WO2013070949A1

WO2013070949A1 - Consolidated bioprocess for biofuel and chemical production from lignocellulosic biomass

Info

Publication number: WO2013070949A1
Application number: PCT/US2012/064201
Authority: WO
Inventors: Zhiliang Fan; Edyta SZEWCZYK
Original assignee: The Regents Of The University Of California
Priority date: 2011-11-08
Filing date: 2012-11-08
Publication date: 2013-05-16

Abstract

The present disclosure realtes to co-culture compositions containing a population of lignocellulolytic cells containing a mutation in one or more β-glucosidase genes, and a population of fermentative microorganisms; and to methods for using such co-culture compositions for producing commodity chemicals, such as biofuels, from lignocellulosic biomass.

Description

CONSOLIDATED BIOPROCESS FOR BIOFUEL AND CHEMICAL PRODUCTION

FROM LIGNOCELLULOSIC BIOMASS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.

61/557,318, filed November 08, 2011, which is hereby incorporated by reference, in its entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 514112005240SeqList.txt, date recorded: November 7, 2012, size: 2 KB).

FIELD

[0003] The present application relates to methods and compositions for producing commodity chemicals, such as biofuels, from lignocellulosic biomass.

BACKGROUND

[0004] Cellulosic biomass, which is available at low cost and in large abundance, is one of the only foreseeable sustainable sources for organic fuels, chemicals and materials. It is estimated that 1.3 billion tons of renewable lignocellulosic biomass can be produced in the United States every year. If all of that biomass could be efficiently converted to biofuels, it could play a significant role in petroleum displacement and greenhouse gas emissions reduction. Hence, production of biofuels from cellulosic biomass can play a significant role in meeting the nation's transportation needs, alleviating dependence on foreign oil, and reducing greenhouse gas emissions. The primary obstacle impeding production of ethanol and other chemicals from cellulosic biomass is the lack of technology for low-cost production.

[0005] Figure 1 depicts a traditional biochemical platform or method for biofuel and chemical production that generates sugars from cellulosic feedstock as reactive intermediates. These sugars can then be fermented to produce fuels and chemicals. There are five key steps involved in the current biochemical platform: (1) pretreatment, (2) cellulase production, (3) enzymatic hydrolysis, (4) fermentation, and (5) product recovery. The first three steps: pretreatment, cellulase production, and enzymatic hydrolysis are the three most costly steps in the production process, constituting approximately 65% of the overall processing cost.

[0006] The first step, pretreatment, is a process to remove hemicellulose and lignin to increase the susceptibility of cellulose to subsequent enzymatic hydrolysis, thus allowing the exposed cellulose to be hydrolyzed into sugars fermentable by cellulases. The pretreatment process tends to be thermochemical. Techniques used in the process include treatment with acid or base, or through steam or ammonia explosions. Most of the techniques are energy- intensive, expensive, and often polluting. In addition, capital cost for pretreatment reactors are extremely high due to specific material requirements for acid or alkali resistance at elevated temperatures. One possible solution is biological pretreatment using lignin- solubilizing organisms, as it has low energy requirements and requires mild reaction.

However, two factors impede the practical application of biological pretreatment: slow reaction rate and severe carbon loss due to the consumption of cellulose, hemicelluloses, and lignin by the microorganism performing the biological pretreatment.

[0007] After the pretreatment step, cellulases are added in a second step to hydrolyze cellulose, resulting in the production of sugars. The cellulases are usually produced in a separate step. While cellulase production costs have dropped significantly due to industrial production of enzymes, costs of this step still remain high. Lowering the processing costs of the two aforementioned steps is crucial for the realization of cost-effective production of biofuels and chemicals from lignocellulosic biomass.

[0008] Biofuels, such as butanol and ethanol, are reduced metabolic products. They are most efficiently produced via an anaerobic process. The key challenges of anaerobic fermentation for biofuel production include the co-fermentation of hexose and pentose sugars from lignocellulosic biomass hydrolysate and alleviation of product inhibition in the fermentation process. Product recovery technologies vary with the specificities of the products. The routine product recovery unit operations for volatile biofuels include gas stripping, distillation, and membrane separation. [0009] While pretreatment , enzymatic hydrolysis cellulase production are the most expensive steps in the entire process, fermentation and product recovery are still substantial contributors to overall processing costs.

[0010] One solution to lowering the overall process cost involves process consolidation. When pretreatment, cellulose hydrolysis, hexose fermentation, pentose fermentation, and product recovery take place in different reactors; the process is called separate hydrolysis and fermentation (SHF). In the SHF configuration, cellulase from the enzyme production step is added to the pretreated material to form glucose from the cellulose fraction. Upon completion of the hydrolysis, a fermentative microorganism is added to convert the glucose to a biofuel, such as ethanol.

[0011] Simultaneous saccharification and fermentation (SSF) consolidates hexose fermentation and enzymatic hydrolysis into one reactor. Cellulases are added to the pretreated materials to hydrolyze the cellulose fraction to glucose, while the fermentative

microorganism converts the glucose to biofuels in the same reactor. Since glucose, which is an inhibitor of cellulase, is converted by the fermenting microorganism as it is formed, SSF can achieve faster rates and higher yields of biofuel as compared to SHF.

[0012] Simultaneous saccharification and co-fermentation (SSCF) consolidates enzymatic hydrolysis, hexose fermentation, and pentose fermentation into one step by engineering the fermentative microorganism to co-ferment both pentose and hexose sugars. Higher product yield and process efficiency can thus be achieved by SSCF as compared to SSF.

[0013] Consolidated bioprocessing (CBP) features the combination of cellulase production, cellulose hydrolysis, hexose fermentation, and pentose fermentation in a single process. One big difference between CBP and the other process configurations is that CBP features cellulase production under anaerobic conditions. Analysis of the economic impact of various potential research-driven processing advances have indicated that greater cost reduction can be achieved with an increased level of process consolidation, and that CBP may offer the largest cost reduction of any process improvement considered to date. Much of the savings projected for CBP results from greatly reduced costs of cellulase production. However, CBP-enabling microorganism engineering is challenging. It is difficult to engineer very robust microorganisms that are capable of degrading cellulose efficiently while producing biofuels at high yields. Part of the reason is the challenge of producing cellulases under ATP-scarce (i.e., anaerobic) conditions.

[0014] Accordingly, a need existed for improved consolidated bioprocessing that incorporates engineered microorganisms that are capable of degrading cellulose efficiently while producing biofuels at high yields.

