nucleic acid reagent is performed.
  • a signal sequence in some embodiments localizes a translated protein or peptide to a cell membrane.
  • signal sequences include, but are not limited to, a nucleus targeting signal (e.g., steroid receptor sequence and N-terminal sequence of SV40 virus large T antigen); mitochondrial targeting signal (e.g., amino acid sequence that forms an amphipathic helix); peroxisome targeting signal (e.g., C-terminal sequence in YFG from S. cerevisiae ); and a secretion signal (e.g., N-terminal sequences from invertase, mating factor alpha, PHO5 and SUC2 in S. cerevisiae ; multiple N-terminal sequences of B. subtilis proteins (e.g., Tjalsma et al., Microbiol. Molec. Biol.
  • a nucleus targeting signal e.g., steroid receptor sequence and N-terminal sequence of SV40 virus large T antigen
  • mitochondrial targeting signal e.g., amino acid sequence that forms an amphipathic helix
  • alpha amylase signal sequence e.g., U.S. Pat. No. 6,288,302
  • pectate lyase signal sequence e.g., U.S. Pat. No. 5,846,8178
  • precollagen signal sequence e.g., U.S. Pat. No. 5,712,114
  • OmpA signal sequence e.g., U.S. Pat. No. 5,470,719
  • Iam beta signal sequence e.g., U.S. Pat. No. 5,389,529
  • B. brevis signal sequence e.g., U.S. Pat. No. 5,232,841
  • P. pastoris signal sequence e.g., U.S. Pat. No. 5,268,273
  • a tag sometimes is directly adjacent to the amino acid sequence encoded by an ORF (i.e., there is no intervening sequence) and sometimes a tag is substantially adjacent to an ORF encoded amino acid sequence (e.g., an intervening sequence is present).
  • An intervening sequence sometimes includes a recognition site for a protease, which is useful for cleaving a tag from a target protein or peptide.
  • the intervening sequence is cleaved by Factor Xa (e.g., recognition site I (E/D)GR), thrombin (e.g., recognition site LVPRGS (SEQ ID NO: 38)), enterokinase (e.g., recognition site DDDDK (SEQ ID NO: 39)), TEV protease (e.g., recognition site ENLYFQG (SEQ ID NO: 40)) or PreScissionTM protease (e.g., recognition site LEVLFQGP (SEQ ID NO: 41)), for example.
  • Factor Xa e.g., recognition site I (E/D)GR
  • thrombin e.g., recognition site LVPRGS (SEQ ID NO: 38)
  • enterokinase e.g., recognition site DDDDK (SEQ ID NO: 39)
  • TEV protease e.g., recognition site ENLYFQG (SEQ ID NO: 40)
  • linker sequence An intervening sequence sometimes is referred to herein as a “linker sequence,” and may be of any suitable length selected by the artisan.
  • a linker sequence sometimes is about 1 to about 20 amino acids in length, and sometimes about 5 to about 10 amino acids in length. The artisan may select the linker length to substantially preserve target protein or peptide function (e.g., a tag may reduce target protein or peptide function unless separated by a linker), to enhance disassociation of a tag from a target protein or peptide when a protease cleavage site is present (e.g., cleavage may be enhanced when a linker is present), and to enhance interaction of a tag/target protein product with a solid phase.
  • a linker can be of any suitable amino acid content, and often comprises a higher proportion of amino acids having relatively short side chains (e.g., glycine, alanine, serine and threonine).
  • a nucleic acid reagent sometimes includes a stop codon between a tag element and an insertion element or ORF, which can be useful for translating an ORF with or without the tag.
  • Mutant tRNA molecules that recognize stop codons (described above) suppress translation termination and thereby are designated “suppressor tRNAs.” Suppressor tRNAs can result in the insertion of amino acids and continuation of translation past stop codons (e.g., U.S. Patent Application No. 60/587,583, filed Jul. 14, 2004, entitled “Production of Fusion Proteins by Cell-Free Protein Synthesis,”; Eggertsson, et al., (1988) Microbiological Review 52(3):354-374, and Engleerg-Kukla, et al.
  • suppressor tRNAs are known, including but not limited to, supE, supP, supD, supF and supZ suppressors, which suppress the termination of translation of the amber stop codon; supB, gIT, supL, supN, supC and supM suppressors, which suppress the function of the ochre stop codon and glyT, trpT and Su-9 suppressors, which suppress the function of the opal stop codon.
  • supE, supP, supD, supF and supZ suppressors which suppress the termination of translation of the amber stop codon
  • supB, gIT, supL, supN, supC and supM suppressors which suppress the function of the ochre stop codon and glyT, trpT and Su-9 suppressors, which suppress the function of the opal stop codon.
  • suppressor tRNAs contain one or more mutations in the anti-codon loop of the tRNA that allows the tRNA to base pair with a codon that ordinarily functions as a stop codon.
  • the mutant tRNA is charged with its cognate amino acid residue and the cognate amino acid residue is inserted into the translating polypeptide when the stop codon is encountered. Mutations that enhance the efficiency of termination suppressors (i.e., increase stop codon read-through) have been identified.
  • mutations in the uar gene also known as the prfA gene
  • mutations in the ups gene mutations in the sueA, sueB and sueC genes
  • mutations in the rpsD ramA
  • rpsE spcA genes
  • mutations in the rplL gene include, but are not limited to, mutations in the uar gene (also known as the prfA gene), mutations in the ups gene, mutations in the sueA, sueB and sueC genes, mutations in the rpsD (ramA) and rpsE (spcA) genes and mutations in the rplL gene.
  • a nucleic acid reagent comprising a stop codon located between an ORF and a tag can yield a translated ORF alone when no suppressor tRNA is present in the translation system, and can yield a translated ORF-tag fusion when a suppressor tRNA is present in the system.
  • Suppressor tRNA can be generated in cells transfected with a nucleic acid encoding the tRNA (e.g., a replication incompetent adenovirus containing the human tRNA-Ser suppressor gene can be transfected into cells, or a YAC containing a yeast or bacterial tRNA suppressor gene can be transfected into yeast cells, for example).
  • Vectors for synthesizing suppressor tRNA and for translating ORFs with or without a tag are available to the artisan (e.g., Tag-On-DemandTM kit (Invitrogen Corporation, California); Tag-On-DemandTM Suppressor Supernatant Instruction Manual, Version B, 6 Jun.
  • Any convenient cloning strategy known in the art may be utilized to incorporate an element, such as an ORF, into a nucleic acid reagent.
  • Known methods can be utilized to insert an element into the template independent of an insertion element, such as (1) cleaving the template at one or more existing restriction enzyme sites and ligating an element of interest and (2) adding restriction enzyme sites to the template by hybridizing oligonucleotide primers that include one or more suitable restriction enzyme sites and amplifying by polymerase chain reaction (described in greater detail herein).
  • Other cloning strategies take advantage of one or more insertion sites present or inserted into the nucleic acid reagent, such as an oligonucleotide primer hybridization site for PCR, for example, and others described hereafter.
  • a cloning strategy can be combined with genetic manipulation such as recombination (e.g., recombination of a nucleic acid reagent with a nucleic acid sequence of interest into the genome of the organism to be modified, as described further below).
  • genetic manipulation such as recombination (e.g., recombination of a nucleic acid reagent with a nucleic acid sequence of interest into the genome of the organism to be modified, as described further below).
  • the cloned ORF(s) can produce (directly or indirectly) a desired product, by engineering a microorganism with one or more ORFs of interest, which microorganism comprises one or more altered activities selected from the group consisting of phosphofructokinase activity, phosphogluconate dehydratase activity, 2-keto-3-deoxygluconate-6-phosphate aldolase activity, xylose isomerase activity, phosphoenolpyruvate carboxylase activity, alcohol dehydrogenase 2 activity, sugar transport activity, phosphoglucoisomerase activity, transaldolase activity, transketolase activity, glucose-6-phosphate dehydrogenase activity, 6-phosphogluconolactonase activity, 6-phosphogluconate dehydrogenase (decarboxylating) activity, and thymidylate synthase activity.
  • the nucleic acid reagent includes one or more recombinase insertion sites.
  • a recombinase insertion site is a recognition sequence on a nucleic acid molecule that participates in an integration/recombination reaction by recombination proteins.
  • the recombination site for Cre recombinase is IoxP, which is a 34 base pair sequence comprised of two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (e.g., FIG. 1 of Sauer, B., Curr. Opin. Biotech. 5:521-527 (1994)).
  • recombination sites include attB, attP, attL, and attR sequences, and mutants, fragments, variants and derivatives thereof, which are recognized by the recombination protein A Int and by the auxiliary proteins integration host factor (IHF), FIS and excisionase (Xis) (e.g., U.S. Pat. Nos. 5,888,732; 6,143,557; 6,171,861; 6,270,969; 6,277,608; and 6,720,140; U.S. patent application Ser. No. 09/517,466, filed Mar. 2, 2000, and 09/732,914, filed Aug. 14, 2003, and in U.S. patent publication no. 2002-0007051-A1; Landy, Curr. Opin. Biotech. 3:699-707 (1993)).
  • IHF auxiliary proteins integration host factor
  • Xis excisionase
  • recombinase cloning nucleic acids are in Gateway® systems (Invitrogen, California), which include at least one recombination site for cloning a desired nucleic acid molecules in vivo or in vitro.
  • the system utilizes vectors that contain at least two different site-specific recombination sites, often based on the bacteriophage lambda system (e.g., att1 and att2), and are mutated from the wild-type (att0) sites.
  • Each mutated site has a unique specificity for its cognate partner att site (i.e., its binding partner recombination site) of the same type (for example attB1 with attP1, or attL1 with attR1) and will not cross-react with recombination sites of the other mutant type or with the wild-type att0 site.
  • Different site specificities allow directional cloning or linkage of desired molecules thus providing desired orientation of the cloned molecules.
  • Nucleic acid fragments flanked by recombination sites are cloned and subcloned using the Gateway® system by replacing a selectable marker (for example, ccdB) flanked by att sites on the recipient plasmid molecule, sometimes termed the Destination Vector. Desired clones are then selected by transformation of a ccdB sensitive host strain and positive selection for a marker on the recipient molecule. Similar strategies for negative selection (e.g., use of toxic genes) can be used in other organisms such as thymidine kinase (TK) in mammals and insects.
  • TK thymidine kinase
  • a recombination system useful for engineering yeast is outlined briefly.
  • the system makes use of the ura3 gene (e.g., for S. cerevisiae and C. albicans , for example) or ura4 and ura5 genes (e.g., for S. pombe , for example) and toxicity of the nucleotide analogue 5-Fluoroorotic acid (5-FOA).
  • the ura3 or ura4 and ura5 genes encode orotine-5′-monophosphate (OMP) dicarboxylase.
  • OMP orotine-5′-monophosphate
  • Yeast with an active ura3 or ura4 and ura5 gene (phenotypically Ura+) convert 5-FOA to fluorodeoxyuridine, which is toxic to yeast cells.
  • Yeast carrying a mutation in the appropriate gene(s) or having a knock out of the appropriate gene(s) can grow in the presence of 5-FOA, if the media is also supplemented
  • a nucleic acid engineering construct can be made which may comprise the URA3 gene or cassette (for S. cerevisiae ), flanked on either side by the same nucleotide sequence in the same orientation.
  • the ura3 cassette comprises a promoter, the ura3 gene and a functional transcription terminator.
  • Target sequences which direct the construct to a particular nucleic acid region of interest in the organism to be engineered are added such that the target sequences are adjacent to and abut the flanking sequences on either side of the ura3 cassette.
  • Yeast can be transformed with the engineering construct and plated on minimal media without uracil. Colonies can be screened by PCR to determine those transformants that have the engineering construct inserted in the proper location in the genome.
  • Checking insertion location prior to selecting for recombination of the ura3 cassette may reduce the number of incorrect clones carried through to later stages of the procedure. Correctly inserted transformants can then be replica plated on minimal media containing 5-FOA to select for recombination of the ura3 cassette out of the construct, leaving a disrupted gene and an identifiable footprint (e.g., nucleic acid sequence) that can be use to verify the presence of the disrupted gene.
  • the technique described is useful for disrupting or “knocking out” gene function, but also can be used to insert genes or constructs into a host organisms genome in a targeted, sequence specific manner. Further detail will be described below in the engineering section and in the example section.
  • a nucleic acid reagent includes one or more topoisomerase insertion sites.
  • a topoisomerase insertion site is a defined nucleotide sequence recognized and bound by a site-specific topoisomerase.
  • the nucleotide sequence 5′-(C/T)CCTT-3′ is a topoisomerase recognition site bound specifically by most poxvirus topoisomerases, including vaccinia virus DNA topoisomerase I.
  • the topoisomerase After binding to the recognition sequence, the topoisomerase cleaves the strand at the 3′-most thymidine of the recognition site to produce a nucleotide sequence comprising 5′-(C/T)CCTT-PO 4 -TOPO, a complex of the topoisomerase covalently bound to the 3′ phosphate via a tyrosine in the topoisomerase (e.g., Shuman, J. Biol. Chem. 266:11372-11379, 1991; Sekiguchi and Shuman, Nucl. Acids Res. 22:5360-5365, 1994; U.S. Pat. No. 5,766,891; PCT/US95/16099; and PCT/US98/12372).
  • a tyrosine in the topoisomerase e.g., Shuman, J. Biol. Chem. 266:11372-11379, 1991; Sekiguchi and Shuman, Nucl. Acids Res. 22:53
  • nucleotide sequence 5′-GCAACTT-3′ is a topoisomerase recognition site for type IA E. coli topoisomerase III.
  • An element to be inserted often is combined with topoisomerase-reacted template and thereby incorporated into the nucleic acid reagent (e.g., http address www.invitrogen.com/downloads/F-13512_Topo_Flyer.pdf; http address at world wide web uniform resource locator invitrogen.com/content/sfs/brochures/710 — 021849%20_B_TOPOCloning_bro.pdf; TOPO TA Cloning® Kit and Zero Blunt® TOPO® Cloning Kit product information).
  • a nucleic acid reagent sometimes contains one or more origin of replication (ORI) elements.
  • a template comprises two or more ORIs, where one functions efficiently in one organism (e.g., a bacterium) and another functions efficiently in another organism (e.g., a eukaryote, like yeast for example).
  • an ORI may function efficiently in one species (e.g., S. cerevisiae , for example) and another ORI may function efficiently in a different species (e.g., S. pombe , for example).
  • a nucleic acid reagent also sometimes includes one or more transcription regulation sites.
  • a nucleic acid reagent can include one or more selection elements (e.g., elements for selection of the presence of the nucleic acid reagent, and not for activation of a promoter element which can be selectively regulated). Selection elements often are utilized using known processes to determine whether a nucleic acid reagent is included in a cell.
  • a nucleic acid reagent includes two or more selection elements, where one functions efficiently in one organism and another functions efficiently in another organism.
  • selection elements include, but are not limited to, (1) nucleic acid segments that encode products that provide resistance against otherwise toxic compounds (e.g., antibiotics); (2) nucleic acid segments that encode products that are otherwise lacking in the recipient cell (e.g., essential products, tRNA genes, auxotrophic markers); (3) nucleic acid segments that encode products that suppress the activity of a gene product; (4) nucleic acid segments that encode products that can be readily identified (e.g., phenotypic markers such as antibiotics (e.g., ⁇ -lactamase), ⁇ -galactosidase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), and cell surface proteins); (5) nucleic acid segments that bind products that are otherwise detrimental to cell survival and/or function; (6) nucleic acid segments that otherwise inhibit the activity of any of the nucleic acid segments described in Nos.
  • antibiotics e.g., ⁇ -lactamase), ⁇ -galacto
  • nucleic acid segments that bind products that modify a substrate e.g., restriction endonucleases
  • nucleic acid segments that can be used to isolate or identify a desired molecule e.g., specific protein binding sites
  • nucleic acid segments that encode a specific nucleotide sequence that can be otherwise non-functional e.g., for PCR amplification of subpopulations of molecules
  • nucleic acid segments that, when absent, directly or indirectly confer resistance or sensitivity to particular compounds (11) nucleic acid segments that encode products that either are toxic or convert a relatively non-toxic compound to a toxic compound (e.g., Herpes simplex thymidine kinase, cytosine deaminase) in recipient cells; (12) nucleic acid segments that inhibit replication, partition or heritability of nucleic acid molecules that contain them; and/or (13) nucleic acid segments that encode condition
  • a nucleic acid reagent is of any form useful for in vivo transcription and/or translation.
  • a nucleic acid sometimes is a plasmid, such as a supercoiled plasmid, sometimes is a yeast artificial chromosome (e.g., YAC), sometimes is a linear nucleic acid (e.g., a linear nucleic acid produced by PCR or by restriction digest), sometimes is single-stranded and sometimes is double-stranded.
  • a nucleic acid reagent sometimes is prepared by an amplification process, such as a polymerase chain reaction (PCR) process or transcription-mediated amplification process (TMA).
  • PCR polymerase chain reaction
  • TMA transcription-mediated amplification process
  • TMA two enzymes are used in an isothermal reaction to produce amplification products detected by light emission (see, e.g., Biochemistry 1996 Jun. 25; 35(25):8429-38 and http address world wide web uniform resource locator devicelink.com/ivdt/archive/00/11/007.html).
  • Standard PCR processes are known (e.g., U.S. Pat. Nos. 4,683,202; 4,683,195; 4,965,188; and 5,656,493), and generally are performed in cycles.
  • Each cycle includes heat denaturation, in which hybrid nucleic acids dissociate; cooling, in which primer oligonucleotides hybridize; and extension of the oligonucleotides by a polymerase (i.e., Taq polymerase).
  • a polymerase i.e., Taq polymerase
  • An example of a PCR cyclical process is treating the sample at 95° C. for 5 minutes; repeating forty-five cycles of 95° C. for 1 minute, 59° C. for 1 minute, 10 seconds, and 72° C. for 1 minute 30 seconds; and then treating the sample at 72° C. for 5 minutes. Multiple cycles frequently are performed using a commercially available thermal cycler.
  • PCR amplification products sometimes are stored for a time at a lower temperature (e.g., at 4° C.) and sometimes are frozen (e.g., at ⁇ 20° C.) before analysis.
  • a nucleic acid reagent, protein reagent, protein fragment reagent or other reagent described herein is isolated or purified.
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring, or a host cell if expressed exogenously), and thus is altered “by the hand of man” from its original environment.
  • purified as used herein with reference to molecules does not refer to absolute purity. Rather, “purified” refers to a substance in a composition that contains fewer substance species in the same class (e.g., nucleic acid or protein species) other than the substance of interest in comparison to the sample from which it originated.
  • nucleic acid or protein refers to a substance in a composition that contains fewer nucleic acid species or protein species other than the nucleic acid or protein of interest in comparison to the sample from which it originated.
  • a protein or nucleic acid is “substantially pure,” indicating that the protein or nucleic acid represents at least 50% of protein or nucleic acid on a mass basis of the composition.
  • a substantially pure protein or nucleic acid is at least 75% on a mass basis of the composition, and sometimes at least 95% on a mass basis of the composition.
  • engineered microorganism refers to a modified organism that includes one or more activities distinct from an activity present in a microorganism utilized as a starting point for modification (e.g., host microorganism or unmodified organism).
  • Engineered microorganisms typically arise as a result of a genetic modification, usually introduced or selected for, by one of skill in the art using readily available techniques.
  • Non-limiting examples of methods useful for generating an altered activity include, introducing a heterologous polynucleotide (e.g., nucleic acid or gene integration, also referred to as “knock in”), removing an endogenous polynucleotide, altering the sequence of an existing endogenous nucleic acid sequence (e.g., site-directed mutagenesis), disruption of an existing endogenous nucleic acid sequence (e.g., knock outs and transposon or insertion element mediated mutagenesis), selection for an altered activity where the selection causes a change in a naturally occurring activity that can be stably inherited (e.g., causes a change in a nucleic acid sequence in the genome of the organism or in an epigenetic nucleic acid that is replicated and passed on to daughter cells), PCR-based mutagenesis, and the like.
  • a heterologous polynucleotide e.g., nucleic acid or gene integration, also referred to as “knock in
  • mutagenesis refers to any modification to a nucleic acid (e.g., nucleic acid reagent, or host chromosome, for example) that is subsequently used to generate a product in a host or modified organism.
  • Non-limiting examples of mutagenesis include, deletion, insertion, substitution, rearrangement, point mutations, suppressor mutations and the like. Mutagenesis methods are known in the art and are readily available to the artisan. Non-limiting examples of mutagenesis methods are described herein and can also be found in Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • genetic modification refers to any suitable nucleic acid addition, removal or alteration that facilitates production of a target product (e.g., phosphogluconate dehydratase activity, 2-keto-3-deoxygluconate-6-phosphate aldolase activity, xylose isomerase activity, or phosphoenolpyruvate carboxylase activity, for example). in an engineered microorganism.
  • a target product e.g., phosphogluconate dehydratase activity, 2-keto-3-deoxygluconate-6-phosphate aldolase activity, xylose isomerase activity, or phosphoenolpyruvate carboxylase activity, for example.
  • Genetic modifications include, without limitation, insertion of one or more nucleotides in a native nucleic acid of a host organism in one or more locations, deletion of one or more nucleotides in a native nucleic acid of a host organism in one or more locations, modification or substitution of one or more nucleotides in a native nucleic acid of a host organism in one or more locations, insertion of a non-native nucleic acid into a host organism (e.g., insertion of an autonomously replicating vector), and removal of a non-native nucleic acid in a host organism (e.g., removal of a vector).
  • heterologous polynucleotide refers to a nucleotide sequence not present in a host microorganism in some embodiments.
  • a heterologous polynucleotide is present in a different amount (e.g., different copy number) than in a host microorganism, which can be accomplished, for example, by introducing more copies of a particular nucleotide sequence to a host microorganism (e.g., the particular nucleotide sequence may be in a nucleic acid autonomous of the host chromosome or may be inserted into a chromosome).
  • a heterologous polynucleotide is from a different organism in some embodiments, and in certain embodiments, is from the same type of organism but from an outside source (e.g., a recombinant source).
  • altered activity refers to an activity in an engineered microorganism that is added or modified relative to the host microorganism (e.g., added, increased, reduced, inhibited or removed activity).