SUMMARY

[0015] In order to meet the above needs, the present disclosure provides co-culture compositions containing a population of mutant lignocellulolytic cells containing a mutation in one or more β-glucosidase genes, and a population of fermentative microorganisms; and to methods of using such co-culture compositions for producing commodity chemicals, such as biofuels. Moreover, the present disclosure is based at least in part on novel processes for biofuel and chemical production from lignocellulosic biomass that overcome the challenges of SSF, SSCF, and CBP. One process is an integrated bioprocessing and separation (IBS) process that can consolidate cellulase production, enzymatic hydrolysis, pentose

fermentation, hexose fermentation, and product recovery into one single step. The novel IBS process is achieved by utilizing a co-culture under ATP-rich (i.e., aerobic) conditions that contains aerobic lignocellulosic microorganisms engineered to preferentially utilize monosaccharides for growth and hydrolysis enzyme production; and anaerobic fermentative microorganisms that preferentially consume oligosaccharides to increase biofuel production. Advantageously, the fermentative microorganisms are immobilized on a gel matrix to achieve a local micro-aerobic or anaerobic environment for oligosaccharide fermentation.

[0016] The second process is a one-step bioconversion and separation (OBS) process that integrates lignocellulosic pretreatment into the IBS process. The novel OBS process is achieved by utilizing or engineering lignocellulolytic microorganisms to produce lignin- solubilizing enzymes that efficiently solubilize or degrade lignin. Advantageously, the IBS and OBS processes have higher levels of consolidation than known CBP processes. Thus, IBS and OBS achieve an even greater reduction in lignocellulosic biomass processing costs.

[0017] Accordingly, certain aspects of the present disclosure relate to a co-culture composition containing: a first population of mutant lignocellulolytic cells, where the mutant cells contain a mutation in one or more β-glucosidase genes; and a second population of fermentative microorganisms. In certain embodiments, the mutant cells are aerobic mutant cells. In other embodiments, the mutant cells are anaerobic mutant cells. In certain embodiments that may be combined with any of the preceding embodiments, the

fermentative microorganisms are aerobic fermentative microorganisms. In other

embodiments that may be combined with any of the preceding embodiments, the

fermentative microorganisms are anaerobic fermentative microorganisms.

[0018] In certain embodiments, the mutant cells contain a mutation in two or more, three or more, four or more, five or more, six or more, or seven β-glucosidase genes. In some embodiments, the β-glucosidase genes encode extracellular β-glucosidases. In other embodiments, the β-glucosidase genes encode intracellular β-glucosidases. In still other embodiments, the β-glucosidase genes are selected from NCU00130, NCU04952,

NCU05577, NCU07487, NCU08054, NCU08755, NCU03641, homologs thereof, orthologs thereof, and paralogs thereof. In yet other embodiments of any of the disclosed co-culture compositions, the mutant cells further contain a mutation in one or more additional cellulose oligosachharide hydrolase genes. In certain preferred embodiments, the one or more additional cellulose oligosachharide hydrolase genes are one or more additional hexose hydrolase genes selected from β-galactosidase gene NCU05956, the β-galactosidase gene NCU05956, the β-mannosidase gene NUC00130, homologs thereof, orthologs thereof, and paralogs thereof. In further embodiments of any of the disclosed co-culture compositions, the mutant cells further contain a mutation in one or more transcription factors. In other embodiments of any of the disclosed co-culture compositions, the mutant cells further contain a mutation in at least one hemicellulose oligosaccharide hydrolase gene. In certain preferred embodiments, the at least one hemicellulose oligosaccharide hydrolase gene is a xylosidase gene. In other embodiments of any of the disclosed co-culture compositions, the mutant cells further contain a mutation in at least one alcohol dehydrogenase gene. In certain preferred embodiments, the at least one alcohol dehydrogenase gene is the adhl gene or the adh3 gene. In other embodiments, the mutant cells further contain a mutation in at least two alcohol dehydrogenase genes. In certain preferred embodiments, the at least two alcohol

dehydrogenase genes are the adhl gene and the adh3 gene. In still other embodiments of any of the disclosed co-culture compositions, the gene mutation contains a partial deletion or a complete deletion of the gene. [0019] In some embodiments of any of the disclosed co-culture compositions, the mutant cells produce one or more of cellulases, hemicellulases, lignin-solubilizing enzymes, or combinations thereof. In other embodiments of any of the disclosed co-culture compositions, the mutant cells have higher cellulase activity as compared to a corresponding cell that does not contain a mutation in one or more β-glucosidase genes and/or hemicellulose

oligosaccharide hydrolase genes. In certain preferred embodiments, the one or more hemicellulases are recombinantly expressed in the mutant cells. In other preferred embodiments, the one or more lignin-solubilizing enzymes are recombinantly expressed in the mutant cells.

[0020] In other embodiments of any of the disclosed co-culture compositions, the mutant cells further contain a recombinant cellulosome. In certain preferred embodiments, the cellulosome is assembled on the surface of the mutant cells. In further embodiments of any of the disclosed co-culture compositions, the mutant cells produce cellodextrin. In certain preferred embodiments, the cellodextrin is cellobiose. In other embodiments of any of the disclosed co-culture compositions, the mutant cells are fungal cells. In still other

embodiments of any of the disclosed co-culture compositions, the fungal cells are

filamentous fungal cells. In certain preferred embodiments of any of the disclosed co-culture compositions, the fungal cells are Neurospora crassa.

[0021] In some embodiments of any of the disclosed co-culture compositions, the fermentative microorganisms contain at least one cellodextrin transporter. In certain preferred embodiments, the cellodextrin transporter is a cellobiose transporter. In some embodiments, the cellodextrin transporter is endogenous to the fermentative microorganisms. In other embodiments, the cellodextrin transporter is a recombinant cellodextrin transporter. In yet other embodiments of any of the disclosed co-culture compositions, the fermentative microorganisms further contain at least one intracellular β-glucosidase or cellobiose phosphorylase. In some embodiments, the intracellular β-glucosidase or cellobiose phosphorylase is endogenous to the fermentative microorganisms. In other embodiments, the intracellular β-galactosidase or cellobiose phosphorylase is a recombinant intracellular β- galactosidase or cellobiose phosphorylase. In still other embodiments of any of the disclosed co-culture compositions, the fermentative microorganisms further contain a mutation in one or more monosaccharide sugar transporter genes. In certain embodiments, the one or more monosaccharide sugar transporter genes are selected from hexose transporter genes, pentose transporter genes, and combinations thereof. In certain embodiments, the one or more monosaccharide sugar transporter genes are selected from glucose transporter genes, xylose transporter genes, galactose transporter genes, mannose transporter genes, arabinose transporter genes, fructose transporter genes, maltose transporter genes, lactose transporter genes, and combinations thereof. In other embodiments of any of the disclosed co-culture compositions, the fermentative microorganisms further contain a mutation in one or more sugar alcohol transporter genes or one or more sugar aldonic transporter genes. In certain embodiments, the one or more sugar alcohol transporter genes are sorbitol transporter genes and the one or more sugar aldonic acid transporter genes are gluconic acid transporter genes. In other preferred embodiments, the gene mutation contains a partial deletion or a complete deletion of the gene. In further embodiments of any of the disclosed co-culture compositions, the fermentative microorganisms further contain at least one recombinant oligosaccharide utilization polypeptide. In certain embodiments, the at least one recombinant oligosaccharide utilization polypeptide is selected from a cellodextrin utilization polypeptide, a cellobiose utilization polypeptide, a xylodextrin utilization polypeptide, a xylobiose utilization polypeptide, a mannobiose utilization polypeptide, a galactobiose utilization polypeptide, a maltose utilization polypeptide, a lactose utilization polypeptide, and combinations thereof. In other embodiments, the at least one recombinant oligosaccharide utilization polypeptide is selected from a cellodextrin transporter, a cellobiose transporter, a xylodextrin transporter, a xylobiose transporter, a mannobiose transporter, a galactobiose transporter, a maltose transporter, a lactose transporter, and combinations thereof.

[0022] In some embodiments of any of the disclosed co-culture compositions, the fermentative microorganisms further contain one or more modifications sufficient for the fermentative microorganisms to produce a commodity chemical from one or more

oligosaccharides. In certain embodiments, the one or more modifications contain a deletion in one or more genes. In other embodiments, the one or more modifications further contain the recombinant expression of one or more genes. In further embodiments, the commodity chemical is a biofuel selected from an alcohol, ethanol, propanol, isopropanol, acetone, butanol, isobutanol, 2-methyl-l-butanol, 3-methyl-l-butanol, phenylethanol, a fatty alcohol, isopentenol, an aldehyde, acetylaldehyde, propionaldehyde, butryaldehyde, isobutyraldehyde, 2-methyl-l-butanal, 3-methyl-l-butanal, phenylacetaldehyde, a fatty aldehyde, a

hydrocarbon, an alkane, an alkene, an isoprenoids, a fatty acid, a wax ester, an ethyl ester, hydrogen, and combinations thereof. [0023] In other embodiments of any of the disclosed co-culture compositions, the fermentative microorganisms are bacteria. In certain preferred embodiments, the bacteria are Klebsiella oxytoca. In still other embodiments of any of the disclosed co-culture

compositions, the fermentative microorganisms are fungi or yeast. In further embodiments of any of the disclosed co-culture compositions, the co-culture composition further includes a third population of aerobic fatty acid-producing and/or hydrocarbon-producing

microorganisms.

[0024] Other aspects of the present disclosure relate to a method for the production of a commodity chemical from lignocellulosic biomass, by: providing a fermentation broth containing any of the disclosed co-culture compositions; contacting lignocellulosic biomass with the fermentation broth; and incubating the fermentation broth and lignocellulosic biomass under conditions sufficient for the co-culture composition to produce a commodity chemical from the lignocellulosic biomass.

[0025] Other aspects of the present disclosure relate to a method for the production of a commodity chemical from lignocellulosic biomass, by: providing a fermentation broth containing a co-culture composition containing: a first population of mutant lignocellulolytic cells, where the mutant cells contain a mutation in one or more β-glucosidase genes, and a second population of fermentative microorganisms; contacting lignocellulosic biomass with the fermentation broth; and incubating the fermentation broth and lignocellulosic biomass under conditions sufficient for the co-culture composition to produce a commodity chemical from the lignocellulosic biomass. In certain embodiments, the lignocellulolytic cells are aerobic lignocellulolytic cells. In other embodiments, the lignocellulolytic cells are anaerobic lignocellulolytic cells. In certain embodiments that may be combined with any of the preceding embodiments, the fermentative microorganisms are aerobic fermentative microorganisms. In other embodiments that may be combined with any of the preceding embodiments, the fermentative microorganisms are anaerobic fermentative microorganisms.

[0026] In certain embodiments of any of the disclosed methods, the co-culture composition is incubated under aerobic conditions. In certain preferred embodiments, air or oxygen is supplied to the fermentation broth to achieve the aerobic conditions. In other embodiments of any of the disclosed methods, the fermentative microorganisms are incubated under a local anaerobic or micro-aerobic environment. In some embodiments, the local anaerobic or micro-aerobic environment is produced by immobilizing the fermentative microorganisms. In certain preferred embodiments, the fermentative microorganisms are immobilized within gel beads. In other preferred embodiments, the fermentative

microorganisms are co-immobilized with the mutant lignocellulolytic cells within gel beads.

[0027] In other embodiments of any of the disclosed methods, the mutant

lignocellulolytic cells produce glucose and cellodextrin from the lignocellulosic biomass. In some embodiments, the cellodextrin is cellobiose. In other embodiments, the mutant lignocellulolytic cells utilize the produced glucose as a carbon source. In still other embodiments, the fermentative microorganisms utilize the cellodextrin to produce the commodity chemical. In yet other embodiments of any of the disclosed methods, the mutant lignocellulolytic cells further produce at least one hemicellulose oligosaccharide from the lignocellulosic biomass. In certain embodiments, the at least one hemicellulose

oligosaccharide is xylobiose. In other embodiments, the fermentative microorganisms further utilize the hemicellulose oligosaccharide to produce the commodity chemical. In further embodiments of any of the disclosed methods, the commodity chemical is extracted from the fermentation broth by gas stripping. In certain preferred embodiments, air or oxygen is utilized to gas strip the commodity chemical. In other embodiments of any of the disclosed methods, the commodity chemical is a biofuel selected from an alcohol, ethanol, propanol, isopropanol, acetone, butanol, isobutanol, 2-methyl-l-butanol, 3-methyl-l-butanol, phenylethanol, a fatty alcohol, isopentenol, an aldehyde, acetylaldehyde, propionaldehyde, butryaldehyde, isobutyraldehyde, 2-methyl-l-butanal, 3-methyl-l-butanal,

phenylacetaldehyde, a fatty aldehyde, a hydrocarbon, an alkane, an alkene, an isoprenoids, a fatty acid, a wax ester, an ethyl ester, hydrogen, and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 depicts a biochemical platform known in the art for biofuel production (Wooley, et al. NREL/TP-580-26157, N. R. E. L., Ed., 1998).

[0029] Figure 2 depicts a schematic of one embodiment of an all-in-one bioprocess for biofuel production.