  • An activity can be altered by introducing a genetic modification to a host microorganism that yields an engineered microorganism having added, increased, reduced, inhibited or removed activity.
  • An added activity often is an activity not detectable in a host microorganism.
  • An increased activity generally is an activity detectable in a host microorganism that has been increased in an engineered microorganism.
  • An activity can be increased to any suitable level for production of a target product (e.g., adipic acid, 6-hydroxyhexanoic acid), including but not limited to less than 2-fold (e.g., about 10% increase to about 99% increase; about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% increase), 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, of 10-fold increase, or greater than about 10-fold increase.
  • a target product e.g., adipic acid, 6-hydroxyhexanoic acid
  • a reduced or inhibited activity generally is an activity detectable in a host microorganism that has been reduced or inhibited in an engineered microorganism.
  • An activity can be reduced to undetectable levels in some embodiments, or detectable levels in certain embodiments.
  • An activity can be decreased to any suitable level for production of a target product (e.g., adipic acid, 6-hydroxyhexanoic acid), including but not limited to less than 2-fold (e.g., about 10% decrease to about 99% decrease; about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% decrease), 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, of 10-fold decrease, or greater than about 10-fold decrease.
  • a target product e.g., adipic acid, 6-hydroxyhexanoic acid
  • An altered activity sometimes is an activity not detectable in a host organism and is added to an engineered organism.
  • An altered activity also may be an activity detectable in a host organism and is increased in an engineered organism.
  • An activity may be added or increased by increasing the number of copies of a polynucleotide that encodes a polypeptide having a target activity, in some embodiments.
  • an activity can be added or increased by inserting into a host microorganism a heterologous polynucleotide that encodes a polypeptide having the added activity.
  • an activity can be added or increased by inserting into a host microorganism a heterologous polynucleotide that is (i) operably linked to another polynucleotide that encodes a polypeptide having the added activity, and (ii) up regulates production of the polynucleotide.
  • an activity can be added or increased by inserting or modifying a regulatory polynucleotide operably linked to another polynucleotide that encodes a polypeptide having the target activity.
  • an activity can be added or increased by subjecting a host microorganism to a selective environment and screening for microorganisms that have a detectable level of the target activity. Examples of a selective environment include, without limitation, a medium containing a substrate that a host organism can process and a medium lacking a substrate that a host organism can process.
  • An altered activity sometimes is an activity detectable in a host organism and is reduced, inhibited or removed (i.e., not detectable) in an engineered organism.
  • An activity may be reduced or removed by decreasing the number of copies of a polynucleotide that encodes a polypeptide having a target activity, in some embodiments.
  • an activity can be reduced or removed by (i) inserting a polynucleotide within a polynucleotide that encodes a polypeptide having the target activity (disruptive insertion), and/or (ii) removing a portion of or all of a polynucleotide that encodes a polypeptide having the target activity (deletion or knock out, respectively).
  • an activity can be reduced or removed by inserting into a host microorganism a heterologous polynucleotide that is (i) operably linked to another polynucleotide that encodes a polypeptide having the target activity, and (ii) down regulates production of the polynucleotide.
  • a heterologous polynucleotide that is (i) operably linked to another polynucleotide that encodes a polypeptide having the target activity, and (ii) down regulates production of the polynucleotide.
  • an activity can be reduced or removed by inserting or modifying a regulatory polynucleotide operably linked to another polynucleotide that encodes a polypeptide having the target activity.
  • An activity also can be reduced or removed by (i) inhibiting a polynucleotide that encodes a polypeptide having the activity or (ii) inhibiting a polynucleotide operably linked to another polynucleotide that encodes a polypeptide having the activity.
  • a polynucleotide can be inhibited by a suitable technique known in the art, such as by contacting an RNA encoded by the polynucleotide with a specific inhibitory RNA (e.g., RNAi, siRNA, ribozyme).
  • An activity also can be reduced or removed by contacting a polypeptide having the activity with a molecule that specifically inhibits the activity (e.g., enzyme inhibitor, antibody).
  • an activity can be reduced or removed by subjecting a host microorganism to a selective environment and screening for microorganisms that have a reduced level or removal of the target activity.
  • an untranslated ribonucleic acid, or a cDNA can be used to reduce the expression of a particular activity or enzyme.
  • a microorganism can be engineered by genetic modification to express a nucleic acid reagent that reduces the expression of an activity by producing an RNA molecule that is partially or substantially homologous to a nucleic acid sequence of interest which encodes the activity of interest.
  • the RNA molecule can bind to the nucleic acid sequence of interest and inhibit the nucleic acid sequence from performing its natural function, in certain embodiments.
  • the RNA may alter the nucleic acid sequence of interest which encodes the activity of interest in a manner that the nucleic acid sequence of interest is no longer capable of performing its natural function (e.g., the action of a ribozyme for example).
  • nucleotide sequences sometimes are added to, modified or removed from one or more of the nucleic acid reagent elements, such as the promoter, 5′UTR, target sequence, or 3′UTR elements, to enhance, potentially enhance, reduce, or potentially reduce transcription and/or translation before or after such elements are incorporated in a nucleic acid reagent.
  • the nucleic acid reagent elements such as the promoter, 5′UTR, target sequence, or 3′UTR elements
  • one or more of the following sequences may be modified or removed if they are present in a 5′UTR: a sequence that forms a stable secondary structure (e.g., quadruplex structure or stem loop stem structure (e.g., EMBL sequences X12949, AF274954, AF139980, AF152961, S95936, U194144, AF116649 or substantially identical sequences that form such stem loop stem structures)); a translation initiation codon upstream of the target nucleotide sequence start codon; a stop codon upstream of the target nucleotide sequence translation initiation codon; an ORF upstream of the target nucleotide sequence translation initiation codon; an iron responsive element (IRE) or like sequence; and a 5′ terminal oligopyrimidine tract (TOP, e.g., consisting of 5-15 pyrimidines adjacent to the cap).
  • a stable secondary structure e.g., quadruplex structure or stem loop stem structure (e.g.
  • a translational enhancer sequence and/or an internal ribosome entry site sometimes is inserted into a 5′UTR (e.g., EMBL nucleotide sequences J04513, X87949, M95825, M12783, AF025841, AF013263, AF006822, M17169, M13440, M22427, D14838 and M17446 and substantially identical nucleotide sequences).
  • EMBL nucleotide sequences J04513, X87949, M95825, M12783, AF025841, AF013263, AF006822, M17169, M13440, M22427, D14838 and M17446 and substantially identical nucleotide sequences.
  • An AU-rich element e.g., AUUUA repeats
  • splicing junction that follows a non-sense codon sometimes is removed from or modified in a 3′UTR.
  • a polyadenosine tail sometimes is inserted into a 3′UTR if none is present, sometimes is removed if it is present, and adenosine moieties sometimes are added to or removed from a polyadenosine tail present in a 3′UTR.
  • some embodiments are directed to a process comprising: determining whether any nucleotide sequences that increase, potentially increase, reduce or potentially reduce translation efficiency are present in the elements, and adding, removing or modifying one or more of such sequences if they are identified.
  • Certain embodiments are directed to a process comprising: determining whether any nucleotide sequences that increase or potentially increase translation efficiency are not present in the elements, and incorporating such sequences into the nucleic acid reagent.
  • an activity can be altered by modifying the nucleotide sequence of an ORF.
  • An ORF sometimes is mutated or modified (for example, by point mutation, deletion mutation, insertion mutation, PCR based mutagenesis and the like) to alter, enhance or increase, reduce, substantially reduce or eliminate the activity of the encoded protein or peptide.
  • the protein or peptide encoded by a modified ORF sometimes is produced in a lower amount or may not be produced at detectable levels, and in other embodiments, the product or protein encoded by the modified ORF is produced at a higher level (e.g., codons sometimes are modified so they are compatible with tRNA's preferentially used in the host organism or engineered organism).
  • the activity from the product of the mutated ORF (or cell containing it) can be compared to the activity of the product or protein encoded by the unmodified ORF (or cell containing it).
  • an ORF nucleotide sequence sometimes is mutated or modified to alter the triplet nucleotide sequences used to encode amino acids (e.g., amino acid codon triplets, for example). Modification of the nucleotide sequence of an ORF to alter codon triplets sometimes is used to change the codon found in the original sequence to better match the preferred codon usage of the organism in which the ORF or nucleic acid reagent will be expressed.
  • the codon usage, and therefore the codon triplets encoded by a nucleic acid sequence from bacteria may be different from the preferred codon usage in eukaryotes like yeast or plants.
  • Preferred codon usage also may be different between bacterial species.
  • an ORF nucleotide sequences sometimes is modified to eliminate codon pairs and/or eliminate mRNA secondary structures that can cause pauses during translation of the mRNA encoded by the ORF nucleotide sequence.
  • Translational pausing sometimes occurs when nucleic acid secondary structures exist in an mRNA, and sometimes occurs due to the presence of codon pairs that slow the rate of translation by causing ribosomes to pause.
  • the use of lower abundance codon triplets can reduce translational pausing due to a decrease in the pause time needed to load a charged tRNA into the ribosome translation machinery.
  • Modification of the nucleotide sequence of an ORF also can be used to correct codon triplet sequences that have diverged in different organisms.
  • certain yeast e.g., C. tropicalis and C. maltosa
  • CUG typically encodes leucine in most organisms.
  • the CUG codon must be altered to reflect the organism in which the nucleic acid reagent will be expressed.
  • the heterologous nucleotide sequence must first be altered or modified to the appropriate leucine codon.
  • the nucleotide sequence of an ORF sometimes is altered or modified to correct for differences that have occurred in the evolution of the amino acid codon triplets between different organisms.
  • the nucleotide sequence can be left unchanged at a particular amino acid codon, if the amino acid encoded is a conservative or neutral change in amino acid when compared to the originally encoded amino acid.
  • an activity can be altered by modifying translational regulation signals, like a stop codon for example.
  • a stop codon at the end of an ORF sometimes is modified to another stop codon, such as an amber stop codon described above.
  • a stop codon is introduced within an ORF, sometimes by insertion or mutation of an existing codon.
  • An ORF comprising a modified terminal stop codon and/or internal stop codon often is translated in a system comprising a suppressor tRNA that recognizes the stop codon.
  • An ORF comprising a stop codon sometimes is translated in a system comprising a suppressor tRNA that incorporates an unnatural amino acid during translation of the target protein or target peptide.
  • Methods for incorporating unnatural amino acids into a target protein or peptide include, for example, processes utilizing a heterologous tRNA/synthetase pair, where the tRNA recognizes an amber stop codon and is loaded with an unnatural amino acid (e.g., World Wide Web URL iupac.org/news/prize/2003/wang.pdf).
  • nucleic acid reagent e.g., Promoter, 5′ or 3′ UTR, ORI, ORF, and the like
  • the modifications described above can alter a given activity by (i) increasing or decreasing feedback inhibition mechanisms, (ii) increasing or decreasing promoter initiation, (iii) increasing or decreasing translation initiation, (iv) increasing or decreasing translational efficiency, (v) modifying localization of peptides or products expressed from nucleic acid reagents described herein, or (vi) increasing or decreasing the copy number of a nucleotide sequence of interest, (vii) expression of an anti-sense RNA, RNAi, siRNA, ribozyme and the like.
  • alteration of a nucleic acid reagent or nucleotide sequence can alter sequences involved in transcription initiation (e.g., promoters, 5′ UTR, and the like).
  • a modification sometimes can be made that can enhance or increase initiation from an endogenous or heterologous promoter element.
  • a modification sometimes can be made that removes or disrupts sequences that increase or enhance transcription initiation, resulting in a decrease or elimination of transcription from an endogenous or heterologous promoter element.
  • alteration of a nucleic acid reagent or nucleotide sequence can alter sequences involved in localization of peptides, proteins or other desired products (e.g., adipic acid, for example).
  • a modification sometimes can be made that can alter, add or remove sequences responsible for targeting a polypeptide, protein or product to an intracellular organelle, the periplasm, cellular membranes, or extracellularly. Transport of a heterologous product to a different intracellular space or extracellularly sometimes can reduce or eliminate the formation of inclusion bodies (e.g., insoluble aggregates of the desired product).
  • alteration of a nucleic acid reagent or nucleotide sequence can alter sequences involved in increasing or decreasing the copy number of a nucleotide sequence of interest.
  • a modification sometimes can be made that increases or decreases the number of copies of an ORF stably integrated into the genome of an organism or on an epigenetic nucleic acid reagent.
  • increasing or decreasing the expression of a nucleotide sequence of interest can also be accomplished by altering, adding or removing sequences involved in the expression of an anti-sense RNA, RNAi, siRNA, ribozyme and the like.
  • the methods described above can be used to modify expression of anti-sense RNA, RNAi, siRNA, ribozyme and the like.
  • Engineered microorganisms can be prepared by altering, introducing or removing nucleotide sequences in the host genome or in stably maintained epigenetic nucleic acid reagents, as noted above.
  • the nucleic acid reagents use to alter, introduce or remove nucleotide sequences in the host genome or epigenetic nucleic acids can be prepared using the methods described herein or available to the artisan.
  • Nucleic acid sequences having a desired activity can be isolated from cells of a suitable organism using lysis and nucleic acid purification procedures available in Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. or with commercially available cell lysis and DNA purification reagents and kits.
  • nucleic acids used to engineer microorganisms can be provided for conducting methods described herein after processing of the organism containing the nucleic acid.
  • the nucleic acid of interest may be extracted, isolated, purified or amplified from a sample (e.g., from an organism of interest or culture containing a plurality of organisms of interest, like yeast or bacteria for example).
  • a sample e.g., from an organism of interest or culture containing a plurality of organisms of interest, like yeast or bacteria for example.
  • isolated refers to nucleic acid removed from its original environment (e.g., the natural environment if it is naturally occurring, or a host cell if expressed exogenously), and thus is altered “by the hand of man” from its original environment.
  • An isolated nucleic acid generally is provided with fewer non-nucleic acid components (e.g., protein, lipid) than the amount of components present in a source sample.
  • a composition comprising isolated sample nucleic acid can be substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free of non-nucleic acid components).
  • the term “purified” as used herein refers to sample nucleic acid provided that contains fewer nucleic acid species than in the sample source from which the sample nucleic acid is derived.
  • a composition comprising sample nucleic acid may be substantially purified (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free of other nucleic acid species).
  • amplified refers to subjecting nucleic acid of a cell, organism or sample to a process that linearly or exponentially generates amplicon nucleic acids having the same or substantially the same nucleotide sequence as the nucleotide sequence of the nucleic acid in the sample, or portion thereof.
  • nucleic acids used to prepare nucleic acid reagents as described herein can be subjected to fragmentation or cleavage.
  • Amplification of nucleic acids is sometimes necessary when dealing with organisms that are difficult to culture. Where amplification may be desired, any suitable amplification technique can be utilized.
  • Non-limiting examples of methods for amplification of polynucleotides include, polymerase chain reaction (PCR); ligation amplification (or ligase chain reaction (LCR)); amplification methods based on the use of Q-beta replicase or template-dependent polymerase (see US Patent Publication Number US20050287592); helicase-dependant isothermal amplification (Vincent et al., “Helicase-dependent isothermal DNA amplification”.
  • Protocols for conducting the various type of PCR listed above are readily available to the artisan. PCR conditions can be dependent upon primer sequences, target abundance, and the desired amount of amplification, and therefore, one of skill in the art may choose from a number of PCR protocols available (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990.
  • PCR often is carried out as an automated process with a thermostable enzyme. In this process, the temperature of the reaction mixture is cycled through a denaturing region, a primer-annealing region, and an extension reaction region automatically. Machines specifically adapted for this purpose are commercially available.
  • a non-limiting example of a PCR protocol that may be suitable for embodiments described herein is, treating the sample at 95° C. for 5 minutes; repeating forty-five cycles of 95° C. for 1 minute, 59° C. for 1 minute, 10 seconds, and 72° C. for 1 minute 30 seconds; and then treating the sample at 72° C. for 5 minutes. Additional PCR protocols are described in the example section. Multiple cycles frequently are performed using a commercially available thermal cycler. Suitable isothermal amplification processes known and selected by the person of ordinary skill in the art also may be applied, in certain embodiments.
  • nucleic acids encoding polypeptides with a desired activity can be isolated by amplifying the desired sequence from an organism having the desired activity using oligonucleotides or primers designed based on sequences described herein
  • nucleic acids can be cloned into the recombinant DNA vectors described in Figures herein or into suitable commercially available recombinant DNA vectors.
  • Cloning of nucleic acid sequences of interest into recombinant DNA vectors can facilitate further manipulations of the nucleic acids for preparation of nucleic acid reagents, (e.g., alteration of nucleotide sequences by mutagenesis, homologous recombination, amplification and the like, for example).
  • Standard cloning procedures e.g., enzymic digestion, ligation, and the like are readily available to the artisan and can be found in Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • nucleic acid sequences prepared by isolation or amplification can be used, without any further modification, to add an activity to a microorganism and thereby generate a genetically modified or engineered microorganism.
  • nucleic acid sequences prepared by isolation or amplification can be genetically modified to alter (e.g., increase or decrease, for example) a desired activity.
  • nucleic acids, used to add an activity to an organism sometimes are genetically modified to optimize the heterologous polynucleotide sequence encoding the desired activity (e.g., polypeptide or protein, for example).
  • optimize as used herein can refer to alteration to increase or enhance expression by preferred codon usage.
  • optimize can also refer to modifications to the amino acid sequence to increase the activity of a polypeptide or protein, such that the activity exhibits a higher catalytic activity as compared to the “natural” version of the polypeptide or protein.
  • Nucleic acid sequences of interest can be genetically modified using methods known in the art. Mutagenesis techniques are particularly useful for small scale (e.g., 1, 2, 5, 10 or more nucleotides) or large scale (e.g., 50, 100, 150, 200, 500, or more nucleotides) genetic modification. Mutagenesis allows the artisan to alter the genetic information of an organism in a stable manner, either naturally (e.g., isolation using selection and screening) or experimentally by the use of chemicals, radiation or inaccurate DNA replication (e.g., PCR mutagenesis).
  • small scale e.g., 1, 2, 5, 10 or more nucleotides
  • large scale e.g., 50, 100, 150, 200, 500, or more nucleotides
  • genetic modification can be performed by whole scale synthetic synthesis of nucleic acids, using a native nucleotide sequence as the reference sequence, and modifying nucleotides that can result in the desired alteration of activity.
  • Mutagenesis methods sometimes are specific or targeted to specific regions or nucleotides (e.g., site-directed mutagenesis, PCR-based site-directed mutagenesis, and in vitro mutagenesis techniques such as transplacement and in vivo oligonucleotide site-directed mutagenesis, for example).
  • Mutagenesis methods sometimes are non-specific or random with respect to the placement of genetic modifications (e.g., chemical mutagenesis, insertion element (e.g., insertion or transposon elements) and inaccurate PCR based methods, for example).
  • Site directed mutagenesis is a procedure in which a specific nucleotide or specific nucleotides in a DNA molecule are mutated or altered.
  • Site directed mutagenesis typically is performed using a nucleic acid sequence of interest cloned into a circular plasmid vector.
  • Site-directed mutagenesis requires that the wild type sequence be known and used a platform for the genetic alteration.
  • Site-directed mutagenesis sometimes is referred to as oligonucleotide-directed mutagenesis because the technique can be performed using oligonucleotides which have the desired genetic modification incorporated into the complement a nucleotide sequence of interest.
  • the wild type sequence and the altered nucleotide are allowed to hybridize and the hybridized nucleic acids are extended and replicated using a DNA polymerase.
  • the double stranded nucleic acids are introduced into a host (e.g., E. coli , for example) and further rounds of replication are carried out in vivo.
  • the transformed cells carrying the mutated nucleic acid sequence are then selected and/or screened for those cells carrying the correctly mutagenized sequence.
  • Cassette mutagenesis and PCR-based site-directed mutagenesis are further modifications of the site-directed mutagenesis technique.
  • Site-directed mutagenesis can also be performed in vivo (e.g., transplacement “pop-in pop-out”, In vivo site-directed mutagenesis with synthetic oligonucleotides and the like, for example).
  • PCR-based mutagenesis can be performed using PCR with oligonucleotide primers that contain the desired mutation or mutations.
  • the technique functions in a manner similar to standard site-directed mutagenesis, with the exception that a thermocycler and PCR conditions are used to replace replication and selection of the clones in a microorganism host.
  • PCR-based mutagenesis also uses a circular plasmid vector, the amplified fragment (e.g., linear nucleic acid molecule) containing the incorporated genetic modifications can be separated from the plasmid containing the template sequence after a sufficient number of rounds of thermocycler amplification, using standard electrophorectic procedures.
  • a modification of this method uses linear amplification methods and a pair of mutagenic primers that amplify the entire plasmid.
  • the procedure takes advantage of the E. coli Dam methylase system which causes DNA replicated in vivo to be sensitive to the restriction endonucleases DpnI.
  • PCR synthesized DNA is not methylated and is therefore resistant to DpnI.
  • This approach allows the template plasmid to be digested, leaving the genetically modified, PCR synthesized plasmids to be isolated and transformed into a host bacteria for DNA repair and replication, thereby facilitating subsequent cloning and identification steps.
  • a certain amount of randomness can be added to PCR-based sited directed mutagenesis by using partially degenerate primers.
  • Recombination sometimes can be used as a tool for mutagenesis.
  • Homologous recombination allows the artisan to specifically target regions of known sequence for insertion of heterologous nucleotide sequences using the host organisms natural DNA replication and repair enzymes.
  • Homologous recombination methods sometimes are referred to as “pop in pop out” mutagenesis, transplacement, knock out mutagenesis or knock in mutagenesis. Integration of a nucleic acid sequence into a host genome is a single cross over event, which inserts the entire nucleic acid reagent (e.g., pop in).
  • a second cross over event excises all but a portion of the nucleic acid reagent, leaving behind a heterologous sequence, often referred to as a “footprint” (e.g., pop out).
  • a heterologous sequence often referred to as a “footprint” (e.g., pop out).
  • Mutagenesis by insertion e.g., knock in
  • double recombination leaving behind a disrupting heterologous nucleic acid (e.g., knock out) both server to disrupt or “knock out” the function of the gene or nucleic acid sequence in which insertion occurs.