[0030] Figure 3 depicts an all-in-one bioprocess disclosed herein for biofuel production. [0031] Figure 4 depicts a schematic of the evolution of biomass processing configurations featuring enzymatic hydrolysis. SHF=Separate Hydrolysis and Fermentation; SSF = Simultaneous Saccharification and Fermentation; SSCF = Simultaneous

Saccharification and co-Fermentation; CBP = Consolidated Bioprocessing; IBS = Integrated Bioprocessing and Separation; and OBS = One-step Bioconversion and Separation.

[0032] Figure 5 depicts a schematic of Integrated Bioprocessing and Separation (IBS) and One-step Bioconversion and Separation (OBS) configurations.

[0033] Figure 6 depicts bgl gene multi-knockout strains of N. crassa, and the genotyping to screen the knockout strains.

[0034] Figure 7 depicts cellobiose production by wild type N. crassa strain (WT) and the six bgl gene knockout N. crassa mutant strain (F5).

[0035] Figure 8 depicts relative expression levels of endoglucanases gh5-l and gh7-l in wild type N. crassa strain (WT) and N. crassa sextuple bgl deletion strains ( Fl, F2, F3, F4, F5, F6, and F7 ) after 4 hours of induction on Avicel^®.

[0036] Figure 9 depicts sugar utilization by wild type N. crassa strain (WT) and the six bgl gene knockout N. crassa mutant strain (F5-BGL5).

[0037] Figure 10 depicts relative gene expression levels of major cellulases genes in the wild type N. crassa strain (WT) and the six bgl gene knockout N. crassa mutant strain (F5- BGL5).

[0038] Figure 11 depicts glucose utilization by the wild type K. Oxytoca strain (wild type) and the AptsG, AmanXYZ, and AptsGAmanXYZ K. Oxytoca strains.

[0039] Figure 12 depicts cellobiose utilization by the wild type K. Oxytoca strain (wild type) and the AptsG, AmanXYZ, and AptsGAmanXYZ K. Oxytoca strains.

[0040] Figure 13 depicts glucose utilization by the wild type K. oxytoca P2 strain (p2), a K. oxytoca mutant strain with deletions in the ptsG gene and manXYZ gene

(AptsGAmanXYZ), and a K. oxytoca mutant strain with deletions in the ptsG gene, the manXYZ gene, the galP gene, and the mglABC gene (AptsGAmanXYZAGalPAmalEFG) or the malEFG gene (AptsGAmanXYZAGalPAmglABC).

[0041] Figure 14 depicts residual cellobiose and cellobiose utilization by a co-culture of the N. crassa F5-BGL5 strain and the K. Oxytoca VlAptsG manXYZ strain.

[0042] Figure 15 depicts ethanol consumption by wild- type N. crassa, the N. crassa AD1 knockout strain, the N. crassa AND3 knockout strain, and the N. crassa F5-BGL strain.

DETAILED DESCRIPTION

Definitions

[0043] Unless defined otherwise, all scientific and technical terms are understood to have the same meaning as commonly used in the art to which they pertain. For the purpose of the present disclosure, the following terms are defined.

[0044] As used herein, a "co-culture composition" refers to a mixed culture of lignocellulolytic microorganisms and fermentative microorganisms.

[0045] As used herein, "lignocellulosic biomass" refers to biomass that is composed primarily of cellulose, hemicellulose, and lignin.

[0046] As used herein, "lignocellulolytic cell" and "lignocellulolytic microorganism" are used interchangeably and refer to a cell that degrades lignocellulose or components thereof. "Lignocellulolytic cells" endogenously express cellulase genes that encode cellulases that degrade cellulose. "Lignocellulolytic cells" may also endogenously express hemicellulase genes that encode hemicellulases the degrade hemicellulose.

[0047] As used herein, "cellodextrin" refers to glucose polymers of varying length and includes, without limitation, cellobiose (2 glucose monomers), cellotriose (3 glucose monomers), cellotetraose (4 glucose monomers), cellopentaose (5 glucose monomers), and cellohexaose (6 glucose monomers). [0048] As used herein, "sugar" refers to monosaccharides (e.g., glucose, fructose, galactose, xylose, arabinose), disaccharides (e.g., cellobiose, sucrose, lactose, maltose), and oligosaccharides (typically containing 2 to 10 component monosaccharides).

[0049] As used herein, a "cellulase" or "cellulase polypeptide" refers to a polypeptide having E.C. 3.2.1.4 activity, which catalyzes the hydrolysis of l,4-P-D-glucosidic linkages in cellulose, lichenin, and cereal β-D-glucans. As used herein, "cellulases" include, without limitation, endocellulases, such as endo-l,4-P-glucanases, endo-l,4-P-D-glucanases, carboxymethyl cellulases (CMCases), P-l,4-glucanases, P-l,4-endoglucan hydrolases, and celludextrinases; exocellulases, such as exoglucanases; cellobiases; oxidative cellulases, such as cellobiose dehydrogenases; and cellulose phosphorylases.

[0050] As used herein, the terms "polynucleotide," "nucleic acid sequence," "sequence of nucleic acids," and variations thereof shall be generic to polydeoxyribonucleotides

(containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing non-nucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence

modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog; inter-nucleotide modifications, such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalkylphosphoramidates,

aminoalkylphosphotriesters); those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalators (e.g., acridine, psoralen, etc.); and those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.). As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature (Biochem. 9:4022, 1970).

[0051] As used herein, a "polypeptide" is an amino acid sequence containing a plurality of consecutive polymerized amino acid residues (e.g., at least about 15 consecutive polymerized amino acid residues, optionally at least about 30 consecutive polymerized amino acid residues, at least about 50 consecutive polymerized amino acid residues). In many instances, a polypeptide contains a polymerized amino acid residue sequence that is a transporter, an enzyme, a predicted protein of unknown function, or a domain or portion or fragment thereof. A transporter is involved in the movement of ions, small molecules, or macromolecules, such as a carbohydrate, across a biological membrane. An enzyme can catalyze a chemical reaction, such as the reduction of a carbohydrate to an alcohol, in a host cell. The polypeptide optionally contains modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, and non-naturally occurring amino acid residues.

[0052] As used herein, "protein" refers to an amino acid sequence, oligopeptide, peptide, polypeptide, or portions thereof whether naturally occurring or synthetic.

[0053] Genes and proteins that may be used in the present disclosure include genes encoding conservatively modified variants and proteins that are conservatively modified variants of those genes and proteins described throughout the application. "Conservatively modified variants" as used herein include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0054] Homologs of the genes and proteins described herein may also be used in the present disclosure. As used herein, "homology" refers to sequence similarity between a reference sequence and at least a fragment of a second sequence. Homologs may be identified by any method known in the art, preferably, by using the BLAST tool to compare a reference sequence to a single second sequence or fragment of a sequence or to a database of sequences. As described below, BLAST will compare sequences based upon percent identity and similarity. [0055] As used herein, "orthologs" are evolutionarily related genes or proteins in different species that have similar sequences and functions, and that develop through a speciation event. Sequences that are orthologs are referred to as being "orthologous" to each other.