  • selectable markers and/or auxotrophic markers By combining selectable markers and/or auxotrophic markers with nucleic acid reagents designed to provide the appropriate nucleic acid target sequences, the artisan can target a selectable nucleic acid reagent to a specific region, and then select for recombination events that “pop out” a portion of the inserted (e.g., “pop in”) nucleic acid reagent.
  • Such methods take advantage of nucleic acid reagents that have been specifically designed with known target nucleic acid sequences at or near a nucleic acid or genomic region of interest. Popping out typically leaves a “foot print” of left over sequences that remain after the recombination event. The left over sequence can disrupt a gene and thereby reduce or eliminate expression of that gene.
  • the method can be used to insert sequences, upstream or downstream of genes that can result in an enhancement or reduction in expression of the gene.
  • new genes can be introduced into the genome of a host organism using similar recombination or “pop in” methods.
  • An example of a yeast recombination system using the ura3 gene and 5-FOA were described briefly above and further detail is presented herein.
  • a method for modification is described in Alani et al., “A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains”, Genetics 116(4):541-545 August 1987.
  • the original method uses a Ura3 cassette with 1000 base pairs (bp) of the same nucleotide sequence cloned in the same orientation on either side of the URA3 cassette.
  • Targeting sequences of about 50 bp are added to each side of the construct.
  • the double stranded targeting sequences are complementary to sequences in the genome of the host organism.
  • the targeting sequences allow site-specific recombination in a region of interest.
  • the modification of the original technique replaces the two 1000 bp sequence direct repeats with two 200 bp direct repeats.
  • the modified method also uses 50 bp targeting sequences.
  • the modification reduces or eliminates recombination of a second knock out into the 1000 bp repeat left behind in a first mutagenesis, therefore allowing multiply knocked out yeast.
  • the 200 bp sequences used herein are uniquely designed, self-assembling sequences that leave behind identifiable footprints.
  • the technique used to design the sequences incorporate design features such as low identity to the yeast genome, and low identity to each other. Therefore a library of the self-assembling sequences can be generated to allow multiple knockouts in the same organism, while reducing or eliminating the potential for integration into a previous knockout.
  • the URA3 cassette makes use of the toxicity of 5-FOA in yeast carrying a functional URA3 gene.
  • Uracil synthesis deficient yeast are transformed with the modified URA3 cassette, using standard yeast transformation protocols, and the transformed cells are plated on minimal media minus uracil.
  • PCR can be used to verify correct insertion into the region of interest in the host genome, and certain embodiments the PCR step can be omitted. Inclusion of the PCR step can reduce the number of transformants that need to be counter selected to “pop out” the URA3 cassette.
  • the transformants (e.g., all or the ones determined to be correct by PCR, for example) can then be counter-selected on media containing 5-FOA, which will select for recombination out (e.g., popping out) of the URA3 cassette, thus rendering the yeast ura3 deficient again, and resistant to 5-FOA toxicity.
  • Targeting sequences used to direct recombination events to specific regions are presented herein.
  • a modification of the method described above can be used to integrate genes in to the chromosome, where after recombination a functional gene is left in the chromosome next to the 200 bp footprint.
  • auxotrophic or dominant selection markers can be used in place of URA3 (e.g., an auxotrophic selectable marker), with the appropriate change in selection media and selection agents.
  • auxotrophic selectable markers are used in strains deficient for synthesis of a required biological molecule (e.g., amino acid or nucleoside, for example).
  • additional auxotrophic markers include; HIS3, TRP1, LEU2, LEU2-d, and LYS2.
  • Certain auxotrophic markers e.g., URA3 and LYS2 allow counter selection to select for the second recombination event that pops out all but one of the direct repeats of the recombination construct.
  • HIS3 encodes an activity involved in histidine synthesis.
  • TRP1 encodes an activity involved in tryptophan synthesis.
  • LEU2 encodes an activity involved in leucine synthesis.
  • LEU2-d is a low expression version of LEU2 that selects for increased copy number (e.g., gene or plasmid copy number, for example) to allow survival on minimal media without leucine.
  • LYS2 encodes an activity involved in lysine synthesis, and allows counter selection for recombination out of the LYS2 gene using alpha-amino adipate ( ⁇ -amino adipate).
  • Dominant selectable markers are useful because they also allow industrial and/or prototrophic strains to be used for genetic manipulations. Additionally, dominant selectable markers provide the advantage that rich medium can be used for plating and culture growth, and thus growth rates are markedly increased.
  • Non-limiting examples of dominant selectable markers include; Tn903 kan r , Cm r , Hyg r , CUP1, and DHFR.
  • Tn903 kan r encodes an activity involved in kanamycin antibiotic resistance (e.g., typically neomycin phosphotransferase II or NPTII, for example).
  • Cm r encodes an activity involved in chloramphenicol antibiotic resistance (e.g., typically chloramphenicol acetyl transferase or CAT, for example).
  • Hyg r encodes an activity involved in hygromycin resistance by phosphorylation of hygromycin B (e.g., hygromycin phosphotransferase, or HPT).
  • CUP1 encodes an activity involved in resistance to heavy metal (e.g., copper, for example) toxicity.
  • DHFR encodes a dihydrofolate reductase activity which confers resistance to methotrexate and sulfanilamde compounds.
  • random mutagenesis does not require any sequence information and can be accomplished by a number of widely different methods. Random mutagenesis often is used to generate mutant libraries that can be used to screen for the desired genotype or phenotype.
  • Non-limiting examples of random mutagenesis include; chemical mutagenesis, UV-induced mutagenesis, insertion element or transposon-mediated mutagenesis, DNA shuffling, error-prone PCR mutagenesis, and the like.
  • Chemical mutagenesis often involves chemicals like ethyl methanesulfonate (EMS), nitrous acid, mitomycin C, N-methyl-N-nitrosourea (MNU), diepoxybutane (DEB), 1, 2, 7, 8-diepoxyoctane (DEO), methyl methane sulfonate (MMS), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4-NQO), 2-methyloxy-6-chloro-9(3-[ethyl-2-chloroethyl]-aminopropylamino)-acridinedihydrochloride (ICR-170), 2-amino purine (2AP), and hydroxylamine (HA), provided herein as non-limiting examples.
  • EMS ethyl methanesulfonate
  • MNU N-methyl-N-nitrosourea
  • DEB diepoxybutane
  • DEO 1,
  • the mutagenesis can be carried out in vivo.
  • the mutagenic process involves the use of the host organisms DNA replication and repair mechanisms to incorporate and replicate the mutagenized base or bases.
  • Another type of chemical mutagenesis involves the use of base-analogs.
  • the use of base-analogs cause incorrect base pairing which in the following round of replication is corrected to a mismatched nucleotide when compared to the starting sequence.
  • Base analog mutagenesis introduces a small amount of non-randomness to random mutagenesis, because specific base analogs can be chose which can be incorporated at certain nucleotides in the starting sequence. Correction of the mispairing typically yields a known substitution.
  • Bromo-deoxyuridine (BrdU) can be incorporated into DNA and replaces T in the sequence.
  • the host DNA repair and replication machinery can sometime correct the defect, but sometimes will mispair the BrdU with a G.
  • the next round of replication then causes a G-C transversion from the original A-T in the native sequence.
  • Ultra violet (UV) induced mutagenesis is caused by the formation of thymidine dimers when UV light irradiates chemical bonds between two adjacent thymine residues.
  • Excision repair mechanism of the host organism correct the lesion in the DNA, but occasionally the lesion is incorrectly repaired typically resulting in a C to T transition.
  • Insertion element or transposon-mediated mutagenesis makes use of naturally occurring or modified naturally occurring mobile genetic elements.
  • Transposons often encode accessory activities in addition to the activities necessary for transposition (e.g., movement using a transposase activity, for example).
  • transposon accessory activities are antibiotic resistance markers (e.g., see Tn903 kan r described above, for example).
  • Insertion elements typically only encode the activities necessary for movement of the nucleic acid sequence. Insertion element and transposon mediated mutagenesis often can occur randomly, however specific target sequences are known for some transposons.
  • Mobile genetic elements like IS elements or Transposons (Tn) often have inverted repeats, direct repeats or both inverted and direct repeats flanking the region coding for the transposition genes.
  • transposase Recombination events catalyzed by the transposase cause the element to remove itself from the genome and move to a new location, leaving behind a portion of an inverted or direct repeat.
  • Classic examples of transposons are the “mobile genetic elements” discovered in maize.
  • Transposon mutagenesis kits are commercially available which are designed to leave behind a 5 codon insert (e.g., Mutation Generation System kit, Finnzymes, World Wide Web URL finnzymes.us, for example). This allows the artisan to identify the insertion site, without fully disrupting the function of most genes.
  • DNA shuffling is a method which uses DNA fragments from members of a mutant library and reshuffles the fragments randomly to generate new mutant sequence combinations.
  • the fragments are typically generated using DNaseI, followed by random annealing and re-joining using self priming PCR.
  • the DNA overhanging ends, from annealing of random fragments, provide “primer” sequences for the PCR process.
  • Shuffling can be applied to libraries generated by any of the above mutagenesis methods.
  • Error prone PCR and its derivative rolling circle error prone PCR uses increased magnesium and manganese concentrations in conjunction with limiting amounts of one or two nucleotides to reduce the fidelity of the Taq polymerase.
  • the error rate can be as high as 2% under appropriate conditions, when the resultant mutant sequence is compared to the wild type starting sequence. After amplification, the library of mutant coding sequences must be cloned into a suitable plasmid.
  • Point mutations are the most common types of mutation in error prone PCR, deletions and frameshift mutations are also possible.
  • error-prone PCR kits available, including those from Stratagene and Clontech (e.g., World Wide Web URL strategene.com and World Wide Web URL clontech.com, respectively, for example).
  • Rolling circle error-prone PCR is a variant of error-prone PCR in which wild-type sequence is first cloned into a plasmid, the whole plasmid is then amplified under error-prone conditions.
  • organisms with altered activities can also be isolated using genetic selection and screening of organisms challenged on selective media or by identifying naturally occurring variants from unique environments.
  • 2-Deoxy-D-glucose is a toxic glucose analog. Growth of yeast on this substance yields mutants that are glucose-deregulated. A number of mutants have been isolated using 2-Deoxy-D-glucose including transport mutants, and mutants that ferment glucose and galactose simultaneously instead of glucose first then galactose when glucose is depleted. Similar techniques have been used to isolate mutant microorganisms that can metabolize plastics (e.g., from landfills), petrochemicals (e.g., from oil spills), and the like, either in a laboratory setting or from unique environments.
  • Similar methods can be used to isolate naturally occurring mutations in a desired activity when the activity exists at a relatively low or nearly undetectable level in the organism of choice, in some embodiments.
  • the method generally consists of growing the organism to a specific density in liquid culture, concentrating the cells, and plating the cells on various concentrations of the substance to which an increase in metabolic activity is desired.
  • the cells are incubated at a moderate growth temperature, for 5 to 10 days.
  • the plates can be stored for another 5 to 10 days at a low temperature.
  • the low temperature sometimes can allow strains that have gained or increased an activity to continue growing while other strains are inhibited for growth at the low temperature.
  • the plates can be replica plated on higher or lower concentrations of the selection substance to further select for the desired activity.
  • a native, heterologous or mutagenized polynucleotide can be introduced into a nucleic acid reagent for introduction into a host organism, thereby generating an engineered microorganism.
  • Standard recombinant DNA techniques (restriction enzyme digests, ligation, and the like) can be used by the artisan to combine the mutagenized nucleic acid of interest into a suitable nucleic acid reagent capable of (i) being stably maintained by selection in the host organism, or (ii) being integrating into the genome of the host organism.
  • nucleic acid reagents comprise two replication origins to allow the same nucleic acid reagent to be manipulated in bacterial before final introduction of the final product into the host organism (e.g., yeast or fungus for example).
  • Standard molecular biology and recombinant DNA methods available to one of skill in the art can be found in Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • Nucleic acid reagents can be introduced into microorganisms using various techniques.
  • methods used to introduce heterologous nucleic acids into various organisms include; transformation, transfection, transduction, electroporation, ultrasound-mediated transformation, particle bombardment and the like.
  • carrier molecules e.g., bis-benzimdazolyl compounds, for example, see U.S. Pat. No. 5,595,899
  • carrier molecules e.g., bis-benzimdazolyl compounds, for example, see U.S. Pat. No. 5,595,899
  • Conventional methods of transformation are readily available to the artisan and can be found in Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • Engineered microorganisms often are cultured under conditions that optimize yield of a target molecule.
  • a non-limiting example of such a target molecule is ethanol.
  • Culture conditions often can alter (e.g., add, optimize, reduce or eliminate, for example) activity of one or more of the following activities: phosphofructokinase activity, phosphogluconate dehydratase activity, 2-keto-3-deoxygluconate-6-phosphate aldolase activity, xylose isomerase activity, phosphoenolpyruvate carboxylase activity, alcohol dehydrogenase 2 activity and thymidylate synthase activities.
  • conditions that may be optimized include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the oxygen level, growth temperature, pH, length of the biomass production phase, length of target product accumulation phase, and time of cell harvest.
  • Fermentation conditions refers to any culture conditions suitable for maintaining a microorganism (e.g., in a static or proliferative state). Fermentation conditions can include several parameters, including without limitation, temperature, oxygen content, nutrient content (e.g., glucose content), pH, agitation level (e.g., revolutions per minute), gas flow rate (e.g., air, oxygen, nitrogen gas), redox potential, cell density (e.g., optical density), cell viability and the like.
  • a change in fermentation conditions e.g., switching fermentation conditions is an alteration, modification or shift of one or more fermentation parameters.
  • increasing or decreasing pH e.g., adding or removing an acid, a base or carbon dioxide
  • increasing or decreasing oxygen content e.g., introducing air, oxygen, carbon dioxide, nitrogen
  • adding or removing a nutrient e.g., one or more sugars or sources of sugar, biomass, vitamin and the like
  • Aerobic conditions often comprise greater than about 50% dissolved oxygen (e.g., about 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than any one of the foregoing).
  • dissolved oxygen e.g., about 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than any one of the foregoing).
  • Anaerobic conditions often comprise less than about 50% dissolved oxygen (e.g., about 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, or less than any one of the foregoing).
  • dissolved oxygen e.g., about 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, or less than any one of the foregoing).
  • Culture media generally contain a suitable carbon source.
  • Carbon sources may include, but are not limited to, monosaccharides (e.g., glucose, fructose, xylose), disaccharides (e.g., lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch, cellulose, hemicellulose, other lignocellulosic materials or mixtures thereof), sugar alcohols (e.g., glycerol), and renewable feedstocks (e.g., cheese whey permeate, cornsteep liquor, sugar beet molasses, barley malt).
  • monosaccharides e.g., glucose, fructose, xylose
  • disaccharides e.g., lactose, sucrose
  • oligosaccharides e.g., polysaccharides (e.g., starch, cellulose, hemicellulose, other lignocellulosic materials or
  • Carbon sources also can be selected from one or more of the following non-limiting examples: linear or branched alkanes (e.g., hexane), linear or branched alcohols (e.g., hexanol), fatty acids (e.g., about 10 carbons to about 22 carbons), esters of fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids and various commercial sources of fatty acids including vegetable oils (e.g., soybean oil) and animal fats.
  • a carbon source may include one-carbon sources (e.g., carbon dioxide, methanol, formaldehyde, formate and carbon-containing amines) from which metabolic conversion into key biochemical intermediates can occur. It is expected that the source of carbon utilized may encompass a wide variety of carbon-containing sources and will only be limited by the choice of the engineered microorganism(s).
  • Nitrogen may be supplied from an inorganic (e.g., (NH.sub.4).sub.2SO.sub.4) or organic source (e.g., urea or glutamate).
  • culture media also can contain suitable minerals, salts, cofactors, buffers, vitamins, metal ions (e.g., Mn.sup.+2, Co.sup.+2, Zn.sup.+2, Mg.sup.+2) and other components suitable for culture of microorganisms.
  • Engineered microorganisms sometimes are cultured in complex media (e.g., yeast extract-peptone-dextrose broth (YPD)).
  • engineered microorganisms are cultured in a defined minimal media that lacks a component necessary for growth and thereby forces selection of a desired expression cassette (e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).
  • Culture media in some embodiments are common commercially prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.).
  • Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism are known.
  • yeast are cultured in YPD media (10 g/L Bacto Yeast Extract, 20 g/L Bacto Peptone, and 20 g/L Dextrose).
  • Filamentous fungi are grown in CM (Complete Medium) containing 10 g/L Dextrose, 2 g/L Bacto Peptone, 1 g/L Bacto Yeast Extract, 1 g/L Casamino acids, 50 mL/L 20 ⁇ Nitrate Salts (120 g/L NaNO 3 , 10.4 g/L KCl, 10.4 g/L MgSO 4 .7H 2 O, 1 mL/L 1000 ⁇ Trace Elements (22 g/L ZnSO 4 .7H 2 O, 11 g/L H 3 BO 3 , 5 g/L MnCl 2 .7H 2 O, 5 g/L FeSO 4 .7H 2 O, 1.7 g/L CoCl 2 .6H 2 O, 1.6 g/L CuSO 4 .5H 2 O, 1.5 g/L Na 2 MoO 4 .2H 2 O, and 50 g/L Na 4 EDTA), and 1
  • a suitable pH range for the fermentation often is between about pH 4.0 to about pH 8.0, where a pH in the range of about pH 5.5 to about pH 7.0 sometimes is utilized for initial culture conditions. Culturing may be conducted under aerobic or anaerobic conditions, where microaerobic conditions sometimes are maintained.
  • a two-stage process may be utilized, where one stage promotes microorganism proliferation and another state promotes production of target molecule. In a two-stage process, the first stage may be conducted under aerobic conditions (e.g., introduction of air and/or oxygen) and the second stage may be conducted under anaerobic conditions (e.g., air or oxygen are not introduced to the culture conditions).
  • a variety of fermentation processes may be applied for commercial biological production of a target product.
  • commercial production of a target product from a recombinant microbial host is conducted using a batch, fed-batch or continuous fermentation process, for example.
  • a batch fermentation process often is a closed system where the media composition is fixed at the beginning of the process and not subject to further additions beyond those required for maintenance of pH and oxygen level during the process.
  • the media is inoculated with the desired organism and growth or metabolic activity is permitted to occur without adding additional sources (i.e., carbon and nitrogen sources) to the medium.
  • additional sources i.e., carbon and nitrogen sources
  • the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated.
  • cells proceed through a static lag phase to a high-growth log phase and finally to a stationary phase, wherein the growth rate is diminished or halted. Left untreated, cells in the stationary phase will eventually die.
  • a variation of the standard batch process is the fed-batch process, where the carbon source is continually added to the fermentor over the course of the fermentation process.
  • Fed-batch processes are useful when catabolite repression is apt to inhibit the metabolism of the cells or where it is desirable to have limited amounts of carbon source in the media at any one time.
  • Measurement of the carbon source concentration in fed-batch systems may be estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases (e.g., CO.sub.2).
  • Batch and fed-batch culturing methods are known in the art. Examples of such methods may be found in Thomas D.
  • Continuous cultures In continuous fermentation process a defined media often is continuously added to a bioreactor while an equal amount of culture volume is removed simultaneously for product recovery.
  • Continuous cultures generally maintain cells in the log phase of growth at a constant cell density.
  • Continuous or semi-continuous culture methods permit the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, an approach may limit the carbon source and allow all other parameters to moderate metabolism.
  • a number of factors affecting growth may be altered continuously while the cell concentration, measured by media turbidity, is kept constant.
  • Continuous systems often maintain steady state growth and thus the cell growth rate often is balanced against cell loss due to media being drawn off the culture.
  • ethanol may be purified from the culture media or extracted from the engineered microorganisms.
  • Culture media may be tested for ethanol concentration and drawn off when the concentration reaches a predetermined level.
  • Detection methods are known in the art, including but not limited to the use of a hydrometer and infrared measurement of vibrational frequency of dissolved ethanol using the CH band at 2900 cm ⁇ 1 .
  • Ethanol may be present at a range of levels as described herein.
  • a target product sometimes is retained within an engineered microorganism after a culture process is completed, and in certain embodiments, the target product is secreted out of the microorganism into the culture medium.
  • culture media may be drawn from the culture system and fresh medium may be supplemented, and/or (ii) target product may be extracted from the culture media during or after the culture process is completed.
  • Engineered microorganisms may be cultured on or in solid, semi-solid or liquid media. In some embodiments media is drained from cells adhering to a plate. In certain embodiments, a liquid-cell mixture is centrifuged at a speed sufficient to pellet the cells but not disrupt the cells and allow extraction of the media, as known in the art. The cells may then be resuspended in fresh media.
  • Target product may be purified from culture media according to methods known in the art.
  • target product is extracted from the cultured engineered microorganisms.
  • the microorganism cells may be concentrated through centrifugation at speed sufficient to shear the cell membranes.
  • the cells may be physically disrupted (e.g., shear force, sonication) or chemically disrupted (e.g., contacted with detergent or other lysing agent).
  • the phases may be separated by centrifugation or other method known in the art and target product may be isolated according to known methods.
  • target product sometimes is provided in substantially pure form (e.g., 90% pure or greater, 95% pure or greater, 99% pure or greater or 99.5% pure or greater).
  • target product may be modified into any one of a number of downstream products.
  • ethanol may be derivatized or further processed to produce ethyl halides, ethyl esters, diethyl ether, acetic acid, ethyl amines, butadiene, solvents, food flavorings, distilled spirits and the like.
  • Target product may be provided within cultured microbes containing target product, and cultured microbes may be supplied fresh or frozen in a liquid media or dried. Fresh or frozen microbes may be contained in appropriate moisture-proof containers that may also be temperature controlled as necessary. Target product sometimes is provided in culture medium that is substantially cell-free. In some embodiments target product or modified target product purified from microbes is provided, and target product sometimes is provided in substantially pure form. In certain embodiments, ethanol can be provided in anhydrous or hydrous forms. Ethanol may be transported in a variety of containers including pints, quarts, liters, gallons, drums (e.g., 10 gallon or 55 gallon, for example) and the like.
  • a target product e.g., ethanol, succinic acid
  • a target product is produced with a yield of about 0.30 grams of target product, or greater, per gram of glucose added during a fermentation process (e.g., about 0.31 grams of target product per gram of glucose added, or greater; about 0.32 grams of target product per gram of glucose added, or greater; about 0.33 grams of target product per gram of glucose added, or greater; about 0.34 grams of target product per gram of glucose added, or greater; about 0.35 grams of target product per gram of glucose added, or greater; about 0.36 grams of target product per gram of glucose added, or greater; about 0.37 grams of target product per gram of glucose added, or greater; about 0.38 grams of target product per gram of glucose added, or greater; about 0.39 grams of target product per gram of glucose added, or greater; about 0.40 grams of target product per gram of glucose added, or greater; about 0.41 grams of target product per gram of glucose added, or greater; 0.42 grams of target product per gram
  • DNA mutagenesis can be accomplished using the Stratagene (San Diego, Calif.) “QuickChange” kit according to the manufacturer's instructions, or by one of the other types of mutagenesis described above.
  • Genomic DNA from Zymomonas mobilis was obtained from the American Type Culture Collection (ATCC accession number 31821 D-5).
  • the genes encoding phosphogluconate dehydratase EC 4.2.1.12 (referred to as “edd”) and 2-keto-3-deoxygluconate-6-phosphate aldolase EC 4.2.1.14 (referred to as “eda”) were isolated from the ZM4 genomic DNA using the following oligonucleotides:
  • the ZM4 eda gene (SEQ ID No: 1) 5′-aactgactagtaaaaaatgcgtgatatcgattcc-3′ (SEQ ID No: 2) 5′-agtaactcgagctactaggcaacagcagcgcgcttg-3′
  • the ZM4 edd gene (SEQ ID NO: 3) 5′-aactgactagtaaaaaaatgactgatctgcattcaacg-3′ (SEQ ID NO: 4) 5′-agtaactcgagctactagataccggcacctgcatatattgc-3′
  • E. coli genomic DNA was prepared using Qiagen DNeasy blood and tissue kit according to the manufacture's protocol.
  • the E. coli edd and eda constructs were isolated from E. coli genomic DNA using the following oligonucleotides:
  • the E. coli eda gene (SEQ ID NO: 5) 5′-aactgactagtaaaaaatgaaaaactggaaaacaagtgcag aatc-3′ (SEQ ID NO: 6) 5′-agtaactcgagctactacagcttagcgccttctacagcttcacg-3′
  • SEQ ID NO: 5′-aactgactagtaaaaaaaatgaaaaactggaaaacaagtgcacg-3′ (SEQ ID NO: 6) 5′-agtaactcgagctactacagcttagcgccttctacagcttcacg-3′
  • oligonucleotides set forth above were purchased from Integrated DNA technologies (“IDT”, Coralville, Iowa). These oligonucleotides were designed to incorporate a SpeI restriction endonuclease cleavage site upstream and a XhoI restriction endonuclease cleavage site downstream of the edd and eda gene constructs such that these sites could be used to clone these genes into yeast expression vectors p426GPD (ATCC accession number 87361) and p425GPD (ATCC accession number 87359).
  • the forward oligonucleotides were designed to incorporate six consecutive AAAAAA nucleotides immediately upstream of the ATG initiation codon. This ensured that there was a conserved kozak sequence important for efficient translation initiation in yeast.
  • Cloning the edd and eda genes from ZM4 and E. coli genomic DNA was accomplished using the following procedure: About 100 ng of ZM4 or E. coli genomic DNA, 1 ⁇ M of the oligonucleotide primer set listed above, 2.5 U of PfuUltra High-Fidelity DNA polymerase (Stratagene), 300 ⁇ M dNTPs (Roche), and 1 ⁇ PfuUltra reaction buffer was mixed in a final reaction volume of 50 ⁇ l.
  • a BIORAD DNA Engine Tetrad 2 Peltier thermal cycler was used for the PCR reactions and the following cycle conditions were used: 5 min denaturation step at 95° C., followed by 30 cycles of 20 sec at 95° C., 20 sec at 55° C., and 1 min at 72° C., and a final step of 5 min at 72° C.
  • the first approach was to remove translational pauses from the polynucleotide sequence by designing the gene to incorporate only codons that are preferred in yeast. This optimization is referred to as the “hot rod” optimization.
  • translational pauses which are present in the native organism gene sequence are matched in the heterologous expression host organism by substituting the codon usage pattern of that host organism. This optimization is referred to as the “matched” optimization.
  • the final gene and protein sequences for edd and eda from the ZM4 native, hot rod (HR) and matched versions, as well as the E. coli native are shown in FIG. 6 .
  • FIG. 6 Certain sequences in FIG. 6 are presented at the end of this Example 1.
  • the matched version of ZM4 edd and ZM4 eda genes were synthesized by IDT, and the hot rod version was constructed using methods described in Larsen et al. ( Int. J. Bioinform. Res. Appl; 2008:4[3]; 324-336).
  • each version of each edd and eda gene was inserted into the yeast expression vector p426GPD (GPD promoter, 2 micron, URA3) (ATCC accession number 87361) between the SpeI and XhoI cloning sites.
  • Each version of the eda gene was also inserted into the SpeI and XhoI sites of the yeast expression vector p425GPD (GPD promoter, 2 micron, LEU3) (ATCC accession number 87359).
  • 3′ His tagged and non tagged p426 GPD constructs were made.
  • Each edd and eda p426GPD construct was transformed into Saccharomyces cerevisiae strain BY4742 (MATalpha his3delta1 leu2delta0 lys2delta0 ura3delta0) (ATCC accession number 201389).
  • This strain has a deletion of the his3 gene, an imidazoleglycerol-phosphate dehydratase which catalyzes the sixth step in histidine biosynthesis; a deletion of leu2 gene, a beta-isopropylmalate dehydrogenase which catalyzes the third step in the leucine biosynthesis pathway; a deletion of the lys2 gene, an alpha aminoadipate reductase which catalyzes the fifth step in biosynthesis of lysine; and a deletion of the ura3 gene, an orotidine-5′-phosphate decarboxylase which catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidines.
  • the genotype of BY4742 makes it an auxotroph for histidine, leucine, lysine and uracil.
  • Transformation of the p426GPD plasmids containing an edd or an eda variant gene into yeast strain BY4742 was accomplished using the Zymo Research frozen-EZ yeast transformation II kit according to the manufacturer's protocol.
  • the transformed BY4742 cells were selected by growth on a synthetic dextrose medium (SD) (0.67% yeast nitrogen base-2% dextrose) containing complete amino acids minus uracil (Krackeler Scientific Inc). Plates were incubated at about 30° C. for about 48 hours. Transformant colonies for each edd and eda variant were inoculated onto 5 ml of SD minus uracil medium and cells were grown at about 30° C. and shaken at about 250 rpm for about 24 hours.
  • SD synthetic dextrose medium
  • Transformants (16 different combinations total including the variant edd and eda combinations plus vector controls) were selected on synthetic dextrose medium (SD) (0.67% yeast nitrogen base-2% dextrose) containing complete amino acids minus uracil and leucine. Transformants of edd and eda variant combinations were inoculated onto 5 ml of SD minus uracil and leucine and cells were grown at about 30° C. in shaker flasks at about 250 rpm for about 24 hours.
  • SD dextrose medium
  • edd and eda variant combinations were inoculated onto 5 ml of SD minus uracil and leucine and cells were grown at about 30° C. in shaker flasks at about 250 rpm for about 24 hours.
  • Fresh overnight culture was used to inoculate about 100 ml of (SD media minus uracil and leucine containing about 0.01 g ergosterol/L and about 400 ⁇ l of Tween80) to an initial inoculum OD 600nm of about 0.1 and grown anaerobically at about 30° C. for approximately 14 hours until cells reached an OD 600nm of 3-4.
  • the cells were centrifuged at about 3000 g for about 10 minutes.
  • the cells were then washed with 25 ml deionized H 2 O and centrifuged at 3000 g for 10 min.
  • the cells were resuspended at about 2 ml/g of cell pellet) in lysis buffer (50 mM TrisCl pH7, 10 mM MgCl 2 1 ⁇ Calbiochem protease inhibitor cocktail set III). Approximately 900 ⁇ l of glass beads were added and cells were lysed by vortexing at maximum speed for 4 ⁇ 30 seconds. Cell lysate was removed from the glass beads, placed into fresh tubes and spun at about 10,000 g for about 10 minutes at about 4° C. The supernatant containing whole cell extract (WCE) was transferred to a fresh tube. WCE protein concentrations were measured using the Coomassie Plus Protein Assay (Thermo Scientific) according to the manufacturer's directions.
  • a total of about 750 ⁇ g of WCE was used for the edd and eda coupled assay.
  • about 750 ⁇ g of WCE was mixed with about 2 mM 6-phosphogluconate and about 4.5 U lactate dehydrogenase in a final volume of about 400 ⁇ l.
  • a total of about 100 ⁇ l of NADH was added to this reaction to a final molarity of about 0.3 mM, and NADH oxidation was monitored for about 10 minutes at about 340 nM using a DU800 spectrophotometer.
  • Saccharomyces cerevisiae strain YGR240CBY4742 was obtained from the ATCC (accession number 4015893). This strain is genetically identical to S. cerevisiae strain BY4742, except that YGR420C, the gene encoding the PFK1 enzyme, which is the alpha subunit of heterooctameric phosphofructokinase, has been deleted. A DNA construct designed to delete the gene encoding the PFK2 enzyme via homologous recombination was prepared. This construct substituted the gene encoding HIS3 (imidazoleglycerol-phosphate dehydratase, an enzyme required for synthesis of histidine) for the PFK2 gene.
  • HIS3 imidazoleglycerol-phosphate dehydratase, an enzyme required for synthesis of histidine
  • the DNA construct comprised, in the 5′ to 3′ direction, 100 bases of the 5′ end of the open reading frame of PFK2, followed by the HIS3 promoter, HIS3 open reading frame, HIS3 terminator, and 100 bp of the 3′ end of the PFK2 open reading frame.
  • This construct was prepared by two rounds of PCR. In the first round, about 100 ng of BY4742 genomic DNA was used as a template. The genomic DNA was prepared from cells using the Zymo Research Yeastar kit according to the manufacturer's instructions. PCR was performed using the following primers:
  • the PCR reaction conditions were the same as those set forth in Example 1 for preparing the edd and eda genes.
  • PCR conditions for this reaction were the same as for the first reaction immediately above.
  • the final PCR product was separated by agarose gel electrophoresis, excised, and purified using MP Biomedicals Geneclean II kit according to the manufacturer's instructions.
  • YGR240CBY4742 Approximately 2 ⁇ g of the purified DNA was used for transformation of the yeast strain YGR240CBY4742 by lithium acetate procedure as described by Shiestl and Gietz with an additional recovery step added after the heat shock step. Essentially after heat shock, cells were centrifuged at 500 ⁇ g for 2 min and resuspended in 1 ml of YP-Ethanol (1% yeast extract-2% peptone-2% ethanol) and incubated at 30° C. for 2 hours prior to plating on selective media containing SC-Ethanol (0.67% yeast nitrogen base-2% ethanol) containing complete amino acids minus histidine.
  • the engineered transformant strain referred to as YGR420CBY4742 ⁇ PFK2 has PFK1 and PFK2 genes deleted and is an auxotroph for leucine, uracil and lysine.
  • the YGR420CBY4742 ⁇ PFK2 strain was used for transformation of the combination of edd-p426 GPD (edd variants in p426 GPD) and eda-p425 GPD (eda variants in p425 GPD) variant constructs.
  • edd-p426 GPD edd variants in p426 GPD
  • eda-p425 GPD eda variants in p425 GPD
  • Each combination was transformed into YGR420CBY4742 ⁇ PFK2. For all transformation, 1 ⁇ g of edd-p426 GPD and 1 ⁇ g of eda-p425 GPD was used.
  • a complementation test for growth of YGR420CBY4742 ⁇ PFK2 strain on YPD 1% yeast extract-2% peptone-2% dextrose
  • YPGluconate 1% yeast extract-2% peptone-2% gluconate
  • Viable colonies of edd-p426 GPD and eda-p425 GPD variant construct combinations grown on SC-Ethanol minus uracil and leucine were patched to plates containing SC-ethanol minus uracil and leucine and incubated at 30° C. for 48 hrs. These patches were used to inoculate 5 ml of YPD media to an initial inoculum OD 600nm of 0.1 and the cells were grown anaerobically at 30° C. for 3 to 7 days.
  • Total genomic DNA from Zymomonas mobilis was obtained from ATCC (ATCC Number 31821).
  • the Z. mobilis gene encoding the enzyme phosphoenolpyruvate carboxylase (“PEP carboxylase”) was isolated from this genomic DNA and cloned using PCR amplification.
  • PCR was performed in a total volume of about 50 micro-liters in the presence of about 20 nanograms of Z. mobilis genomic DNA, about 0.2 mM of 5′ forward primer, about 0.2 mM of 3′ reverse primer, about 0.2 mM of dNTP, about 1 micro-liter of pfu UltraII DNA polymerase (Stratagene, La Jolla, Calif.), and 1 ⁇ PCR buffer (Stratagene, La Jolla, Calif.).
  • PCR was carried out in a thermocycler using the following program: Step One “95° C. for 10 minutes” for 1 cycle, followed by Step Two “95° C. for 20 seconds, 65° C. for 30 seconds, and 72° C. for 45 seconds” for 35 cycles, followed by Step Three “72° C. for 5 minutes” for 1 cycle, and then Step Four “4° C. Hold” to stop the reaction.
  • the primers for the PCR reaction were:
  • the DNA sequence of native Z. Mobilis PEP carboxylase is set forth as SEQ ID NO:20.
  • the cloned gene was inserted into the vector pGPD426 (ATCC Number: 87361) in between the SpeI and XhoI sites.
  • the final plasmid containing the PEP carboxylase gene was named pGPD426 PEPC.
  • pGPD426 N-his PEPC was constructed to insert a six-histidine tag (SEQ ID NO: 35) at the N-terminus of the PEPC sequence for protein expression verification in yeast.
  • This plasmid was constructed using two rounds of PCR to extend the 5′ end of the PEPC gene to incorporate a six-histidine tag (SEQ ID NO: 35) at the N-terminus of the PEPC protein.
  • the two 5′ forward primers used sequentially were:
  • the same 3′ primer was used as described above.
  • the PCR was performed in a total volume of about 50 micro-liters in the presence of about 20 nanograms of Z Mobilis PEP carboxylase polynucleotide, about 0.2 mM of 5′ forward primer, about 0.2 mM of 3′ reverse primer, about 0.2 mM of dNTP, about 1 micro-liter of pfu UltraII DNA polymerase (Stratagene, La Jolla, Calif.), and 1 ⁇ PCR buffer (Stratagene, La Jolla, Calif.).
  • the PCR was carried out in a thermocycler using the following program: Step One “95° C. for 10 minutes” for 1 cycle, followed by Step Two “95° C. for 20 seconds, 65° C. for 30 seconds, and 72° C. for 45 seconds” for 35 cycles, followed Step Three “72° C. for 5 minutes” for 1 cycle, and then Step Four “4° C. Hold” to stop the reaction.
  • the PEPC coding sequence was optimized to incorporate frequently used codons obtained from yeast glycolytic genes.
  • the resulting PEP carboxylase amino acid sequence remains identical to the wild type.
  • the codon optimized PEP carboxylase DNA sequence was ordered from IDT and was inserted into the vector pGPD426 at the SpeI and XhoI site.
  • the final plasmid containing the codon optimized PEP carboxylase gene was named pGPD426 PEPC_opti.
  • a similar plasmid, named pGPD426 N-his PEPC_opti was constructed to insert a six-histidine tag (SEQ ID NO: 35) at the N-terminus of the optimized PEPC gene for protein expression verification in yeast.
  • Both PCR reactions were performed in a total volume of about 50 micro-liters in the presence of about 20 nanograms of the codon optimized PEP carboxylase polynucleotide, about 0.2 mM of 5′ forward primer, about 0.2 mM of 3′ reverse primer, about 0.2 mM of dNTP, about 1 micro-liter of pfu UltraII DNA polymerase (Stratagene, La Jolla, Calif.), and 1 ⁇ PCR buffer (Stratagene, La Jolla, Calif.).
  • PCR reactions were carried out in a thermocycler using the following program: Step One “95° C. for 10 minutes” for 1 cycle, followed by Step Two “95° C. for 20 seconds, 65° C. for 30 seconds, and 72° C. for 45 seconds” for 35 cycles, followed Step Three “72° C. for 5 minutes” for 1 cycle, and then Step Four “4° C. Hold” to stop the reaction.
  • Saccharomyces cerevisiae strain BY4742 was cultured in YPD medium to an OD of about 1.0, and then prepared for transformation using the Frozen-EZ Yeast Transformation II kit (Zymo Research, Orange, Calif.) and following the manufacturer's instructions. Approximately 500 micrograms of each plasmid was added to the cells, and transformation was accomplished by addition of PEG solution (“Solution 3” in the Frozen-EZ Yeast Transformation II kit) and incubation at about 30° C. for an hour. After transformation, the cells were plated on synthetic complete medium (described in Example IV below) minus uracil (sc-ura) medium, grown for about 48 hours at about 30° C., and transformants were selected based on auxotrophic complementation.
  • synthetic complete medium described in Example IV below
  • sc-ura minus uracil
  • YKR097W ATCC Number 4016013, APCK, in the phosphoenolpyruvate carboxykinase gene is deleted
  • YGL062W ATCC Number 4014429, ⁇ PYC1, in which the pyruvate carboxylase 1 gene is deleted
  • YBR218C ATCC Number 4013358, ⁇ PYC2, in which the pyruvate carboxylase 2 gene is deleted.
  • the full length gene encoding the enzyme xylose isomerase from Ruminococcus flavefaciens strain 17 also known as Ruminococcus flavefaciens strain Siijpesteijn 1948
  • a substitution at position 513 in which cytidine was replaced by guanidine
  • IDT Integrated DNA Technologies, Inc.
  • SEQ ID NO:22 The sequence of this gene is set forth below as SEQ ID NO:22.
  • PCR was used to engineer a unique SpeI restriction site into the 5′ end of each of the xylose isomerase genes, and to engineer a unique XhoI restriction site at the 3′ end.
  • a version of each gene was created that contained a 6-HIS tag (SEQ ID NO: 35) at the 3′ end of each gene to enable detection of the proteins using Western analysis.
  • PCR amplifications were performed in about 50 ⁇ l reactions containing 1 ⁇ PfuI I Ultra reaction buffer (Stratagene, San Diego, Calif.), 0.2 mM dNTPs, 0.2 ⁇ M specific 5′ and 3′ primers, and 1 U PfuUltra II polymerase (Stratagene, San Diego, Calif.). The reactions were cycled at 95° C. for 10 minutes, followed by 30 rounds of amplification (95° C. for 30 seconds, 62° C. for 30 seconds, 72° C. for 30 seconds) and a final extension incubation at 72° C. for 5 minutes. Amplified PCR products were cloned into pCR Blunt II TOPO (Life Sciences, Carlsbad, Calif.) and confirmed by sequencing (GeneWiz, La Jolla, Calif.). The PCR primers for these reactions were:
  • the xylose isomerase gene from Piromyces , strain E2 was synthesized by IDT.
  • the sequence of this gene is set forth below as SEQ ID NO: 24.
  • Two hot rod (“HR”) versions of the Piromyces xylose isomerase gene were prepared using the method of Larsen et al., supra.
  • One version contained DNA sequence encoding a 6-histidine tag (SEQ ID NO: 35) at the 5′ terminus and the other did not.
  • the annealing temperature for the self-assembling oligonucleotides was about 48 degrees Celsius. The sequence of this gene is set forth below as
  • a unique SpeI restriction site was engineered at the 5′ end of each of the XI genes, and a unique XhoI restriction site was engineered at the 3′ end.
  • a 6-HIS tag (SEQ ID NO: 35) was engineered at the 3′ end of each gene sequence to enable detection of the proteins using Western analysis.
  • the primers are listed in Table X. PCR amplifications were performed in 50 ⁇ l reactions containing 1 ⁇ PfuI I Ultra reaction buffer (Stratagene, San Diego, Calif.), 0.2 mM dNTPs, 0.2 ⁇ M specific 5′ and 3′ primers, and 1 U PfuUltra II polymerase (Stratagene, San Diego, Calif.).
  • the reactions were cycled at 95° C. for 10 minutes, followed by 30 rounds of amplification (95° C. for 30 seconds, 62° C. for 30 seconds, 72° C. for 30 seconds) and a final extension incubation at 72° C. for 5 minutes.
  • Amplified PCR products were cloned into pCR Blunt II TOPO (Life Sciences, Carlsbad, Calif.) and confirmed by sequencing (GeneWiz).
  • the primers used for PCR were:
  • the genes encoding the native and HR versions of xylose isomerase were separately inserted into the vector p426GDP (ATCC catalog number 87361).
  • Saccharomyces cerevisiae strain BY4742 cells (ATCC catalog number 201389) were cultured in YPD media (10 g Yeast Extract, 20 g Bacto-Peptone, 20 g Glucose, 1 L total) at about 30° C. Separate aliquots of the cells were transformed with the plasmid constructs containing the various xylose isomerase constructs or with the vector alone. Transformation was accomplished using the Zymo kit (Catalog number T2001; Zymo Research Corp., Orange, Calif.
  • transformed cells containing the various xylose isomerase constructs were selected from the cultures and grown in about 100 ml of SC-Dextrose (minus uracil) to an OD 600 of about 4.0.
  • the S. cerevisiae cultures that were transformed with the various xylose isomerase-histidine constructs were then lysed using YPER-Plus reagent (Thermo Scientific, catalog number 78999) according to the manufacturer's directions. Protein quantitation of the lysates was performed using the Coomassie-Plus kit (Thermo Scientific, catalog number 23236) as directed by the manufacturer. Denaturing and native Western blot analyses were then conducted.
  • the secondary antibody [Dnk pAb to Ms IgG (HRP), AbCam, Cambridge, Mass.] was used at 1:15000 dilution in 0.3% BSA and allowed to incubate for about 90 minutes at room temperature with gentle shaking. The membrane was washed three times for about 5 minutes using 1 ⁇ PBS and 0.05% Tween-20 with gentle shaking. The membrane was then incubated with 5 ml of Supersignal West Pico Chemiluminescent substrate (Thermo Scientific, San Diego, Calif.) for 1 minute and then was exposed to a phosphorimager (Bio-Rad Universal Hood II, Bio-Rad, Hercules, Calif.) for about 10-100 seconds. The results are shown in FIG. 7 . As can be seen, both Piromyces (“P” in FIG. 7 ) and Ruminococcus (“R” in FIG. 7 ) xylose isomerases are expressed in both the soluble and insoluble fractions of the yeast cells.