[0056] As used herein, "paralogs" are evolutionarily related genes or proteins in the same organism that have similar sequences and functions, and that develop through a gene duplication event. Sequences that are paralogs are referred to as being "paralogous" to each other.

[0057] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200, or more amino acids) in length.

[0058] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. When comparing two sequences for identity, it is not necessary that the sequences be contiguous, but any gap would carry with it a penalty that would reduce the overall percent identity. For blastn, the default parameters are Gap opening penalty=5 and Gap extension penalty=2. For blastp, the default parameters are Gap opening penalty=l 1 and Gap extension penalty=l. [0059] A "comparison window," as used herein, includes reference to a segment of any one of the number of contiguous positions including, but not limited to from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970) J Mol Biol 48(3):443-453, by the search for similarity method of Pearson and Lipman (1988) Proc Natl Acad Sci USA

85(8):2444-2448, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection [see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou Ed)].

[0060] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol 215(3)-403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the

accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix [see Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA 89(22): 10915-10919] alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0061] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, (1993) Proc Natl Acad Sci USA 90(12):5873-5877). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0062] Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

Overview

[0063] The present disclosure relates to the production and use of a co-culture of:

lignocellulolytic microorganisms that are engineered to preferentially produce cellodextrins and other hemicellulose oligosaccharides (e.g., xylodextrins, etc.) from lignocellulosic biomass without utilizing the cellodextrin for growth; and fermentative microorganisms that preferentially utilize cellodextrins and other hemicellulose oligosaccharides (e.g., soluble hemicellulose hydrolysate compound containing more than one sugar unit) for growth and for the production of commodity chemicals, such as biofuels (Fig. 2). Advantageously, the lignocellulolytic microorganisms of the present disclosure may be either aerobic microorganisms or anaerobic microorganisms. Additionally, the fermentative

microorganisms of the present disclosure may be either aerobic microorganisms or anaerobic microorganisms. Moreover, the present disclosure is based, at least in part, on the novel strategy of using such a co-culture in an integrated bioprocessing and separation (IBS) process that consolidates cellulase and hemicellulase production, enzymatic hydrolysis, pentose fermentation, hexose fermentation, and product recovery into one single step (Fig. 3). Such an IBS process can achieve a greater reduction in lignocellulosic biomass processing costs as compared to known consolidated bioprocessing (CBP) processes. Advantageously, a higher level of consolidation can be achieved by integrating the lignocellulosic pretreatment step into the IBS process by utilizing or engineering lignocellulolytic microorganisms that degrade or solubilize lignin.

[0064] Accordingly, certain aspects of the present disclosure provide co-culture compositions containing a first population of mutant lignocellulolytic cells, where the mutant cells contain a mutation in one or more β-glucosidase genes, and where the mutant cells are either aerobic cells or anaerobic cells; and a second population of fermentative

microorganisms, where the fermentative microorganisms are either aerobic microorganisms or anaerobic microorganisms. Other aspects of the present disclosure provide methods for the production of a commodity chemical from lignocellulosic biomass, by providing a fermentation broth containing any co-culture composition of the present disclosure;

contacting lignocellulosic biomass with the fermentation broth; and incubating the

fermentation broth and lignocellulosic biomass under conditions sufficient for the co-culture composition to produce a commodity chemical from the lignocellulosic biomass.

Lignocellulolytic and Fermentative Microorganisms of the Present Disclosure

[0065] Certain aspects of the present disclosure relate to a co-culture composition containing a population of lignocellulolytic microorganisms and a population of fermentative microorganisms. Such microorganisms may be either aerobic microorganisms or anaerobic microorganisms. Moreover, such microorganisms may be used for an integrated

bioprocessing and separation system for producing commodity chemicals form

lignocellulosic biomass. [0066] The lignocellulolytic and fermentative microorganisms of the present disclosure may be transformed via insertion of recombinant DNA or RNA. Such recombinant DNA or RNA can be in an expression vector. Thus, a microorganism as described herein may be a prokaryotic organism (e.g., an organism of the kingdom Eubacteria) or a eukaryotic microorganism. As will be appreciated by one of ordinary skill in the art, a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus.

[0067] Any prokaryotic or eukaryotic lignocellulolytic and/or fermentative

microorganism may be used in the present disclosure so long as it remains viable after being transformed with a sequence of nucleic acids. Preferably, the microorganism is not adversely affected by the transduction of the necessary nucleic acid sequences, the subsequent expression of the proteins (e.g., lignin-solubilizing enzymes, transporters, etc.), or the resulting intermediates. Suitable eukaryotic microorganism include, without limitation, fungi and yeast.

[0068] The lignocellulolytic and fermentative microorganisms of the present disclosure may be genetically modified in that recombinant nucleic acids have been introduced into the microorganisms or endogenous genes have been modified, and as such the genetically modified microorganisms do not occur in nature. A suitable microorganism of the present disclosure is one capable of expressing one or more nucleic acid constructs encoding one or more proteins for different functions.

[0069] "Recombinant nucleic acid" or "heterologous nucleic acid" or "recombinant polynucleotide" as used herein refers to a polymer of nucleic acids wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host cell; (b) the sequence may be naturally found in a given host cell, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a recombinant nucleic acid sequence will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid.

Specifically, the present disclosure describes the introduction of an expression vector into a host cell, where the expression vector contains a nucleic acid sequence coding for a protein that is not normally found in a host cell or contains a nucleic acid coding for a protein that is normally found in a cell but is under the control of different regulatory sequences. With reference to the microorganism's genome, then, the nucleic acid sequence that codes for the protein is recombinant. A protein that is referred to as recombinant generally implies that it is encoded by a recombinant nucleic acid sequence in the microorganism.

[0070] In some embodiments, the genes encoding the desired proteins in the

microorganism may be heterologous to the microorganism or these genes may be endogenous to the microorganism but are operatively linked to heterologous promoters and/or control regions which result in the higher expression of the gene(s) in the microorganism. In certain embodiments, the microorganism does not naturally produce the desired proteins, and contains heterologous nucleic acid constructs capable of expressing one or more genes necessary for producing those molecules.

[0071] "Endogenous" as used herein with reference to a nucleic acid molecule or polypeptide and a particular cell or microorganism refers to a nucleic acid sequence or polypeptide that is in the microorganism and was not introduced into the microorganism using recombinant engineering techniques; for example, a gene that was present in the microorganism when the microorganism was originally isolated from nature.