  • P Piromyces
  • the yeast gene cdc21 encodes thymidylate synthase, which is required for de novo synthesis of pyrimidine deoxyribonucleotides.
  • a cdc 21 mutant, strain 17206, (ATCC accession number 208583) has a point mutation G139S relative to the initiating methionine.
  • the restrictive temperature of this temperature sensitive mutant is 37° C., which arrests cell division at S phase, so that little or no cell growth and division occurs at or above this temperature.
  • Saccharomyces cerevisiae strain YGR420CBY4742 ⁇ PFK2 was used as the starting cell line to create the cdc21 growth sensitive mutant.
  • a construct for homologous recombination was prepared to replace the wild type thymidylate synthase YGR420CBY4742 ⁇ PFK2 for the cdc21 mutant. This construct was made in various steps. First, the cdc21 mutant region from Saccharomyces cerevisiae strain 17206 was PCR amplified using the following primers:
  • CDC21_fwd (SEQ ID NO: 52) 5′-aatcgatcaaagcttctaaatacaagacgtgcgatgacgactatac tggac-3′
  • CDC21_rev (SEQ ID NO: 53) 5′-taccgtactacccgggtatatagtctttttgccctggtgttcctt aataatttc-3′
  • PCR amplification reaction Saccharomyces cerevisiae 17206 genomic DNA was used.
  • the genomic DNA was extracted using Zymo research YeaStar Genomic DNA kit according to instructions.
  • 100 ng of 17206 genomic DNA 1 ⁇ M of the oligonucleotide primer set listed above, 2.5 U of PfuUltra High-Fidelity DNA polymerase (Stratagene), 300 ⁇ M dNTPs (Roche), and 1 ⁇ PfuUltra reaction buffer was mixed in a final reaction volume of 500.
  • the genomic DNA of BR214-4a (ATTC accession number 208600) was extracted using Zymo research YeaStar Genomic DNA kit according to instructions.
  • the lys2 gene with promoter and terminator regions was PCR amplified from BR214-4a genomic DNA using the following primers:
  • Lys2Fwd (SEQ ID NO: 54) 5′-tgctaatgacccgggaattccacttgcaattacataaaaattcc ggcgg-3′
  • Lys2Rev (SEQ ID NO: 55) 5′-atgatcattgagctcagcttcgcaagtattcattttagacccat ggtgg-3′.
  • the PCR cycle was identical to that just described above but with genomic DNA of BR214-4a instead.
  • XmaI and SacI restriction sites were designed to flank this DNA construct to clone it into the XmaI and SacI sites of the PUC19-cdc21 vector according to standard cloning procedures described by Maniatis in Molecular Cloning.
  • the new construct with the cdc21 mutation with a lys2 directly downstream of that will be referred to as PUC19-cdc2′-lys2.
  • the final step involved the cloning of the downstream region of thymidylate synthase into the PUC19-cdc2′-lys2 vector immediately downstream of the lys2 gene.
  • the downstream region of the thymidylate synthase was amplified from BY4742 genomic DNA (ATCC accession number 201389D-5 using the following primers:
  • ThymidylateSynthase_DownFwd (SEQ ID NO: 56) 5′-tgctaatgagagctctcattttttggtgcgatatgtttttggtt gatg-3′ and ThymidylateSynthatse_DownRev: (SEQ ID NO: 57) 5′-aatgatcatgagctcgtcaacaagaactaaaaaattgttcaaaa atgc-3′.
  • This final construct is referred as PUC19-cdc2′-lys2-ThymidylateSynthase_down.
  • the sequence is set forth in the tables.
  • a final PCR amplification reaction of this construct was performed using the following PCR primers:
  • ThymidylateSynthase::cdc21 fwd (SEQ ID NO: 58) 5′-ctaaatacaagacgtgcgatgacgactatactgg-3′ and ThymidylateSynthase::cdc21 rev: (SEQ ID NO: 59) 5′-gtcaacaagaactaaaaaattgttcaaaaatgcaattgtc-3′.
  • the PCR reaction was identical to that described above but using 100 ng of the PUC19-cdc2′-lys2-ThymidylateSynthase_down construct as a template.
  • the final PCR product was separated by agarose gel electrophoresis, excised, and purified using MP Biomedicals Geneclean II kit as recommended. Homologous recombination of YGR420CBY4742 ⁇ PFK2 to replace the wt thymidylate synthase for the cdc21 mutant was accomplished using 10 ⁇ g of the purified PCR product to transform YGR420CBY4742 ⁇ PFK2 strain using same transformation protocol described above. Transformants were selected by culturing the cells on selective media containing SC-Ethanol (0.67% yeast nitrogen base-2% ethanol) containing complete amino acids minus lysine.
  • SC-Ethanol 0.67% yeast nitrogen base-2% ethanol
  • This final engineered strain contains the mutated cdc21 gene, and has both the PFK1 and PFK2 genes deleted.
  • This final engineered strain will be transformed with the best combination of edd-p426 GPD and eda-p425 GPD variant constructs. Ethanol and glucose measurements will be monitored during aerobic and anaerobic growth conditions using Roche ethanol and glucose kits according to instructions.
  • regulator polynucleotides that can be utilized in embodiments herein. Such polynucleotides may be utilized in native form or may be modified for use herein. Examples of regulatory polynucleotides include those that are regulated by oxygen levels in a system (e.g., up-regulated or down-regulated by relatively high oxygen levels or relatively low oxygen levels)
  • PacC RDR1 Transcriptional repressor involved in the control of multidrug resistance; negatively regulates expression of the PDR5 gene; member of the Gal4p family of zinc cluster proteins SUM1 Transcriptional repressor required for mitotic repression of middle sporulation-specific genes; also acts as general replication initiation factor; involved in telomere maintenance, chromatin silencing; regulated by pachytene checkpoint XBP1 Transcriptional repressor that binds to promoter sequences of the cyclin genes, CYS3, and SMF2; expression is induced by stress or starvation during mitosis, and late in meiosis; member of the Swi4p/Mbp1p family; potential Cdc28p substrate NRG2 Transcriptional repressor that mediates glucose repression and negatively regulates filamentous growth; has similarity to Nrg1p NRG1 Transcriptional repressor that recruits the Cyc8p-Tup1p complex to promoters; media
  • SKT5 Activator of Chs3p (chitin synthase III), recruits Chs3p to the bud neck via interaction with Bni4p; has similarity to Shc1p, which activates Chs3p during sporulation MSA1 Activator of G1-specific transcription factors, MBF and SBF, that regulates both the timing of G1-specific gene transcription, and cell cycle initiation; potential Cdc28p substrate AMA1 Activator of meiotic anaphase promoting complex (APC/C); Cdc20p family member; required for initiation of spore wall assembly; required for Clb1p degradation during meiosis STB5 Activator of multidrug resistance genes, forms a heterodimer with Pdr1p; contains a Zn(II)2Cys6 zinc finger domain that interacts with a PDRE (pleotropic drug resistance element) in vitro; binds Sin3p in a two-hy
  • nidulans developmental regulator potential Cdc28p substrate FHL1 Transcriptional activator with similarity to DNA-binding domain of Drosophila forkhead but unable to bind DNA in vitro; required for rRNA processing; isolated as a suppressor of splicing factor prp4 VHR1 Transcriptional activator, required for the vitamin H-responsive element (VHRE) mediated induction of VHT1 (Vitamin H transporter) and BIO5 (biotin biosynthesis intermediate transporter) in response to low biotin concentrations CDC20 Cell-cycle regulated activator of anaphase-promoting complex/cyclosome (APC/C), which is required for metaphase/anaphase transition; directs ubiquitination of mitotic cyclins, Pds1p, and other anaphase inhibitors; potential Cdc28p substrate CDH1 Cell-cycle regulated activator of the anaphase-promoting complex/cyclosome (APC/C), which directs ubiquitination of cyclins resulting in mit
  • nucleic acids that encode a polypeptide having xylose isomerase activity can be obtained.
  • Certain nucleic acid encoded polypeptides having active xylose isomerase activity can be expressed in an engineered yeast ( S. cerevisiae ).
  • Pseudomonas aeruginosa strain PAO1 DNA was prepared using Qiagen DNeasy Blood and Tissue kit (Qiagen, Valencia, Calif.) according to the manufacture's instructions.
  • the P. aeruginosa edd and eda constructs were isolated from P. aeruginosa genomic DNA using the following oligonucleotides:
  • the P. aeruginosa edd gene (SEQ ID NO: 63) 5′-aactgaactgactagtaaaaaatgcaccctcgtgtgctcgaagt- 3′ (SEQ ID NO: 64) 5′-agtaaagtaaaagcttctactagcgccagccgttgaggctct-3′
  • SEQ ID NO: 64 5′-agtaaagtaaaagcttctactagcgccagccgttgaggctct-3′
  • aeruginosa edd gene with 6-HIS c-terminal tag (SEQ ID NO: 35): (SEQ ID NO 63) 5′-aactgaactgactagtaaaaaatgcaccctcgtgtgctcgaagt- 3′ (SEQ ID NO: 65) 5′-agtaaagtaaagcttctactaatgatgatgatgatgatggcgccag ccgttgaggctc-3′ The P.
  • aeruginosa eda gene (SEQ ID NO: 66) 5′-aactgaactgactagtaaaaaatgcacaaccttgaacagaagacc- 3′ (SEQ ID NO: 67) 5′-agtaaagtaactcgagctattagtgtctgcggtgctcggcgaa-3′ The P.
  • aeruginosa eda gene with 6-HIS c-terminal tag (SEQ ID NO: 35): (SEQ ID NO: 66) 5′-aactgaactgactagtaaaaaatgcacaaccttgaacagaagacc- 3′ (SEQ ID NO: 68) 5′-taaagtaactcgagctactaatgatgatgatgatgatgatggtgtctgcgg gtgcggcgaa-3′
  • oligonucleotides set forth above were purchased from Integrated technologies (“IDT”, Coralville, Iowa). These oligonucleotides were designed to incorporate a SpeI restriction endonuclease cleavage site upstream of a HindIII restriction endonuclease cleavage site or downstream of an XhoI restriction endonuclease cleavage site, with respect to the edd and eda gene constructs. These restriction endonuclease sites could be used to clone the edd and eda genes into yeast expression vectors p426GPD (ATCC accession number 87361) and p425GPD (ATCC accession number 87359).
  • the forward oligonucleotides also incorporate six consecutive A nucleotides (e.g., AAAAAA) immediately upstream of the ATG initiation codon.
  • the six consecutive A nucleotides ensured that there was a conserved ribosome binding sequence for efficient translation initiation in yeast.
  • PCR amplification of the genes were performed as follows: about 100 ng of the genomic P. aeruginosa PAO1 DNA was added to 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers (SEQ. ID. NOS: 63-68, and combinations as indicated), and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix. This was cycled as follows: 95° C. 10 minutes followed by 30 rounds of 95° C. for 20 seconds, 50° C. (eda amplifications) or 53° C. (edd amplifications) for 30 seconds, and 72° C.
  • nucleotide and amino acid sequences of the P. aeruginosa edd and eda genes are given below as SEQ ID NOS. 69-72.
  • aeruginosa edd amino sequence SEQ ID NO: 70 MHPRVLEVTRRIQARSAATRQRYLEMVRAAASKGPHRGTLPCGNLAHGVAACGESDKQTLRLMN QANVAIVSAYNDMLSAHQPFERFPGLIKQALHEIGSVGQFAGGVPAMCDGVTQGEPGMELSLASR DVIAMSTAIALSHNMFDAALCLGVCDKIVPGLLIGSLRFGHLPTVFVPAGPMPTGISNKEKAAVRQL FAEGKATREELLASEMASYHAPGTCTFYGTANTNQLLVEVMGLHLPGASFVNPNTPLRDELTREA ARQASRLTPENGNYVPMAEIVDEKAIVNSVVALLATGGSTNHTLHLLAIAQAAGIQLTWQDMSELS HVVPTLARIYPNGQADINHFQAAGGMSFLIRQLLDGGLLHEDVQTVAGPGLRRYTREPFLEDGRLV WREGPERSLDEAILRPLDKPFSAEGGLRLMEGNLGRGVMKVSAVA
  • aeruginosa eda nucleotide sequence SEQ ID NO: 71 ATGCACAACCTTGAACAGAAGACCGCCCGCATCGACACGCTGTGCCGGGAGGCGCGCATCC TCCCGGTGATCACCATCGACCGCGAGGCGGACATCCTGCCGATGGCCGATGCCCTCGCCGC CGGCGGCCTGACCGCCCTGGAGATCACCCTGCGCACGGCGCACGGGCTGACCGCCATCCG GCGCCTCAGCGAGGAGCGCCCGCACCTGCGCATCGGCCGGCACCGTGCTCGACCCGCG GACCTTCGCCGCCGCGCGGAAAAGGCCGGGGCGAGCTTCGTGGTCACCCCGGGTTGCACCGA CGAGTTGCTGCGCTTCGCCCTGGACAGCGAAGTCCCGCTGTTGCCCGGCGTGGCCAGCGCT TCCGAGATCATGCTCGCCTACCGCCATGGCTACCGCCGCTTCAAGCTGTTTCCCGCCGAAGT CAGCGGCGGCCCGGCGGCTGAAGGCGTTCTCGGGACCATTCCCCG
  • aeruginosa eda amino sequence SEQ ID NO: 72 MHNLEQKTARIDTLCREARILPVITIDREADILPMADALAAGGLTALEITLRTAHGLTAIRRLSEERPH LRIGAGTVLDPRTFAAAEKAGASFVVTPGCTDELLRFALDSEVPLLPGVASASEIMLAYRHGYRRF KLFPAEVSGGPAALKAFSGPFPDIRFCPTGGVSLNNLADYLAVPNVMCVGGTWMLPKAVVDRGD WAQVERLSREALERFAEHRRH
  • a haploid Saccharomyces cerevisiae strain (BY4742; ATCC catalog number 201389) was cultured in YPD media (10 g Yeast Extract, 20 g Bacto-Peptone, 20 g Glucose, 1 L total) at about 30° C. Separate aliquots of these cultured cells were transformed with a plasmid construct(s) containing the eda gene alone, the eda and edd genes, or with vector alone. Transformation was accomplished using the Zymo frozen yeast transformation kit (Catalog number T2001; Zymo Research Corp., Orange, Calif.).
  • SC drop out media with glucose minus leucine (eda), minus uracil and minus leucine (eda and edd) (about 20 g glucose; about 2.21 g SC drop-out mix [described below], about 6.7 g yeast nitrogen base, all in about 1 L of water); this mixture was cultured for 2-3 days at about 30° C.
  • SC drop-out mix contained the following ingredients (Sigma); all indicated weights are approximate:
  • Cell lysates of the various EDD and EDA expressing strains were prepared as follows. About 50 to 100 ml of SCD-ura-leu media containing 10 mM MnCl 2 was used to culture strains containing the desired plasmid constructs. When cultured aerobically, strains were grown in a 250 ml baffled shaker flask. When grown anaerobically, 400 ⁇ l/L Tween-80 (British Drug Houses, Ltd., West Chester, Pa.) plus 0.01 g/L Ergosterol (Alef Aesar, Ward Hill, Mass.) were added and the culture was grown in a 250 ml serum bottle outfitted with a butyl rubber stopper with an aluminum crimp cap. Each strain was inoculated at an initial OD 600 of about 0.2 and grown to an OD 600 of about 3-4. Cells were grown at 30° C. at 200 rpm.
  • Yeast cells were harvested by centrifugation at 1046 ⁇ g (e.g., approximately 3000 rpm) for 5 minutes at 4° C. The supernatant was discarded and the cells were resuspended in 25 mL cold sterile water. This wash step was repeated once. Washed cell pellets were resuspended in 1 mL sterile water, transferred to 1.5 mL screw cap tube, and centrifuged at 16,100 ⁇ g (e.g., approximately 13,200 rpm) for 3 minutes at 4° C.
  • Cell pellets were resuspended in about 800-1000 ⁇ l of freshly prepared lysis buffer (50 mM Tris-Cl pH 7.0, 10 mM MgCl2, 1 ⁇ protease inhibitor cocktail EDTA-free (Thermo Scientific, Waltham, Mass.) and the tube filled with zirconia beads to avoid any headspace in the tube.
  • the tubes were placed in a Mini BeadBeater (Bio Spec Products, Inc., Bartlesville, Okla.) and vortexed twice for 30 seconds at room temperature.
  • the supernatant was transferred to a new 1.5 mL microcentrifuge tube and centrifuged twice to remove cell debris at 16,100 ⁇ g (e.g., approximately 13,200 rpm) for 10 minutes, at 4° C. Quantification of the lysates was performed using the Coomassie-Plus kit (Thermo Scientific, San Diego, Calif.) as directed by the manufacturer ('6-HIS' below disclosed as SEQ ID NO: 35).
  • E. coli native E. coli native BF460 E. coli native with 6-HIS E. coli native with 6-HIS BF591
  • PAO1 native PAO1 native BF568
  • PAO1 native with 6-HIS PAO1 native with 6-HIS BF592
  • PAO1 native E. coli native BF603
  • PAO1 native E. coli native BF604
  • SDS-PAGE gels were performed according to the manufacturer's recommendation using NuPage MES-SDS Running Buffer at 1 ⁇ concentration with the addition of NuPage antioxidant into the cathode chamber at a 1 ⁇ concentration.
  • Novex Sharp Protein Standards (Life Technologies, Carlsbad, Calif.) were used as standards.
  • Gels were transferred onto a nitrocellulose membrane (0.45 micron, Thermo Scientific, San Diego, Calif.) using Western blotting filter paper (Thermo Scientific) using a Bio-Rad Mini Trans-Blot Cell (BioRad, Hercules, Calif.) system for approximately 90 minutes at 40V.
  • the membrane was washed in 1 ⁇ PBS (EMD, San Diego, Calif.), 0.05% Tween-20 (Fisher Scientific, Fairlawn, N.J.) for 2-5 minutes with gentle shaking.
  • the membrane was blocked in 3% BSA dissolved in 1 ⁇ PBS and 0.05% Tween-20 at room temperature for about 2 hours with gentle shaking.
  • the membrane was washed once in 1 ⁇ PBS and 0.05% Tween-20 for about 5 minutes with gentle shaking.
  • the membrane was then incubated at room temperature with the 1:5000 dilution of primary antibody (Ms mAB to 6 ⁇ His Tag (SEQ ID NO: 35), AbCam, Cambridge, Mass.) in 0.3% BSA (Fraction V, EMD, San Diego, Calif.) dissolved in 1 ⁇ PBS and 0.05% Tween-20 with gentle shaking.
  • primary antibody Ms mAB to 6 ⁇ His Tag (SEQ ID NO: 35
  • BSA Fraction V, EMD, San Diego, Calif.
  • the membrane incubated with 5 ml of Supersignal West Pico Chemiluminescent substrate (Thermo Scientific, San Diego, Calif.) for 1 minute and then was exposed to a phosphorimager (Bio-Rad Universal Hood II, Bio-Rad, Hercules, Calif.) for about 10-100 seconds.
  • Supersignal West Pico Chemiluminescent substrate Thermo Scientific, San Diego, Calif.
  • a phosphorimager Bio-Rad Universal Hood II, Bio-Rad, Hercules, Calif.
  • FIGS. 8A and 8B The results of the Western blots, shown in FIGS. 8A and 8B . Included in the expression data are engineered and/or optimized versions of certain eda and edd genes. The genes were modified to include a C-terminal HIS tag to facilitate purification. The two letters refer to the EDD and EDA source, respectively.
  • P is from P. aeruginosa
  • PAO1 E is from E. coli
  • Z is from Zymomonas mobilis ZM4
  • hot rod is the optimized version of Zymomonas mobilis
  • Harmonized is the codon harmonized version of Zymomonas mobilis
  • V refers to the vector(s). Both total crude extract and the solubilized extract are shown. The results presented in FIGS.
  • Cell lysates of the various EDD and EDA expressing strains were prepared as follows. About 50 to 100 ml of SCD-ura-leu media containing 10 mM MnCl 2 was used. When cultured aerobically, strains were grown in a 250 ml baffled shake flask. When grown anaerobically, 4000/L Tween-80 (British Drug Houses, Ltd., West Chester, Pa.) plus 0.01 g/L Ergosterol (Alef Aesar, Ward Hill, Mass.) were added and the culture was grown in a 250 ml serum bottle outfitted with a butyl rubber stopper with an aluminum crimp cap. Each strain was inoculated at an initial OD 600 of about 0.2 and grown to an OD 600 of about 3-4. Cells were grown at 30° C. at 200 rpm.
  • Yeast cells were harvested by centrifugation at 1046 ⁇ g (3000 rpm) for 5 minutes at 4° C. The supernatant was discarded and the cells were resuspended in 25 mL cold sterile water. This wash step was repeated once. Washed cell pellets were resuspended in 1 mL sterile water, transferred to 1.5 mL screw cap tube, and centrifuged at 16,100 ⁇ g (13,200 rpm) for 3 minutes at 4° C.
  • Cell pellets were resuspended in about 800-1000 ⁇ l of freshly prepared lysis buffer (50 mM Tris-Cl pH 7.0, 10 mM MgCl 2 , 1 ⁇ protease inhibitor cocktail EDTA-free (Thermo Scientific, Waltham, Mass.) and the tube filled with zirconia beads to avoid any headspace in the tube.
  • the tubes were placed in a Mini BeadBeater (Bio Spec Products, Inc., Bartlesville, Okla.) and vortexed twice for 30 seconds at room temperature. The supernatant was transferred to a new 1.5 mL microcentrifuge tube and centrifuged twice to remove cell debris at 16,100 ⁇ g (13,200 rpm) for 10 minutes, at 4° C. Quantification of the lysates was performed using the Coomassie-Plus kit (Thermo Scientific, San Diego, Calif.) as directed by the manufacturer.
  • the table below presents the relative specific activities for BY4742 strains expressing EDD and EDA from either P. aeruginosa (PAO1) or E. coli sources.
  • the results presented in the table below indicate that each of the listed combinations of EDD and EDA genes, when expressed in S. cerevisiae strain BY4742, confers activity.
  • FIG. 9 graphically displays the relative activities of the various EDD/EDA combinations presented in the table above, as measured in assays using 750 micrograms of crude extract. From the height of the PE bar in FIG. 9 , and the data presented in the table above, it is evident that the combinations conferring the highest level of activity were the EDD-P/EDA-E (e.g., PE) and EDD-P/EDA-P (e.g., PP) combinations.
  • EDD-P/EDA-E e.g., PE
  • EDD-P/EDA-P e.g., PP
  • Strains BF428 vector control
  • BF591 ED-PAO1/EDA-PAO1
  • BF592 ELD-PAO1/EDA- E. coli
  • BF603 ED- E. coli /EDA-PAO1
  • BF604 ED- E. coli /EDA- E.
  • coli were inoculated into 15 ml SCD-ura-leu media containing 400 ⁇ l/L Tween-80 (British Drug Houses, Ltd., West Chester, Pa.) plus 0.01 g/L Ergosterol (EMD, San Diego, Calif.) in 20 ml Hungate tubes outfitted with a butyl rubber stopper and sealed with an aluminum crimped cap to prevent oxygen from entering the culture at an initial OD 600 of 0.5 and grown for about 20 hours.
  • Glucose and ethanol in the culture media were assayed using YSI 2700 BioAnalyzer instruments (world wide web uniform resource locator ysi.com), according to the manufacturer's recommendations at 0 and 20 hours post inoculation.
  • the results of the fermentation of glucose to ethanol are showing graphically in FIG. 10 .
  • the results presented in FIG. 9 indicate that the presence of the EDD/EDA combinations in S. cerevisiae increase the yield of ethanol produced, when compared to a vector-only control.
  • the EDD/EDA combinations that showed the greatest fermentation efficiency in yeast were EDD-P/EDA-E (e.g., PE) and EDD-E/EDA-P (e.g., EP).
  • a fermentation test of the strain BF591 [BY4742 with plasmids pBF290 (p426GPD-EDD_PAO1) and pBF292 (p425GPD-EDA_PAO1)] was conducted against BF428 (BY4742 p426GPD/p425GPD) control strain in 700 ml w.v. Multifors multiplexed fermentors.
  • the fermentation medium was SC-Ura-Leu with about 2% glucose. Vessels were inoculated with about a 6.25% inoculum from overnight cultures grown in about 50 ml SC-Ura-Leu with about 2% glucose.
  • the cultures were grown aerobically at about 30° C. with about 250 rpm agitation, 1 vvm sparge of process air, (21% O2).
  • the pH was controlled at around 5.0 with 0.25 N NaOH.
  • glucose concentrations dropped below 0.5 g/L the fermentation was switched to anaerobic conditions.
  • samples were taken to measure glucose concentrations and biomass by OD 600 as reported in Table B. Ethanol and glucose concentrations in the fermentation broth were monitored using YSI 2700 BioAnalyzer instruments.
  • the table below presents the elapsed fermentation time (EFT), the biomass and glucose at the start of anaerobic fermentation in a 400 ml fermentor.
  • EFT elapsed fermentation time
  • the edd and eda combinations carried by the strains are described above.
  • a bolus of 20 g/L glucose plus 3.35 g/L of yeast nitrogen base without amino acids was added to the fermentors.
  • 4 ml/L of 2.5 g/L ergosterol in ethanol, 0.4 ml/L Tween 80, and 0.01% AF-204 were added to each fermentor.
  • Oxygen was purged with 100% N2 sparged at about 1 vvm until pO2 was below 1%.
  • FIGS. 11A and 11B The data presented in the table above also is presented graphically in FIGS. 11A and 11B .
  • FIG. 11A presents the fermentation data from strain BF428 (BY4742 with vector controls) and
  • FIG. 11B presents the fermentation data from strain BF591 (BY4742 with EDD-PAO1/EDA-PAO1). Fermentation profiles for strains BF 428 and BF 591, grown on 2% dextrose, were calculated and are presented in the table below.
  • the results from the fermentation show that the BF591 has a higher ethanol yield (triangles, compare FIG. 11A and FIG. 11B ) than the control BF428 strain.
  • the calculated yield of ethanol was also determined to be higher in the engineered BF591 strain (0.43 g ethanol/g glucose) than that of the BF428 control strain (0.40 g ethanol/g glucose).
  • Oligonucleotides (SEQ ID NO: 276) #350 - 5′-TAAAACGACGGCCAGTGAAT-3′ (SEQ ID NO: 277) #351 - 5′-TGCAGGTCGACTCTAGAGGAT-3′ (SEQ ID NO: 278) #352 - 5′-GTGTGCGTGTATGTGTACACCTGTATTTAATTTCCTTACT CGCGGGTTTTTCTAAAACGACGGCCAGTGAAT-3′ (SEQ ID NO: 279) #353 - 5′-TGTACCAGTCTAGAATTCTACCAACAAATGGGGAAATCAA AGTAACTTGGGCTGCAGGTCGACTCTAGAGGA-3′
  • oligonucleotides set forth above were purchased from Integrated Technologies (“IDT”, Coralville, Iowa). PCR amplification of the genes were performed as follows: about 50 ng of the pBFU-719 DNA (e.g., plasmid with unique 200-mer sequence) was added to 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers (#350/#351 in the first round), and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix. The reaction mixture was cycled as follows: 95° C. 10 minutes followed by 30 rounds of 95° C. for 20 seconds, 60° C. for 30 seconds, and 72° C.
  • a final 5 minute extension reaction at 72° C. was also included.
  • a second round of PCR amplification was done using 50 ng of the first round PCR amplification with 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers (#352/#353 in the second round), and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix.
  • the second reaction mixture was cycled as follows: 95° C. 10 minutes followed by 30 rounds of 95° C. for 20 seconds, 60° C. for 30 seconds, and 72° C. for 45 seconds.
  • a final 5 minute extension reaction at 72° C. was also included.
  • the final PCR product was purified using the Zymo Research DNA Clean & Concentrator-25 kit (Zymo Research, Orange, Calif.).
  • Transformation was accomplished by a high-efficiency competency method.
  • a 5 ml culture of the BY4742 or BY4741 strain was grown overnight at about 30° C. with shaking at about 200 rpm.
  • a suitable amount of this overnight culture was added to 60 ml of YPD media to obtain an initial OD600 of about 0.2 (approximately 2 ⁇ 10 6 cells/ml).
  • the cells were allowed to grow at 30° C. with agitation (about 200 rpm) until the OD 600 was about 1.
  • the cells were then centrifuged at 3000 rpm for 5 min, washed with 10 ml sterile water and re-centrifuged.
  • the cell pellet was resuspended in 1 ml sterile water, transferred to a 1.5 ml sterile microcentrifuge tube and spun down at 4000 ⁇ g for about 5 minutes.
  • This cell pellet was resuspended in 1 ml sterile 1 ⁇ TE/LiOAC solution (10 mM Tris-HCl, 1 mM EDTA, 100 mM LiOAc, pH7.5) and re-centrifuged at about 4000 ⁇ g for about 5 minutes.
  • the cell pellet was resuspended in 0.25 ml 1 ⁇ TE/LiOAc solution.
  • the cells were pelleted in a microcentrifuge at 13000 rpm for about 30 seconds and the supernatant removed.
  • the cells were resuspended in 1 ml 1 ⁇ TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), centrifuged at 13000 rpm for about 30 seconds and resuspended in 1 ml 1 ⁇ TE.
  • About 100-200 ⁇ l of cells were plated onto SCD-URA media, as described above, and allowed to grow at about 30° C. for about 3 days. After 3 days, transformed colonies were streaked for single colonies on SCD-URA plates and allowed to grow at about 30° C. for about 3 days.
  • SCD agar plates (20 g/L agar in SCD media) containing 1 g/L 5-FOA (Research Products International Corp, Mt. Prospect, Ill.), and also inoculated into YPD liquid broth.
  • the plates were allowed to grow at about 30° C. for about 4 days and the liquid culture was grown overnight at about 30° C. with agitation of about 200 rpm.
  • genomic DNA was prepared from the YPD overnight cultures. Briefly, the yeast cells were pelleted by centrifugation at room temperature for 5 minutes at approximately 3000 rpm. The cell pellet was resuspended in 200 ⁇ l of breaking buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH8, 1 mM EDTA) and placed into a 1.5 ml microcentrifuge tube containing about 200 ⁇ l glass beads and about 200 ⁇ l of phenol:chloroform:isoamyl alcohol (Ambion, Austin, Tex.). The mixture was vortexed for about 2 to 5 minutes at room temperature.
  • breaking buffer 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH8, 1 mM EDTA
  • the genomic DNA was then precipitated by placing the tubes at ⁇ 80° C. for at least one hour (or in a dry ice/ethanol bath for about 30 minutes). The tubes were then centrifuged at about 13000 rpm for 5 minutes at about 4° C. to pellet the DNA. The DNA pellet was then washed two times or more times with about 200 ⁇ l of 70% ethanol and re-centrifuged. The DNA pellet was dried using vacuum assisted air drying and resuspended in about 50 to 200 ⁇ l 1 ⁇ TE.
  • the genomic DNA isolated as described above was used in a PCR amplification reaction consisting of about 50 ng of the genomic DNA was added to 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers (#276/#277), and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix.
  • the reaction mix was cycled as follows: 95° C. 10 minutes followed by 30 rounds of 95° C. for 20 seconds, 60° C. for 30 seconds, and 72° C. for 45 seconds. A final 5 minute extension reaction at 72° C. was also included.
  • a second round of PCR amplification was done using 50 ng of the first round PCR amplification with 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers (#352/#353 in the second round), and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix.
  • the second mixture was cycled as follows: 95° C. 10 minutes followed by 30 rounds of 95° C. for 20 seconds, 55° C. for 30 seconds, and 72° C. for about 30 seconds. A final 5 minute extension reaction at 72° C. was also included.
  • Oligonucleotides (SEQ ID NO: 280) #276 - 5′-GTCGACTGGAAATCTGGAAGGTTGGT-3′ (SEQ ID NO: 281) #277 - 5′-GTCGACGCTTTGCTGCAAGGATTCAT-3′
  • the BY4742 tal1 strain was then made competent using the high efficiency competent method as described above. About 500 ng of plasmids pBF290 and pBF292 or with plasmids p426GPD and p425GPD were used to transform the BY4742 tal1 strain. The final transformation mixture was plated onto SCD-ura-leu plates and grown at about 30° C. for about 3 days. Strain BF716 (BY4742 tal1) with p426GPD/p425GPD was labeled as BF738. Strain BF716 with pBF290/pBF292 was labeled as BF741.
  • a fermentation test of the BF738 was conducted against BF741 in a 400 ml multiplexed fermentor.
  • the fermentation medium utilized was SC-Ura-Leu with 2% glucose. Cultures were grown overnight in 50 ml SC-Ura-Leu 2% glucose and used to inoculate the fermentors at 4 to 5% inoculum. OD 600 readings of the inoculum are shown in the table below.
  • the cultures were grown aerobically at about 30° C. with about 250 rpm agitation, 0.5 vvm sparge of process air, 21% O 2 . pH was controlled at 5.0 with 1N NaOH.
  • Glucose concentrations in the fermentation broth were monitored by YSI 2700 BioAnalyzers during aerobic fermentation. Once glucose was depleted the fermentation was switched to anaerobic conditions. Before changing to anaerobic conditions samples were taken to measure glucose usage. Biomass was measured by monitoring the optical density of the growth medium at 600 nanometers (e.g., OD 600 ). EFT at glucose depletion, glucose concentrations and OD 600 are shown in the table below. The table below reports the amount of biomass in the fermentor and the amount of ethanol produced in grams per liter, after the specified amount of time (EFT), by the respective strains.
  • FIGS. 12A and 12B illustrate the fermentation data for strain BF738 (BY4742 tal1 with vector controls p426GPD and p425GPD) and FIG. 12B illustrates the fermentation data for strain BF741 (BY4742 tal1 with plasmids pBF290 (EDD-PAO1) and pBF292 (EDA-PAO1).
  • strain BF741 which expresses the activities encoded by the eda and edd genes, yields more ethanol than control strain BF738.
  • Strain BF741 produced about 0.43 g ethanol per gram of glucose consumed whereas strain BF738 produced only 0.36 g ethanol per gram of glucose consumed. Fermentation profiles were calculated for strains BF738 and BF741 and are presented below.
  • Strain BF205 (YGR240C/BY4742, ATCC Cat. No. 4015893; PubMed: 10436161) was transformed with plasmids p426GPD and p425GPD or with plasmids pBF290 (p426GPD/EDD-PAO1) and pBF292 (p426GPD/EDA-PAO1), generating strains BF740 (vector controls) and BF743, respectively. Transformation was accomplished by a high-efficiency competency method using 500 ng of plasmids p426GPD and p425GPD or plasmids pBF290 and pBF292.
  • Transformants were plated onto SCD-ura-leu agar plates and grown at about 30° C. for about 3 days.
  • the final strains were named BF740 (BY4742 pfk1 with plasmids p426GPD and p425GPD) and BF743 (BY4742-pfk1, pBF290/pBF292).
  • a fermentation test of the control strain BF740 (BY4742 pfk1 with plasmids p426GPD and p425GPD) was conducted against BF743 (BY4742-pfk1, pBF290/pBF292) in 400 ml w.v. Multifors multiplexed fermentors.
  • the fermentation medium was SC-Ura-Leu with 2% glucose.
  • Vessels were inoculated with about a 10% inoculum from overnight cultures grown in about 50 ml SC-Ura-Leu with about 2% glucose and normalized to 0.5 OD 600 .
  • the actual inoculated ODs for the fermentations are shown in the table below.
  • the cultures were grown aerobically at about 30° C. with about 250 rpm agitation, 1 vvm sparge of process air, (21% O 2 ).
  • the pH was controlled at around 5.0 with 0.25 N NaOH.
  • glucose concentrations dropped below 0.5 g/L the fermentation was switched to anaerobic conditions.
  • samples were taken to measure glucose concentrations and biomass by OD 600 as shown in the table below.
  • the table below shows the beginning cell biomass and glucose concentration (in grams per liter of nutrient broth). Ethanol and glucose concentrations in the fermentation broth were monitored using a YSI 2700 BioAnalyzer.
  • a bolus of about 18 g/L glucose plus about 4 ml/L of 2.5 g/L ergosterol in Ethanol, 0.4 ml/L Tween 80, and 0.01% AF-204 were added to each fermentor.
  • Oxygen was purged with 100% N 2 sparged at about 1 vvm until pO 2 was below 1%.
  • Samples were taken every 4 to 8 hours and measured for ethanol and glucose concentrations and biomass (OD 600 ). The fermentation was harvested when the glucose concentration was below 0.05 g/L, at about 42 hours elapsed fermentation time (EFT). Ethanol and glucose concentrations and OD 600 of the final sample are shown in the table below.
  • the EDD and EDA genes also have been isolated from additional sources and tested for the ability to direct fermentation in yeast.
  • the additional EDD and EDA genes have been isolated from Shewanella oneidensis, Gluconobacter oxydans , and Ruminococcus flavefaciens .
  • Genomic DNA was purchased from ATCC for both S. oneidensis (Cat. No. 700550D) and G. oxydans (621 HD-5).
  • R. flavefaciens , strain C94 (NCDO 2213) was also purchased from ATCC (Cat. No. 19208).
  • To prepare genomic DNA R. flavefaciens was grown in cooked meat media (Becton Dickinson, Franklin Lakes, N.J. USA) overnight at 37° C.
  • eda and edd genes were PCR amplified from the corresponding genomic DNA using the following sets of PCR oligonucleotides.
  • the nucleotide and amino acid sequences of eda and edd genes PCR amplified using the following sets of PCR oligonucleotide primers, also is given below.
  • the S. oneidensis edd gene (SEQ. ID. NO: 73) 5′-GTTCACTGCactagtaaaaaATGCACTCAGTCGTTCAATCTG-3′ (SEQ. ID. NO: 74) 5′-CTTCGAGATCTCGAGTTAGTAAAGTTCATCGATGGC-3′
  • the S. oneidensis eda gene (SEQ. ID. NO: 75) 5′-GTTCACTGCactagtaaaaaaATGCTTGAGAATAACTGGTC-3′ (SEQ. ID. NO: 76) 5′-CTTCGAGATCTCGAGTTAAAGTCCGCCAATCGCCTC-3′
  • the G. oxydans edd gene (SEQ.
  • oneidensis 6-phosphogluconate dehydratase (edd)-Amino Acid sequence (SEQ. ID. NO: 84) MHSVVQSVTDRIIARSKASREAYLAALNDARNHGVHRSSLSCGNLAHGFAACNPDDKNALRQLTK ANIGIITAFNDMLSAHQPYETYPDLLKKACQEVGSVAQVAGGVPAMCDGVTQGQPGMELSLLSRE VIAMATAVGLSHNMFDGALLLGICDKIVPGLLIGALSFGHLPMLFVPAGPMKSGIPNKEKARIRQQF AQGKVDRAQLLEAEAQSYHSAGTCTFYGTANSNQLMLEVMGLQLPGSSFVNPDDPLREALNKMA AKQVCRLTELGTQYSPIGEVVNEKSIVNGIVALLATGGSTNLTMHIVAAARAAGIIVNWDDFSELSD AVPLLARVYPNGHADINHFHAAGGMAFLIKELLDAGLLHEDVNTVAGYGLRRYT
  • oxydans 6-phosphogluconate dehydratase (edd)-Amino Acid sequence (SEQ. ID. NO: 86) MSLNPVVESVTARIIERSKVSRRRYLALMERNRAKGVLRPKLACGNLAHAIAASSPDKPDLMRPTG TNIGVITTYNDMLSAHQPYGRYPEQIKLFAREVGATAQVAGGAPAMCDGVTQGQEGMELSLFSRD VIAMSTAVGLSHGMFEGVALLGICDKIVPGLLMGALRFGHLPAMLIPAGPMPSGLPNKEKQRIRQL YVQGKVGQDELMEAENASYHSPGTCTFYGTANTNQMMVEIMGLMMPDSAFINPNTKLRQAMTR SGIHRLAEIGLNGEDVRPLAHCVDEKAIVNAAVGLLATGGSTNHSIHLPAIARAAGILIDWEDISRLSS AVPLITRVYPSGSEDVNAFNRVGGMPTVIAELTRAGMLHKDILTVSRGGFSDYARRASLEGDEIVY TH
  • ClustalW is a free alignment tool available at the European Bioinformatics Institute website (e.g., world wide web uniform resource locator ebi.ac.uk, specific ClustalW location is ebi.ac.uk/Tools/clustalw2/index.html).
  • PAO1 Pseudomonas aeruginosa
  • E.C. Eschericia coli
  • S.O. S. oneidensis
  • G.O. G. oxydans
  • R.F. Ruminococcus flavefaciens .
  • oligonucleotides set forth above were purchased from Integrated technologies (“IDT”, Coralville, Iowa). These oligonucleotides were designed to incorporate a SpeI restriction endonuclease cleavage site upstream and an XhoI restriction endonuclease cleavage site downstream of the edd and eda gene constructs, such that the sites could be used to clone the genes into yeast expression vectors p426GPD (ATCC accession number 87361) and p425GPD (ATCC accession number 87359). In addition to incorporating restriction endonuclease cleavage sites, the forward oligonucleotides were designed to incorporate six consecutive A nucleotides immediately upstream of the ATG initiation codon.
  • PCR amplification of the genes were performed as follows: about 100 ng of the genomic DNA was added to 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix. The reaction mixture was cycled as follows: 95° C. 10 minutes followed by 30 rounds of 95° C. for 20 seconds, 50° C. (eda amplifications) or 53° C. (edd amplifications) for 30 seconds, and 72° C. for 15 seconds (eda amplifications) or 30 seconds (edd amplifications). A final 5 minute extension reaction at 72° C. was also included. Each amplified product was TOPO cloned into the pCR Blunt II TOPO vector (Life Technologies, Carlsbad, Calif.) according to the manufacturer's recommendations and the sequences verified (GeneWiz, La Jolla, Calif.
  • each of the sequence-verified eda and edd fragments were subcloned into the corresponding restriction sites in plasmids p425GPD and p426GPD vectors (ATCC #87361; PubMed: 7737504). Briefly, about 50 ng of SpeI-XhoI-digested p425GPD vector was ligated to about 50 ng of SpeI/XhoI-restricted eda or edd fragment in a 10 ⁇ l reaction with 1 ⁇ T4 DNA ligase buffer and 1 U T4 DNA ligase (Fermentas) overnight at 16° C.
  • a yeast strain was developed to enable in vivo gene combination evaluation. Growth on glucose was impaired in this strain by disrupting both copies of phosphofructokinase (PFK), however, the strain could grow normally on galactose due to the presence of a single plasmid copy of the PFK2 gene under the control of a GAL1 promoter. The strain can only grow on glucose if a functional EDD/EDA is present in the cell.
  • the strain was generated using strain BF205 (YGR240C/BY4742, ATCC Cat. No. 4015893; Winzeler E A, et al. Science 285: 901-906, 1999, PubMed: 10436161) as the starting strain.
  • the primers used were designed to include a unique SpeI restriction site at the 5′ end of the gene and a unique XhoI restriction site at the 3′ end of the gene.
  • This SpeI-XhoI fragment (approximately 2900 bp) was cloned into the SpeI-XhoI sites of the yeast vector p416GAL (ATCC Cat. No. 87332; Mumberg D, et al., Nucleic Acids Res. 22: 5767-5768, 1994.
  • a knockout cassette for the PFK2 gene was constructed by first PCR amplifying about 300 bp of the 5′ and 3′ flanking regions of the PFK2 gene from S. cerevisiae , strain BY4742 using primers JML/85 and JML/87 and primers JML/86 and JML/88, respectively. These flanking regions were designed such that the 5′ flanking region had a HindIII site at its 5′ edge and a BamHI site at its 3′ end. The 3′ flanking region had a BamHI site at its 5′ edge and a EcoRI site at its 3′ edge.