[0072] "Genetically engineered" or "genetically modified" refers to any recombinant DNA or RNA method used to create a prokaryotic or eukaryotic microorganism that expresses a protein at elevated levels, at lowered levels, or in a mutated form. In other words, the microorganism has been transfected, transformed, or transduced with a recombinant polynucleotide molecule, and thereby been altered so as to cause the microorganism to alter expression of a desired protein. Methods and vectors for genetically engineering

microorganisms are well known in the art; for example various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates). Genetically engineering techniques include, without limitation, expression vectors, and targeted homologous recombination and gene activation (see, for example, U.S. Pat. No. 5,272,071).

Lignocellulolytic Cells

[0073] Other aspects of the present disclosure relate to a co-culture composition containing a population of lignocellulolytic cells having reduced expression of one or more β- glucosidase genes. As disclosed herein, the lignocellulolytic cells may be either aerobic cells or anaerobic cells. In certain preferred embodiments, the lignocellulolytic cells are aerobic cells.

[0074] Lignocellulolytic cells of the present disclosure produce enzymes that degrade lignocellulose or components thereof. The lignocellulolytic cells may degrade the

lignocellulose or components thereof under aerobic (i.e., oxygen rich), or anaerobic (i.e., oxygen deficient) conditions. In certain embodiments, the lignocellulolytic cells of the present disclosure are capable of pretreating lignocellulosic biomass. Such lignocellulolytic cells simultaneously degrade lignin, solubilize lignin, or change lignin to a revised form, such as de-methylized lignin. Lignin is an energy-rich compound that can be utilized for energy production (e.g. electricity). In other embodiments, lignocellulolytic cells of the present disclosure produce one or more cellulases, hemicellulases, lignin-solubilizing enzymes, or combinations thereof. In certain embodiments, the one or more hemicellulases and/or lignin- solubilizing enzymes are recombinantly expressed in the lignocellulolytic cells. Accordingly, lignocellulolytic cells of the present disclosure can produce monosaccharides (e.g., glucose) and cellodextrins (e.g., cellobiose, cellotriose, cellotetrose, cellopentose, etc.) from

lignocellulosic biomass. Additionally, lignocellulolytic cells of the present disclosure can also produce hemicellulose oligosaccharides, such as xylobiose, from lignocellulosic biomass.

[0075] Lignocellulolytic cells of the present disclosure include, without limitation, fungi and bacteria. Suitable lignocellulolytic fungi of the present disclosure include, without limitation, White Rot Fungi, Brown Rot Fungi, Soft Rot Fungi, and ascomycetes fungi.

Suitable lignocellulolytic bacteria of the present disclosure include, without limitation, Clostridium sp. and Thermanaerobacterium sp. Additional examples of suitable

lignocellulolytic cells include, without limitation, Trichoderma reesei, Clostridium

thermocellum, Clostridium papyrosolvens C7, Podospera anserine, Chaetomium globosum, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Phanerochaete chrysosporium, Sporotrichum thermophile (Myceliophthora thermophila), Gibberella zeae, Sclerotinia sclerotiorum, Botryotinia fuckelian, Aspergillus niger, Thielavia terrestris, Fusarium spp., Rhizopus spp.,

Neocallimastix frontalis, Orpinomyces sp., Piromyces sp., Penicillium chrysogenum cells, Schizophyllum commune, Postia placenta, Acremonium cellulolyticus, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Chrysosporium lucknowense, Aspergillus sp., Trichoderma sp., Caldocellulosiruptor sp., Butyrivibrio sp., Butyrivibrio sp., Eubacterium sp., Clostridium sp., Bacteroides sp., icetivibrio sp., Thermoactinomyces sp., Caldibacillus sp., Bacillus sp., Acidothermus sp., Cellulomonas sp., Micromonospora sp., Actinoplanes sp., Streptomyces sp., Thermobifida sp., Thermomonospora sp., Microbispora sp., Microbispora sp., Fibrobacter sp., Sporocytophaga sp., Cytophaga sp., Flavobacterium sp., Achromobacter sp., Xanthomonas sp., Cellvibrio sp., Pseudomonas sp., Myxobacter sp., Clostridium phytofermentans, Clostridium japonicas, and Thermoanaerobacterium saccharolyticum cells.

[0076] In certain embodiments, lignocellulolytic cells of the present disclosure are filamentous fungal cells including, without limitation, Neurospora, Trichoderma, and Aspergillus cells. In certain preferred embodiments, the filamentous fungal cells are

Neurospora crassa cells. β-Glucosidase genes

[0077] Lignocellulolytic cells of the present disclosure may have reduced expression of one or more β-glucosidase genes, by containing a modification, such as a mutation, in one or more β-glucosidase genes. Mutations in one or more extracellular β-glucosidase genes that reduce expression of the genes or that delete the genes result in lignocellulolytic cells that preferentially produce oligosaccharides (e.g., cellodextrins, cellobiose, etc.) rather than monosaccharides (e.g., glucose) as a major product. Mutations in one or more intracellular β- glucosidase genes that reduce expression of the genes or that delete the genes result in lignocellulolytic cells that preferentially utilize monosaccharides (e.g., glucose), rather than oligosaccharides (e.g., cellodextrins, cellobiose, etc.) for growth and hydrolysis enzyme production.

[0078] β-Glucosidase (bgl) genes of the present disclosure encode β-glucosidase enzymes. As used herein, "β-glucosidase" refers to a β-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis of terminal non-reducing β-D-glucose residues with the release of β-D-glucose. A β-glucosidase is any enzyme that catalyzes the hydrolysis of terminal non-reducing residues in β-D-glucosides, such as cellodextrins, with release of glucose. [0079] β-glucosidases of the present disclosure may be either intracellular β-glucosidases or extracellular β-glucosidases. As used herein "intracellular β-glucosidases" are expressed within lignocellulolytic cells and hydrolyze cellodextrins transported into the cell. As used herein "extracellular β-glucosidases" are expressed and secreted from lignocellulolytic cells or expressed on the surface of lignocellulolytic cells.

[0080] In certain embodiments, the β-glucosidase is a glycosyl hydrolase family 1 member. Members of this group can be identified by the motif, [LIVMFSTC] - [LIVFYS] - [LIV] - [LIVMST] - E - N - G - [LIVMFAR] - [CSAGN]. Here, E is the catalytic glutamate (webpage expasy.org/cgi-bin/prosite-search-ac?PDOC00495). In some embodiments, the β- glucosidase is from N. crassa. Other β-glucosidases may include those from the glycosyl hydrolase family 3. These β-glucosidases can be identified by the following motif according to PROSITE: [LIVM](2) - [KR] - x - [EQKRD] - x(4) - G - [LIVMFTC] - [LIVT] - [LIVMF] - [ST] - D - x(2) - [SGADNIT] . Here D is the catalytic aspartate. Typically, any

β-glucosidase may be used that contains the conserved domain of β-glucosidase/6-phospho- β-glucosidase/β-galactosidase found in NCBI sequence COG2723.