  • the nucleotide sequence of the PFK2 gene and the primers used for amplification of the PFK2 gene are given below.
  • the nucleic acid fragments were amplified using the following conditions; about 100 ng of the BY4742 genomic DNA was added to 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers, and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix. The reaction was cycled at 95° C. for 10 minutes, followed by 30 rounds of 95° C. for 20 seconds, 58° C. for 30 seconds, and 72° C. for 20 seconds. A final 5 minute extension reaction at 72° C. was also included.
  • a three fragment ligation was performed using about 100 ng of the 5′ flanking region HindIII-BamHI fragment, about 100 ng of the 3′ flanking region BamHI-EcoRI fragment and about 50 ng of pUC19 digested with HindIII and EcoRI in a 5 ⁇ l ligation reaction containing 1 ⁇ ligation buffer and 1 U T4 DNA ligase (Fermentas). This reaction was incubated at room temperature for about one hour. About 2 ⁇ l of this reaction mix was used to transform competent DH5 ⁇ cells (Zymo Research, Orange, Calif.) and plated onto LB agar media containing 100 ⁇ g/ml ampicillin. The final construct was confirmed by restriction endonuclease digests and sequence verification (GeneWiz, San Diego, Calif.), resulting in plasmid pBF653.
  • the Lys2 gene was isolated by PCR amplification from pRS317 (ATCC Cat. No. 77157; Sikorski R S, Boeke J D. Methods Enzymol. 194: 302-318, 1991. PubMed: 2005795) using primers JML/93 and JML/94.
  • PCR amplification was performed as follows: about 25 ng of the pRS317 plasmid DNA was added to 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers, and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix. The reactions were cycled at: 95° C.
  • the amplified product was TOPO cloned into the pCR Blunt II TOPO vector as described herein, resulting in plasmid pBF656.
  • the nucleotide sequence of Lys2 gene and the primers used for amplification of the Lys2 gene are given below.
  • the knockout cassette was fully assembled by cloning the NotI-FseI LYS2 fragment from plasmid pBF656 into the NotI-FseI sites located between the 5′ and 3′ flanking PFK2 regions in plasmid pBF653. About 50 ng of plasmid pBF653 digested with NotI and FseI was ligated to about 100 ng of the NotI-FseI LYS2 fragment from plasmid pBF656 in a 5 ⁇ l reaction containing 1 ⁇ ligation buffer and 1 U T4 DNA ligase (Fermentas) for about 1 hour at room temperature.
  • Strain BF1477 was transformed with the about 5 kbp PacI fragment using the method described above (LiOAc/PEG method) generating strain BF1411.
  • Strain BF1411 has the ability to grow on galactose as a carbon source, but cannot grow on glucose.
  • Various combinations of the EDD and EDA constructs can be expressed in this strain and monitored for growth on glucose.
  • Strains which show growth on glucose (or the highest growth rate on glucose) can be further characterized to determine which combination of EDD and EDA genes is present.
  • libraries of EDD and EDA genes can be screened for improved activities and activity combinations in a host organism.
  • a single plasmid system expressing EDD and EDA for industrial yeast was constructed as follows: The approximately 2800 bp fragment containing the GPD1 promoter, EDD-PAO1 gene and CYC1 terminator from plasmid pBF291 (p426GPD with EDD-PAO1) was PCR amplified using primers KAS/5′-BamHI-Pgpd and KAS/3′-NdeI-CYCt, described below.
  • the amplified product was TOPO cloned into the pCR Blunt II TOPO vector, as described herein, and the final plasmid was sequence verified and designated, pBF475.
  • KAS/5′-BamHI-Pgpd (SEQ ID NO: 290) GGATCCgtttatcattatcaatactcgccatttcaag KAS/3′-Ndel-CYCt (SEQ ID NO: 291) CATATGttgggtaccggccgcaaattaaagccttcgagcg
  • KANMX4 cassette was PCR amplified from plasmid pBF413 HO-poly-KanMX4-HO (ATCC Cat. No. 87804) using primers KAS/5′-Bam_NdeI-KANMX4 and KAS/3′-Sal_NheI-KANMX4, described below.
  • KAS/5′-Bam_Ndel-KANMX4 (SEQ ID NO: 292) GGATTCagtcagatCATATGggtacccccgggttaattaaggcgcgccag atctg KAS/3′-Sal_Nhel-KANMX4 (SEQ ID NO: 293) GTCGACaggcctactgtacgGCTAGCgaattcgagctcgttttcgacact ggatggcggc
  • plasmid pBF413 HO-poly-KanMX4-HO DNA was added to 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix.
  • the reaction was cycled at 95° C. for 10 minutes, followed by 30 rounds of 95° C. for 20 seconds, 55° C. for 30 seconds, and 72° C. for 30 seconds. A final 5 minute extension reaction at 72° C. was also included.
  • the amplified product was TOPO cloned into the pCR Blunt II TOPO vector, as described herein. The resulting plasmid was sequence verified and designated, pBF465.
  • An approximately 225 bp ADH1 terminator was PCR amplified from the genome of BY4742 using primers KAS/5′-Xba-XhoI-ADHt and KAS/3′-StuI-ADHS.
  • the sequence of primers KAS/5′-Xba-XhoI-ADHt and KAS/3′-StuI-ADHS is given below.
  • genomic DNA from BY4742 was added to 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix.
  • the reaction was cycled at 95° C. for 10 minutes, followed by 30 rounds of 95° C. for 20 seconds, 55° C. for 30 seconds, and 72° C. for 15 seconds. A final 5 minute extension reaction at 72° C. was also included.
  • the amplified product was TOPO cloned into the pCR Blunt II TOPO vector according to the manufacturer's recommendations and sequence verified. The resulting plasmid was designated pBF437.
  • the TEF2 promoter was PCR amplified from the genome of BY4742 using primers KAS/5′-Xba-XhoI-ADHt and KAS/3′-StuI-ADHS, described below.
  • genomic DNA from BY4742 was added to 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers, and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix. This was cycled at 95° C. for 10 minutes, followed by 30 rounds of 95° C. for 20 seconds, 55° C. for 30 seconds, and 72° C. for 15 seconds. A final 5 minute extension reaction at 72° C. was also included.
  • the amplified product was TOPO cloned into the pCR Blunt II TOPO vector (Life Technologies, Carlsbad, Calif.) according to the manufacturer's recommendations and sequence verified (GeneWiz, San Diego, Calif.).
  • the resulting plasmid was called pBF440.
  • the EDA gene cassettes were constructed as follows: First the TEF2 promoter from the plasmid pBF440 was digested with BamHI and XbaI and was cloned into the BamHI and XbaI sites of pUC19 creating plasmid pBF480. Plasmid pBF480 was then digested with XbaI and HindIII and was ligated to the XbaI-HindIII fragment from plasmid pBF437 containing the ADH1 terminator, creating plasmid pBF521.
  • Plasmid pBF521 was then digested with SpeI and XhoI and then ligated to either SpeI-XhoI fragment containing either the PAO1 eda gene from plasmid pBF292 or the E. coli eda gene from plasmid pBF268.
  • the 2 plasmids generated, depending on the eda gene chosen, were designated pBF523 (e.g., containing the PAO1-eda) and pBF568 (e.g., containing the E. coli -eda), respectively.
  • the approximately 1386 bp TEF-EDA-ADHt cassette from either plasmid pBF 523 or pBF568 was then gel extracted using the NheI-StuI sites.
  • the final vector was generated by first altering the Nde1 site in pUC19 using the mutagenesis primers described below.
  • the PCR reaction mixture was then digested with 30 U of DpnI for about 2 hours and 5 ⁇ l of the digested PCR reaction mixture was used to transform competent DH5 ⁇ (Zymo Research, Orange, Calif.) and plated onto LB plates containing 100 ⁇ g/ml ampicillin.
  • Plasmid pBF429 was then digested with BamHI and SalI and ligated to the BamHI-SalI KANMX4 cassette described above.
  • the resultant plasmid, designated pBF515, was digested with BamHI and NdeI and ligated to the BamHI-NdeI fragment containing the 2802 bp GPD-EDD-CYCt fragment from pBF475.
  • the resulting plasmid designated pBF522, was digested with NheI-StuI and was ligated to the 1386 bp NheI-StuI TEF-EDA-ADHt fragment from plasmids pBF523 or pBF568, creating final plasmids pBF524 and pBF612.
  • activities can result in increased ethanol production due to an increase in the utilization of the fermentation substrate, sometimes due to an increase in transport and/or metabolism of a desired sugar.
  • activities that can be over expressed to increase ethanol production by increasing sugar transport and/or metabolism include activities encoded by the genes gxf1, gxs1, hxt7, zwf1, gal2, sol3, sol4, the like, homologs thereof (e.g., Candida albicans Sol1p, Schizosaccharomyces pombe Sol1p, human PGLS and human H6PD), that can be expressed in a desired host organism, and combinations thereof. Nucleotide and amino acid sequences for some of these additional activities are given below.
  • 1, 2, 3, 4, 5, 6 or more of the non-limiting additional activities can be increased in expression or over expressed in an engineered host, thereby increasing transport and/or metabolism of a desired carbon source, wherein increased transport and/or metabolism of a desired carbon source results in increased ethanol production.
  • ZWF1 genes were cloned from S. cerevisiae, Zymomonas mobilis, Pseudomonas fluorescens (zwf1 and zwf2), and P. aeruginosa strain PAO1. The sequences of these additional ZWF1 genes are given below.
  • Strain BY4742 zwf1 (ATCC Cat. No. 4011971; Winzeler E A, et al. Science 285: 901-906, 1999. PubMed: 10436161) was used as the base strain for all ZWF1 assays. The assays were performed as follows: A 5 ml overnight of the strain expressing the ZWF1 gene was grown in SCD-ura. A 50 ml culture of the strain was then grown for about 18 hours from an initial (OD 600 of about 0.2 until it had reached about OD 600 of about 4.
  • the cells were centrifuged at 1046 ⁇ g washed twice with 25 ml cold sterile water, and resuspended in 2 ml/g Yper Plus (Thermo Scientific) plus 1 ⁇ protease inhibitors (EDTA-free). The cells were allowed to lyse at room temperature for about 30 minutes with constant rotation of the tubes. The lysate was centrifuged at 16,100 ⁇ g for 10 minutes at 4° C. and the supernatants were transferred to a new 1.5 ml microcentrifuge tube. Quantification of the lysates was performed using the Coomassie-Plus kit (Thermo Scientific, San Diego, Calif.) as directed by the manufacturer.
  • Each kinetic assay was done using approximately 50 to 60 ⁇ g of crude extract in a reaction mixture containing 50 mM Tris-HCl, pH 8.9, and 1 mM NADP+ or NAD+.
  • the reaction was started with 20 mM glucose-6-phosphate and the reaction was monitored at A340.
  • the specific activity was measured as the ⁇ mol substrate/min/mg protein.
  • the results of the assays are presented in the table below.
  • ZWF1 from S. cerevisiae is an NADP + -only utilizing enzyme.
  • Site-directed mutagenesis was used to alter of ZWF1 so that the altered ZWF1 could also utilize NAD+, thereby improving the REDOX balance within the cell.
  • Site directed mutagenesis reactions were performed in the same manner for all mutations, and for mutants which include more than one mutation, each mutation was performed sequentially.
  • About 50 ng of plasmid DNA was added to 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol site directed mutagenesis specific primers, and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix.
  • the reaction was cycled at 95° C. for 10 minutes, followed by 15 rounds of 95° C. for 15 seconds, 55° C. for 40 seconds, and 72° C. for 3 minutes. A final 10 minute extension reaction at 72° C. was also included.
  • the PCR reaction mixture was then digested with 30 U of DpnI for about 2 hours and 5 ⁇ l of the digested PCR reaction mixture was used to transform competent DH5 ⁇ (Zymo Research, Orange, Calif.) and plated onto LB plates containing the appropriate antibiotics.
  • the table below lists mutants generated in a first round of mutagenesis.
  • Mutant # zwf1_sc Codon changes 1 A24G GCA -> GGT 2 A24G/T28G GCA -> GGT, ACT -> GGT 3 A51N GCC -> AAT 4 A51D GCC -> GAT 5 T28F ACT -> TTT 6 K46R AAG -> AGA 7 Y40L TAC -> TTG 8 F33Y TTT -> TAC 9 T28L ACT -> TTG 10 V16L GTC -> TTG 11 V13T GTC -> ACT 12 L66E CTA -> GAA 13 A24G/A51D GCA -> GGT, GCC -> GAT 14 A24G/T28G/A51D GCA -> GGT, ACT -> GGT, GCC -> GAT 15 R52D CGG -> GAT 16 A51D/R52A GCC -> GAT, CGG -> GCT 17 A24G/A51D/R52A GCA -> GGT, GCC -> G
  • oligonucleotides utilized to generate the mutants listed in the table above, are listed in the table below. All oligonucleotides were purchased from Integrated DNA Technologies (IDT).
  • Mutants 4 (A51D) and 13 (A24G/A51D) were identified as mutants which enabled NAD+ utilization with concomitant loss of NADP+ utilization.
  • the SOL3 gene from S. cerevisiae was cloned as follows. The approximately 750 bp SOL3 gene was PCR amplified from the BY4742 genome using primers KAS/5-SOL3-NheI and KAS/3′-SOL3-SalI, shown below.
  • genomic DNA from S. cerevisiae strain BY4742 was added to 1 ⁇ Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 ⁇ mol gene-specific primers, and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 ⁇ l reaction mix.
  • the reaction was cycled at 95° C. for 10 minutes, followed by 30 rounds of 95° C. for 20 seconds, 55° C. for 30 seconds, and 72° C. for 15 seconds. A final 5 minute extension reaction at 72° C. was also included.
  • the amplified product was TOPO cloned into the pCR Blunt II TOPO vector (Life Technologies, Carlsbad, Calif.) according to the manufacturer's recommendations and sequence verified (GeneWiz, San Diego, Calif.).
  • the resultant plasmid was designated pBF301.
  • the sequence of the S. cerevisiae SOL3 gene is given below.
  • NheI-SalI SOL3 gene fragment from plasmid pBF301 will be cloned into the SpeI-XhoI site in plasmids p413GPD and p423GPD (HIS3 marker-based plasmids; ATCC 87354 and ATCC 87355).
  • a URA blaster cassette was digested with NotI and ligated into the MET17 integration cassette plasmid pBF691 to generate the Met17 knockout plasmid pBF772.
  • Plasmid pBF772 was digested with PacI and linear fragments were purified by Zymo PCR purification kit (Zymo Research, Orange, Calif.) and concentrated in 10 ⁇ l ddH2O. LiCl2 high efficiency transformation was performed as shown described. About 1 ⁇ g linear MET17 knockout fragment was transformed into 50 ⁇ l fresh made BY4742 competent cells and cells were plated onto SCD-Ura plates at 30° C. for about 2-3 days.
  • a single URA+ colony was streaked out on a SCD-Ura plate and grown at 30° C. for about 2-3 days.
  • a single colony was inoculated overnight in YPD medium at 30° C. 50 ⁇ l of the overnight culture was then plated onto SCD complete ⁇ 5FOA plates and incubated at 30° C. for about 3 days.
  • Yeast genomic DNA was extracted by YeaStar genomic extraction kit (Zymo Research, Orange, Calif.) and confirmation of the strain was confirmed by PCR using primers JML/237 and JML/238, shown below.
  • JML/237 (SEQ ID NO: 340) CCAACACTAAGAAATAATTTCGCCATTTCTTG JML/238: (SEQ ID NO: 341) GCCAACAATTAAATCCAAGTTCACCTATTCTG
  • the PCR amplification was performed as follows: 10 ng of yeast genomic DNA with 0.1 ⁇ mol gene specific primers, 1 ⁇ Pfu Ultra II buffer, 0.2 mmol dNTPs, and 0.2 U Taq DNA polymerase. The PCR mixture was cycled at 95° C. for 2 minutes, followed by 30 cycles of 95° C. for 20 seconds, 55° C. for 30 seconds and 72° C. for 45 seconds. A final step of 72° C. for 5 minutes was also included. The resultant strain was designated BF1618.
  • Strain BF1618 is undergoing transformation with the following plasmid combinations. Additionally, the affect of the ZWF1 mutant constructs will also be evaluated with and without SOL3 constructs. The table below shows the plasmid combinations being transformed into strain BF1618.
  • Strains with improved ethanol production may benefit from two or more copies of the ZWF1 gene due to increased flux of the carbon towards the alternative pathway.
  • a strain embodiment currently under construction has the phenotype; pfk1, ZWF1, SOL3, tal1, EDD-PAO1*, EDA- E. coli *, where the “*” represents additional copies of the gene. It is believed that multiple copies of the EDD and EDA genes may provide additional increases in ethanol production.
  • the primers used for amplification of nucleic acids utilized to generate the disruption cassette are described in the table below.
  • JML/ ACTAGTATGTCTGACAAGGAACAAACGAGC (SEQ ID NO: 5′ScAto1SpeI 51 342) JML/ CTCGAGTTAAAAGATTACCCTTTCAGTAGATGGTAATG 3′ScAto1XhoI 52 (SEQ ID NO: 343) JML/ caagcctttggtggtacccagaatccagggttagctcc ScATO(L75Q)_For 55 (SEQ ID NO: 344) JML/ ggagctaaccctggattctgggtaccaccaaaggcttg ScATO(L75Q)_Rev 56 (SEQ ID NO: 345) JML/ ggtacaacgcatatgcagatgttgctacaagcagaa (SEQ ScATO1G259D_For 57 ID NO: 346) JML/ ttctgcttgtagcaacatctgcatatgcgttt
  • ScATO1 was amplified from genomic DNA (gDNA) isolated from BY4742 with primers oJML51 and oJML52 and cloned into pCR Blunt II-TOPO (Invitrogen, Carlsbad, Calif.). Site Directed Mutagenesis (SDM) was performed on that plasmid with oJML55 and oJML56, as described herein. The mutagenized clone was re-amplified with primers oJML51 and oJML52 and cloned into pCR Blunt II-TOPO (Invitrogen, Carlsbad, Calif.), and designated ATO1-L75Q. ATO1-L75Q was subcloned into p416GPD using SpeI/XhoI restriction enzyme sites. The resulting plasmid was designated pJLV048.
  • SDM Site Directed Mutagenesis
  • the 5′ and 3′ flanking regions of URA3 were amplified via PCR of the 5′ regions with primers oJML63 and oJML65, the 3′ region with primers oJML64 and oJML66.
  • the amplified nucleic acids were annealed and re-amplified with oligonucleotides oJML63 and oJML66.
  • the template used was TURBO gDNA.
  • the PCR product was Topo cloned into pCR-Blunt II. The desired sequence was moved as an EcoRI-SphI fragment into vector pUC19 and designated pJLV63.
  • the R-KanMX fragment was made as follows: The KANMX fragment was first amplified from pBF524 with primers oJML71 and oJML73. The R-200-mer from plasmid pBF32 was then amplified using primers oJML72 and oJML74. The two fragments were annealed together and PCR amplified using primers oJML67 and oJML70 and topo cloned using pCR-Blunt II. The final plasmid construct was designated pJLV062.
  • the R-P TDH3 -ATO1-L75Q construct was generated by amplifying a mixture of PCR oJML67-oJM L69 (pBF32)+PCR oJML68-oJML70 (pJLV048). The resulting plasmid was designated pJLV065.
  • the R-PT DH3 -ATO1 L75Q (SalI/SphI) fragment from pJLV065 was ligated in a 3 piece ligation to the SalI/BamHI (R-KanMX) fragment from pJLV063 into the BamHI/SphI site of pUC19.
  • R-KanMX-P TDH3 -ATO1-L75Q-R fragment was ligated as a NotI piece into the NotI site of pJLV63 and designated pJLV74.
  • the unique sequence tags are described in Example 28. A table describing the intermediate and final plasmids is presented below.
  • Haploid yeast strains were transformed with 2 to 3 ⁇ g of a PvuII, SphI digested ura3::R-KanMX-ATO1-L75Q-R disruption cassette using the high-efficiency Li-PEG procedure with a heat shock time of 8 minutes.
  • Transformants were plated on YPD plus G418 (200 ⁇ g/ml) plates. Colonies were re-streaked onto ScD FOA plates. Single colonies were replica plated on ScD-ura, ScD+FOA, YPD, and YPD G418 200 ⁇ g/ml plates. Ura-FOA R G418 R colonies were grown overnight in YPD.
  • the absence of the URA3 loci was verified by PCR that amplifies a 500 bp region of the Actin gene open reading frame and a 300 bp region of the URA3 open reading frame.
  • the primers utilized for amplification and verification are presented, respectively, in the tables below.
  • Plasmid DNA was digested with PacI using manufacturers suggestions. The digestions were purified using the GeneJETTM Gel Extraction Kit I (Fermentas). Each column was eluted with 20 ⁇ l of Elution buffer and multiple digests were combined. S. cerevisiae was transformed using the high-efficiency Li-PEG procedure with 2 to 3 ⁇ g of DNA and transformants were selected on ScD-ura solid media. Correct integrations were confirmed by PCR analysis with primers outside the flanking regions used as the disruption cassette and primers complementary to either the open reading frame of EDA or the 200-mer repeat. Oligonucleotide primers utilized for verification are described in the tables below.
  • JML/276 CCTACCCGCCTCGGATCCCAGCTACC R-repeat (SEQ ID NO: 373) JML/277 GGTAGCTGGGATCCGAGGCGGGTAGG R-repeat (SEQ ID NO: 374) JML/278 CCTCCCGGCACAGCGTGTCGATGC R at the 5′EDA (SEQ ID NO: 375)
  • JML/ CGAAGCCCTGGAGCGCTTCGC PCR for PaEDA 297 (SEQ ID NO: 376) going out at the 3′ of the ORF JML/ GTGGTCAGGATTGATTCTGCACTTGTTTT PCR for EcEDA 298 CCAG (SEQ ID NO: 377) Reverse at the 5′ end JML/ CGCGTGAAGCTGTAGAAGGCGCTAAG PCR for EcEDA 299 (SEQ ID NO: 378) Forward at the 3′ end
  • the PCR reactions were performed in a final reaction volume of 25 ⁇ l using the following amplification profile; 1 cycle at 94 degrees C. for 2 minutes, followed by 35 cycles of 94 degrees C. for 30 seconds, 52 degrees C. for 30 second and 72 degrees C. for 2 minutes.
  • P TDH3 -PaEDA was amplified from pBF292 using primers oJML225 and oJML226, shown in the table below and Topo cloned in pCR Blunt II to make pJLV95.
  • the desired fragment was moved as a FseI-SacI piece into pBF730 or pBF731 (the integration cassette of either YBR110.5 or YDL075.5, respectively) to make plasmids pJLV114 and pJLV115, respectively.
  • YBR110.5 is located in between loci YBR110 and YBR111
  • YDL075.5 is located in between loci YDL075 and YDL076.
  • the R-URA3-R sequence was moved into these plasmids as a NotI fragment to make pJLV119 and pJLV120.
  • the resultant plasmids are described in the table below.
  • EDA genes isolated from a variety of sources were expressed in yeast and evaluated independently of EDA activity, to identify EDA activities suitable of inclusion in an engineered yeast strain.
  • the EDA activities were was independently assessed by adding saturating amounts of over expressed E. coli EDD extracts to S. cerevisiae EDA extracts lacking EDD (Chemyan et al., Protein Science 16:2368-2377, 2007).
  • the relative activities of EDAs, expressed in S. cerevisiae were compared and ranked in this way.
  • the activity of integrated EDAs in Thermosacc-Gold haploids were also evaluated in this manner.
  • the table below describes oligonucleotide primers used to isolate the various EDA genes.
  • EDA extracts were prepared using the following protocol.
  • Each reaction contains 50 mM Tris-HCl, pH 7, 10 mM MgCl 2 , 0.15 mM NADH, 15 ⁇ g LDH, saturating amounts of EDD determined empirically (usually ⁇ 100 ⁇ g), 1-50 ⁇ g EDA (depending on level of activity), and 1 mM 6-phosphogluconate. Reactions are started by the addition of 6-phosphogluconate and monitored for 5 mins at 30° C.
  • yCH strains are Thermosacc-based (Lallemand).
  • BF strains are based on BY4742.
  • EDA BF1721 pBF909 Bacilluis licheniformis EDA BF1722 pBF910 Bacillus subtilis EDA BF1723 pBF911 Pseudomonas fluorescens EDA BF1724 pBF912 Pseudomonas syringae EDA BF1725 pBF913 Saccharaophagus degradans EDA BF1726 pBF914 Xanthamonas axonopodis EDA BF1727 pBF766 Escherichia coli EDA BF1728 pBF764 Pseudomonas aeruginosa EDA BF1729 pBF729 Gluconobacter oxydans EDA BF1730 pBF727 Shewanella oneidensis EDA BF1775 pBF87 p425GPD (empty vector) BF1776 pBF928 PAO1 EDA codon optimized for S.
  • E. coli expressed EDD was prepared and confirmed by western blot analysis as shown in FIG. 15 .
  • the expected size of EDD is approximately 66 kilodaltons (kDa).
  • a band of approximately that size was identified by western blot.
  • the E. coli expressed EDD was used with S. cerevisiae expressed EDA's to evaluate the EDA activities. The results of EDA kinetic assays are presented in the table below.
  • EDD/EDA slope % max EC/EC 0.3467 100.00 EC/SO 0.1907 55.00 EC/BS 0.0897 25.87 EC/GO 0.0848 24.46 EC/PCO 0.084 24.23 EC/PA 0.0533 15.37 EC/PE5 0.0223 6.43 EC/PE10 0.0218 6.29 EC/SD 0.015 4.33 EC/PS 0.0135 3.89 EC/BL 0.0112 3.23 EC/ZM 0.0109 3.14 EC/PF 0.0082 2.37 EC/V 0.0074 2.13 EC/XA 0.0065 1.87 EC/PE15 0.005 1.44
  • the slope of the E. coli (EC) EDA is outside the linear range for accurate detection, and is therefore underestimated.
  • the calculated percentage of maximum activity e.g., % max
  • the slopes are accurate.
  • the results of this experiment indicate that the E. coli EDA has higher activity as compared to the other EDA activities evaluated herein, and is approximately 16-fold more active than the EDA from P. aeruginosa .
  • EDA's from X. anoxopodis and a chimera between E. coli EDA and P. aeruginosa show less activity than the vector control.
  • Codon-optimized EDA from P. aeruginosa showed a slight improvement over the native sequence, however chimeric versions (e.g., PE5, PE10, PE15) showed less activity than native.
  • the experiments were repeated using 100 ⁇ g of EDD and 25 ⁇ g of EDA cell lysates in each reaction (unless otherwise noted, such as 5 ⁇ g of E. coli EDA).
  • the reactions in the repeated experiment all were in the linear range of detection and the results of these additional kinetic assays are shown graphically in FIG. 16 , and in the table below. E. coli EDA was again found to be the most active of those EDA's tested.
  • Phosphoglucose isomerase (PGI1) activity was decreased or disrupted, in some embodiments, to favor the conversion of glucose-6-phosphate to gluconolactone-6-phosphate by the activity of ZWF1 (e.g., glucose-6-phosphate dehydrogenase).
  • ZWF1 e.g., glucose-6-phosphate dehydrogenase
  • the nucleotide sequence of the S. cerevisiae PGI1 gene altered to decrease or disrupt phosphoglucose isomerase activity is shown below.
  • 6-phosphogluconate dehydrogenase (decarboxylating) (GND1) activity was decreased or disrupted, in some embodiments, to minimize or eliminate the conversion of gluconate-6-phophate to ribulose-5-phosphate.
  • the nucleotide sequence of the S. cerevisiae GND1 and GND2 genes altered to decrease or disrupt 6-phosphogluconate dehydrogenase (decarboxylating) activity is shown below.
  • Transaldolase (TAL1) activity was increased in some embodiments, and in certain embodiments transaldolase activity was decreased or disrupted.
  • Transaldolase converts sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate to erythrose 4-phosphate and fructose 6-phosphate.
  • the rationale for increasing or decreasing transaldolase activity is described herein with respect to various embodiments.
  • the nucleotide sequence of the S. cerevisiae TAL1 gene altered to increase or decrease transaldolase activity, and the encoded amino acid sequence are shown below.
  • Transketolase (TKL1 and TKL2) activity was increased in some embodiments, and in certain embodiments transaldolase activity was decreased or disrupted.
  • Transketolase converts xylulose-5-phosphate and ribose-5-phosphate to sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate.
  • the rationale for increasing or decreasing transketolase activity is described herein with respect to various embodiments.
  • the nucleotide sequence of the S. cerevisiae TKL1 gene altered to increase or decrease transketolase activity, and the encoded amino acid sequence are shown below.
  • TKL1 nucleotide sequence (SEQ ID NO: 428) ATGACTCAATTCACTGACATTGATAAGCTAGCCGTCTCCACCATAAGAA TTTTGGCTGTGGACACCGTATCCAAGGCCAACTCAGGTCACCCAGGTGC TCCATTGGGTATGGCACCAGCTGCACACGTTCTATGGAGTCAAATGCGC ATGAACCCAACCAACCCAGACTGGATCAACAGAGATAGATTTGTCTTGT CTAACGGTCACGCGGTCGCTTTGTTGTATTCTATGCTACATTTGACTGG TTACGATCTGTCTATTGAAGACTTGAAACAGTTCAGACAGTTGGGTTCC AGAACACCAGGTCATCCTGAATTTGAGTTGCCAGGTGTTGAAGTTACTA CCGGTCCATTAGGTCAAGGTATCTCCAACGCTGTTGGTATGGCCATGGC TCAAGCTAACCTGCCACTTACAACAACAAGCCGGGCTTTACCTTGTCT GACAACTACACCTATGTTTTCTTGGGTGACGGTTGTTTGCAAGAAGG
  • a composition comprising a nucleic acid that includes heterologous polynucleotides that encode a phosphogluconate dehydratase enzyme, a 2-keto-3-deoxygluconate-6-phosphate aldolase enzyme and a nucleotide sequence identification tag selected from the group of six (6) nucleotide sequences consisting of
  • composition of embodiment A1, wherein the yeast is a Saccharomyces spp. yeast.
  • composition of embodiment A2, wherein the yeast is a Saccharomyces cerevisiae yeast strain.
  • composition of any one of embodiments A1 to A3, wherein the polynucleotides encoding the phosphogluconate dehydratase enzyme and the 3-deoxygluconate-6-phosphate aldolase enzyme independently are from an Escherichia spp. microbe or Psuedomonas spp. microbe.
  • composition of embodiment A3, wherein the Escherichia spp. microbe is an Escherichia coli strain.
  • composition of embodiment A3 or A4, wherein the Pseudomonas spp. microbe is a Pseudomonas aeruginosa strain.
  • composition of any one of embodiments A1 to A5, wherein the polynucleotide that encodes the 2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA gene.
  • composition of embodiment A8, wherein the SOL gene is a SOL3 gene.
  • composition of embodiment A10, wherein the polynucleotide that encodes the glucose-6-phosphate dehydrogenase enzyme is from a yeast.
  • composition of embodiment A11, wherein the yeast is a Saccharomyces spp. yeast.
  • composition of embodiment A12, wherein the yeast is a Saccharomyces cerevisiae strain.
  • composition of embodiment A17, wherein the promoter is selected from promoters that regulate glucose phosphate dehydrogenase (GPD), translation elongation factor (TEF-1), phosphoglucokinase (PGK-1) and triose phosphate dehydrogenase (TDH-1).
  • GPD glucose phosphate dehydrogenase
  • TEZ-1 translation elongation factor
  • PGK-1 phosphoglucokinase
  • TSH-1 triose phosphate dehydrogenase
  • PFK phosphofructokinase
  • PKI phosphoglucoisomerase
  • 6-phosphogluconate dehydrogenase decarboxylating
  • transketolase enzyme transaldolase enzyme, or combination thereof.
  • composition of embodiment A19, wherein the transketolase enzyme is encoded by a TKL-1 coding sequence or a TKL-2 coding sequence.
  • composition of embodiment A19, wherein the transaldolase is encoded by a TAL-1 coding sequence is encoded by a TAL-1 coding sequence.
  • composition of embodiment A19, wherein the phosphofructokinase (PFK) enzyme is a PFK-2 enzyme or PFK-1 enzyme.
  • composition of embodiment A19, wherein the 6-phosphogluconate dehydrogenase (decarboxylating) enzyme is encoded by a GND-1 gene or a GND-2 gene.
  • composition of embodiment A19, wherein the PGI is encoded by a PGI-1 gene is provided.
  • composition of any one of embodiments A1 to A24, wherein the nucleic acid is one or two separate nucleic acid molecules.
  • composition of embodiment A25, wherein each nucleic acid molecule includes one or two or more of the polynucleotide subsequences, one or two or more of the promoters, or one or two or more of the polynucleotide subsequences and one or two or more of the promoters.
  • a composition comprising an engineered yeast that includes an alteration that adds or increases a phosphogluconate dehydratase activity and a 2-keto-3-deoxygluconate-6-phosphate aldolase activity, and a nucleotide sequence identification tag having a nucleotide sequence selected from the group of six (6) nucleotide sequences consisting of
  • composition of embodiment B1 wherein the yeast is a Saccharomyces spp. yeast.
  • composition of embodiment B2, wherein the yeast is a Saccharomyces cerevisiae yeast strain.
  • composition of embodiment B4, wherein the polynucleotides encoding the phosphogluconate dehydratase enzyme and the 3-deoxygluconate-6-phosphate aldolase enzyme independently are from an Escherichia spp. microbe or Psuedomonas spp. microbe.
  • composition of embodiment B5, wherein the Escherichia spp. microbe is an Escherichia coli strain.
  • composition of embodiment B5, wherein the Pseudomonas spp. microbe is a Bseudomonas aeruginosa strain.
  • composition of any one of embodiments B4 to B7, wherein the polynucleotide that encodes the phosphogluconate dehydratase enzyme is an EDD gene.
  • composition of any one of embodiments B4 to B7, wherein the polynucleotide that encodes the 2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA gene.
  • composition of embodiment B12, wherein the yeast comprises a heterologous polynucleotide that encodes a glucose-6-phosphate dehydrogenase enzyme, or wherein the yeast comprises multiple copies of an endogenous polynucleotide that encodes a glucose-6-phosphate dehydrogenase enzyme.
  • composition of embodiment B13, wherein the polynucleotide that encodes the glucose-6-phosphate dehydrogenase enzyme is from a yeast.
  • composition of embodiment B14, wherein the yeast is a Saccharomyces spp. yeast.
  • PFK phosphofructokinase
  • PKI phosphoglucoisomerase
  • 6-phosphogluconate dehydrogenase decarboxylating
  • transketolase enzyme transaldolase enzyme, or combination thereof.
  • composition of embodiment B22, wherein the phosphofructokinase (PFK) enzyme is a PFK-2 enzyme or PFK-1 enzyme.
  • composition of embodiment B22, wherein the PGI is encoded by a PGI-1 gene is provided.
  • composition of embodiment B30 wherein the polynucleotides, the promoters, or the polynucleotides and the promoters are integrated in a transposition integration event, in a homologous recombination integration event, or in a transposition integration event and a homologous recombination integration event.
  • composition of embodiment B31, wherein the transposition integration event includes transposition of an operon comprising two or more of the polynucleotide subsequences, the promoters, or the polynucleotide subsequences and the promoters.
  • composition of embodiment B31, wherein the homologous recombination integration event includes homologous recombination of an operon comprising two or more of the polynucleotide subsequences, the promoters, or the polynucleotide subsequences and the promoters.
  • a method comprising contacting an engineered yeast of any one of embodiments B1 to B33 with a feedstock that contains one or more hexose sugars under conditions in which the microbe synthesizes ethanol.
  • a microorganism comprising a polynucleotide that includes a sequence selected from the group consisting of
  • a method comprising detecting the presence or absence of a nucleotide sequence identification tag in a microorganism, wherein the nucleotide sequence is selected from the group consisting of
  • a composition comprising a nucleic acid comprising (i) heterologous polynucleotides that encode a phosphogluconate dehydratase enzyme and a 2-keto-3-deoxygluconate-6-phosphate aldolase enzyme, (ii) one or more polynucleotides that homologously combine in a gene of a host that encodes a 6-phosphogluconate dehydrogenase (decarboxylating) enzyme, and (iii) a nucleotide sequence identification tag selected from the group consisting
  • composition of embodiment E1 wherein the yeast is a Saccharomyces spp. yeast.
  • composition of embodiment E2, wherein the yeast is a Saccharomyces cerevisiae yeast strain.
  • composition of any one of embodiments E1 to E3, wherein the polynucleotides encoding the phosphogluconate dehydratase enzyme and the 3-deoxygluconate-6-phosphate aldolase enzyme independently are from an Escherichia spp. microbe or Psuedomonas spp. microbe.
  • composition of embodiment E3, wherein the Escherichia spp. microbe is an Escherichia coli strain.
  • composition of embodiment E3 or E4, wherein the Pseudomonas spp. microbe is a Pseudomonas aeruginosa strain.
  • composition of any one of embodiments E1 to E5, wherein the polynucleotide that encodes the 2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA gene.
  • composition of embodiment E8, wherein the polynucleotide that encodes the 6-phosphogluconolactonase enzyme is from a yeast.
  • composition of embodiment E10, wherein the polynucleotide that encodes the glucose-6-phosphate dehydrogenase enzyme is from a yeast.
  • composition of embodiment E11, wherein the yeast is a Saccharomyces spp. yeast.
  • composition of embodiment E12, wherein the yeast is a Saccharomyces cerevisiae strain.
  • composition of embodiment E17, wherein the promoter is selected from promoters that regulate glucose phosphate dehydrogenase (GPD), translation elongation factor (TEF-1), phosphoglucokinase (PGK-1) and triose phosphate dehydrogenase (TDH-1).
  • GPD glucose phosphate dehydrogenase
  • TEZ-1 translation elongation factor
  • PGK-1 phosphoglucokinase
  • TSH-1 triose phosphate dehydrogenase
  • PFK phosphofructokinase
  • PKI phosphoglucoisomerase
  • transketolase enzyme phosphoaldolase enzyme, or combination thereof.
  • composition of embodiment E19, wherein the transketolase enzyme is encoded by a TKL-1 coding sequence or a TKL-2 coding sequence.
  • composition of embodiment E19, wherein the transaldolase is encoded by a TAL-1 coding sequence is encoded by a TAL-1 coding sequence.
  • composition of embodiment E19, wherein the phosphofructokinase (PFK) enzyme is a PFK-2 enzyme or PFK-1 enzyme.
  • composition of any one of embodiments E1 to E22, wherein the 6-phosphogluconate dehydrogenase (decarboxylating) enzyme is encoded by a GND-1 gene or a GND-2 gene.
  • composition of embodiment E25, wherein each nucleic acid molecule includes one or two or more of the polynucleotide subsequences, one or two or more of the promoters, or one or two or more of the polynucleotide subsequences and one or two or more of the promoters.
  • E29 The composition of any one of embodiments E25 to E28, wherein each of the one or two nucleic acid molecules functions as an expression vector.
  • a composition comprising an engineered yeast that includes (i) an alteration that adds or increases a phosphogluconate dehydratase activity and a 2-keto-3-deoxygluconate-6-phosphate aldolase activity, (ii) an alteration that reduces a 6-phosphogluconate dehydrogenase (decarboxylating) activity, and (iii) a nucleotide sequence identification tag selected from the group consisting of
  • composition of embodiment F1 wherein the yeast is a Saccharomyces spp. yeast.
  • composition of embodiment F2 wherein the yeast is a Saccharomyces cerevisiae yeast strain.
  • F4.1 The composition of any one of embodiments F1 to F4 where the yeast includes heterologous polynucleotides, or multiple copies of endogenous polynucleotides, that encode a phosphogluconate dehydratase enzyme and a 2-keto-3-deoxygluconate-6-phosphate aldolase enzyme.
  • composition of embodiment F4 wherein the polynucleotides encoding the phosphogluconate dehydratase enzyme and the 3-deoxygluconate-6-phosphate aldolase enzyme independently are from an Escherichia spp. microbe or Psuedomonas spp. microbe.
  • composition of embodiment F5 wherein the Pseudomonas spp. microbe is a Pseudomonas aeruginosa strain.
  • composition of any one of embodiments F4 to F7, wherein the polynucleotide that encodes the phosphogluconate dehydratase enzyme is an EDD gene.
  • composition of any one of embodiments F4 to F7, wherein the polynucleotide that encodes the 2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA gene.
  • composition of embodiment F10 wherein the yeast comprises a heterologous polynucleotide that encodes a 6-phosphogluconolactonase enzyme, or wherein the yeast comprises multiple copies of an endogenous polynucleotide that encodes a 6-phosphogluconolactonase enzyme.
  • composition of embodiment F10.1, wherein the polynucleotide that encodes the 6-phosphogluconolactonase enzyme is from a yeast.
  • F10.3 The composition of embodiment F10.2, wherein the yeast is a Saccharomyces spp. yeast.
  • composition of embodiment F10.3, wherein the yeast is a Saccharomyces cerevisiae strain.
  • F10.5. The composition of any one of embodiments F10 to F10.4, wherein the 6-phosphogluconolactonase enzyme is expressed from a SOL gene.
  • composition of embodiment F10.4, wherein the SOL gene is a SOL3 gene.
  • F12 The composition of any one of embodiments F4 to F11, wherein a glucose-6-phosphate dehydrogenase activity is added or increased.
  • composition of embodiment F12, wherein the yeast comprises a heterologous polynucleotide that encodes a glucose-6-phosphate dehydrogenase enzyme, or wherein the yeast comprises multiple copies of an endogenous polynucleotide that encodes a glucose-6-phosphate dehydrogenase enzyme.
  • composition of embodiment F13, wherein the polynucleotide that encodes the glucose-6-phosphate dehydrogenase enzyme is from a yeast.
  • composition of embodiment F14, wherein the yeast is a Faccharomyces spp. yeast.
  • composition of embodiment F15, wherein the yeast is a Faccharomyces cerevisiae strain.
  • composition of embodiment F19, wherein the promoter is selected from promoters that regulate glucose phosphate dehydrogenase (GFD), translation elongation factor (TEF-1), phosphoglucokinase (FGK-1) and triose phosphate dehydrogenase (TDH-1).
  • GFD glucose phosphate dehydrogenase
  • TEZ-1 translation elongation factor
  • FGK-1 phosphoglucokinase
  • TSH-1 triose phosphate dehydrogenase
  • PFK phosphofructokinase
  • PKI phosphoglucoisomerase
  • transketolase activity transaldolase activity, or combination thereof.
  • composition of embodiment F21, wherein the yeast includes an alteration in one or more polynucleotides that inhibits production of one or more enzymes selected from the group consisting of phosphofructokinase (PFK) enzyme, phosphoglucoisomerase (PGI) enzyme, 6-phosphogluconate dehydrogenase (decarboxylating) enzyme, transketolase enzyme, transaldolase enzyme, or combination thereof.
  • PFK phosphofructokinase
  • PKI phosphoglucoisomerase
  • 6-phosphogluconate dehydrogenase decarboxylating
  • transketolase enzyme transaldolase enzyme, or combination thereof.
  • composition of embodiment F22, wherein the transketolase enzyme is encoded by a TKL-1 coding sequence or a TKL-2 coding sequence.
  • composition of embodiment F22, wherein the transaldolase is encoded by a TAL-1 coding sequence is encoded by a TAL-1 coding sequence.
  • composition of embodiment F22, wherein the phosphofructokinase (PFK) enzyme is a PFK-2 enzyme or PFK-1 enzyme.
  • composition of any one of embodiments F4 to F25, wherein the 6-phosphogluconate dehydrogenase (decarboxylating) enzyme is encoded by a GND-1 gene or GND-2 gene.
  • composition of embodiment F22, wherein the PGI is encoded by a PGI-1 gene is provided.
  • F28 The composition of any one of embodiments F1 to F27, wherein the polynucleotides, the promoters, or the polynucleotides and the promoters are not integrated in the yeast nucleic acid.
  • composition of embodiment F30 wherein the polynucleotides, the promoters, or the polynucleotides and the promoters are integrated in a transposition integration event, in a homologous recombination integration event, or in a transposition integration event and a homologous recombination integration event.

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