[0081] In certain embodiments, β-glucosidases of the present disclosure include, without limitation, N. crassa β-glucosidases encoded by NCU00130, NCU04952, NCU05577, NCU07487, NCU08054, NCU08755, and NCU03641. Suitable β-glucosidases of the present disclosure also include homologs, orthologs, and paralogs of NCU00130, NCU04952, NCU05577, NCU07487, NCU08054, NCU08755, and NCU03641.

[0082] Intracellular β-glucosidases of the present disclosure include, without limitation, those encoded by NCU00130, NCU05577, NCU07487, NCU08054, homologs thereof, orthologs thereof, and paralogs thereof. Extracellular β-glucosidases of the present disclosure include, without limitation, those encoded by NCU04952, NCU08755, NCU03641, homologs thereof, orthologs thereof, and paralogs thereof.

Cellulose oligosaccharide hydrolase genes

[0083] Lignocellulolytic cells of the present disclosure may also have reduced expression of one or more cellulose oligosaccharide hydrolase genes, by containing a mutation in one or more cellulose oligosaccharide hydrolase genes. Mutations in one or more cellulose oligosaccharide hydrolase genes that reduce expression of the genes or that delete the genes result in lignocellulolytic cells with an increased ability to convert cellulose to cellodextrins, such as cellobiose.

[0084] Cellulose oligosaccharide hydrolase genes of the present disclosure include, without limitation, hexose hydrolase genes that encode hexose hydrolase enzymes. As used herein, "hexose hydrolase" refers to a glycoside hydrolase (E.C. 3.2.1), which catalyzes the hydrolysis of the glycosidic linkage in a hexose, such galactose or mannose, to release smaller chain oligosaccharides.

[0085] Hexose hydrolases of the present disclosure include, without limitation,

β-galactosidases and β-mannosidases. In certain embodiments β-galactosidases of the present disclosure include, without limitation, the N. crassa β-galactosidase encoded by NCU05956, the N. crassa β-galactosidase encoded by and NCU05956, homologs thereof, orthologs thereof, and paralogs thereof. An example of a suitable pentose hydrolase includes, without limitation, β-mannosidase. In certain embodiments β-mannosidases of the present disclosure include, without limitation, the N. crassa β-mannosidase encoded by NUC00130, homologs thereof, orthologs thereof, and paralogs thereof.

Hemicellulose oligosaccharide hydrolase genes

[0086] Lignocellulolytic cells of the present disclosure may further have reduced expression of one or more hemicellulose oligosaccharide hydrolase genes, by containing a mutation in one or more hemicellulose oligosaccharide hydrolase genes. Mutations in one or more hemicellulose oligosaccharide hydrolase genes that reduce expression of the genes or that delete the genes result in lignocellulolytic cells that preferentially convert hemicellulose to hemicellulose oligosaccharides. As used herein, "hemicellulose oligosaccharides" refers to shot chain saccharides derived from hemicellulose.

[0087] Hemicellulose oligosaccharide hydrolase genes of the present disclosure encode proteins that allow lignocellulolytic cells to metabolize hemicellulose oligosaccharides to produce hemicellulose monosaccharides. Suitable hemicellulose oligosaccharide hydrolase genes of the present disclosure include, without limitation, genes necessary for

lignocellulolytic cells to hydrolyze hemicellulose oligosaccharides to monosaccharides, such as xylose and arabinose. In certain preferred embodiments, hemicellulose oligosaccharide hydrolase genes include, without limitation, xylosidase genes, β-xylosidase genes, β-arabinosidase genes, etc.

Alcohol dehydrogenase genes

[0088] Lignocellulolytic cells of the present disclosure may further have reduced expression of one or more alcohol dehydrogenase genes, by containing a mutation in one or more alcohol dehydrogenase genes. Mutations in one or more alcohol dehydrogenase genes that reduce expression of the genes or that delete the genes result in lignocellulolytic cells that do not utilize (i.e., consume) ethanol.

[0089] Alcohol dehydrogenase genes of the present disclosure encode proteins that allow lignocellulolytic cells to metabolize alcohol, such as ethanol. Suitable alcohol dehydrogenase genes of the present disclosure include, without limitation, adhl genes, such as the N. crassa adhl gene NCU01754, homologs thereof, orthologs thereof, and paralogs thereof; and adh3 genes, such as the N. crassa adh3 gene NCU02476, homologs thereof, orthologs thereof, and paralogs thereof.

Methods of reducing gene expression

[0090] Certain aspects of the present disclosure relate to reducing the expression of one more oligosaccharide utilization genes in lignocellulolytic cells of the present disclosure. Oligosaccharide utilization genes of the present disclosure include, without limitation, β- glucosidase genes, β-galactosidase genes, β-mannosidase genes, hemicellulose

oligosaccharide hydrolase genes, xylose utilization genes, and xylosidase genes. The expression of oligosaccharide utilization genes found in lignocellulolytic cells can be reduced by any method known to those of skill in the art.

[0091] In some embodiments reduced expression of oligosaccharide utilization genes is achieved by, for example, promoter modification or RNAi.

[0092] In other embodiments, reduced expression of oligosaccharide utilization genes is achieved by modifying the oligosaccharide utilization genes. Examples of such

modifications include, without limitation, a deletion mutation, a knock-out mutation, a truncation mutation, a point mutation, a missense mutation, a substitution mutation, a frameshift mutation, an insertion mutation, a duplication mutation, an amplification mutation, a translocation mutation, or an inversion mutation. Methods of generating at least one mutation in one or more oligosaccharide utilization genes are well known in the art and include, without limitation, random mutagenesis and screening, site-directed mutagenesis, PCR mutagenesis, insertional mutagenesis, chemical mutagenesis, and irradiation.

[0093] In certain embodiments, a portion of the oligosaccharide utilization gene is modified, such as the region encoding the catalytic domain, the coding region, or a control sequence required for expression of the coding region. Such a control sequence of the gene may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the gene. For example, a promoter sequence may be inactivated resulting in no expression or a weaker promoter may be substituted for the native promoter sequence to reduce expression of the coding sequence. Other control sequences for possible modification include, without limitation, a leader sequence, a propeptide sequence, a signal sequence, a transcription terminator, and a transcriptional activator.

[0094] Oligosaccharide utilization genes of the present disclosure that are present in lignocellulolytic cells may also be modified by utilizing gene deletion techniques to eliminate or reduce expression of the gene. Gene deletion techniques enable the partial or complete removal of the gene thereby eliminating their expression. In such methods, deletion of the gene may be accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5' and 3' regions flanking the gene.

[0095] Oligosaccharide utilization genes of the present disclosure that are present in lignocellulolytic cells may also be modified by introducing, substituting, and/or removing one or more nucleotides in the gene, or a control sequence thereof required for the transcription or translation of the gene. For example, nucleotides may be inserted or removed for the introduction of a stop codon, the removal of the start codon, or a frame-shift of the open reading frame. Such a modification may be accomplished by methods known in the art, including without limitation, site-directed mutagenesis and peR generated mutagenesis (see, for example, Botstein and Shortie, 1985, Science 229: 4719; Lo et al., 1985, Proceedings of the National Academy of Sciences USA 81: 2285; Higuchi et al., 1988, Nucleic Acids

Research 16: 7351; Shimada, 1996, Meth. Mol. Biol 57: 157; Ho et al., 1989, Gene 77: 61; Horton et al., 1989, Gene 77: 61; and Sarkar and Sommer, 1990, BioTechniques 8: 404). [0096] Additionally, oligosaccharide utilization genes of the present disclosure that are present in lignocellulolytic cells may be modified by gene disruption techniques by inserting into the gene a disruptive nucleic acid construct containing a nucleic acid fragment homologous to the gene that will create a duplication of the region of homology and incorporate construct DNA between the duplicated regions. Such a gene disruption can eliminate gene expression if the inserted construct separates the promoter of the gene from the coding region or interrupts the coding sequence such that a nonfunctional gene product results. A disrupting construct may be simply a selectable marker gene accompanied by 5' and 3' regions homologous to the gene. The selectable marker enables identification of transformants containing the disrupted gene.

[0097] Oligosaccharide utilization genes of the present disclosure that are present in lignocellulolytic cells may also be modified by the process of gene conversion (see, for example, Iglesias and Trautner, 1983, Molecular General Genetics 189:5 73-76). For example, in the gene conversion a nucleotide sequence corresponding to the gene is mutagenized in vitro to produce a defective nucleotide sequence, which is then transformed into a lignocellulolytic cell of the present disclosure to produce a defective gene. By homologous recombination, the defective nucleotide sequence replaces the endogenous gene. It may be desirable that the defective nucleotide sequence also contains a marker for selection of transformants containing the defective gene.

[0098] Oligosaccharide utilization genes of the present disclosure that are present in lignocellulolytic cells may also be modified by established anti-sense techniques using a nucleotide sequence complementary to the nucleotide sequence of the gene (see, for example, Parish and Stoker, 1997, FEMS Microbiology Letters 154: 151-157). In particular, expression of the gene by lignocellulolytic cells may be reduced or inactivated by introducing a nucleotide sequence complementary to the nucleotide sequence of the gene, which may be transcribed in the cells and is capable of hybridizing to the mRNA produced in the cells. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated.

[0099] In addition, oligosaccharide utilization genes of the present disclosure that are present in lignocellulolytic cells may also be modified by established RNA interference (RNAi) techniques (see, for example, WO 2005/056772 and WO 2008/080017). [0100] Oligosaccharide utilization genes of the present disclosure that are present in lignocellulolytic cells may also be modified by random or specific mutagenesis using methods well known in the art, including without limitation, chemical mutagenesis (see, for example, Hopwood, The Isolation of Mutants in Methods in Microbiology (J.R. Norris and D.W. Ribbons, eds.) pp. 363-433, Academic Press, New York, 25 1970). Modification of the gene may be performed by subjecting lignocellulolytic cells to mutagenesis and screening for mutant cells in which expression of the gene has been reduced or inactivated. The

mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide, subjecting the DNA sequence to peR generated mutagenesis, or any combination thereof. Examples of physical and chemical mutagenizing agents include, without limitation, ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosogaunidine (NTG) O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the lignocellulolytic cells to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and then selecting for mutants exhibiting reduced or no expression of the gene.

[0101] Accordingly, in certain embodiments, lignocellulolytic cells of the present disclosure may contain a modification, such as a mutation, in one or more, two or more, three or more, four or more, five or more, six or more, or seven β-glucosidase genes. In certain embodiments, the β-glucosidase genes include extracellular β-glucosidase genes. In other embodiments, the β-glucosidase genes include intracellular β-glucosidase genes.

[0102] Advantageously, lignocellulolytic cells of the present disclosure containing a modification, such as a mutation, in one or more β-glucosidase genes have higher cellulase activity as compared to a corresponding cell that does not contain a mutation in one or more β-glucosidase genes. Accordingly, in certain embodiments, lignocellulolytic cells of the present disclosure containing a mutation in one or more β-glucosidase genes have a level of cellulase activity that is at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, at least 12 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 21 times, at least 22 times, at least 23 times, at least 25 times, or more higher than the level of activity of a corresponding lignocellulolytic cells that do not contain a modification, such as a mutation, in one or more β-glucosidase genes.

[0103] Lignocellulolytic cells of the present disclosure may also contain a modification, such as a mutation, in one or more additional hexose hydrolase genes. Examples of hexose hydrolase genes that may contain a mutation include, without limitation, the β-galactosidase gene NCU05956, the β-galactosidase gene NCU00810, the β-mannosidase gene NUC00130, homologs thereof, orthologs thereof, and paralogs thereof.

[0104] Lignocellulolytic cells of the present disclosure may further contain a

modification, such as a mutation, in at least one hemicellulose oligosaccharide hydrolase gene. In certain embodiments, the hemicellulose oligosaccharide hydrolase gene is a xylosidase gene.

[0105] Lignocellulolytic cells of the present disclosure may further contain a

modification, such as a mutation, in at least one or more genes that code for transcription factors, etc. In certain embodiments, the transcription factor is selected from ACRE1, CRE1, and combinations thereof.

Cellulosomes

[0106] Further aspects of the present disclosure relate to lignocellulolytic cells that further contain a recombinant cellulosome, which allows the lignocellulolytic cells to utilize less carbon source for growth and enzyme production.

[0107] As used herein, a "cellulosome" refers to a complex of cellulolytic enzymes created by bacteria such as Clostridium. Cellulosomes may contain catalytic subunits such as glycoside hydrolases, polysaccharide lyases, and carboxyl esterases bound together by scaffoldins that consist of cohesins connected to other functional units such as the enzymes and carbohydrate binding modules via dockerins. As disclosed herein, cellulosomes are much more efficient than cellulases at degrading cellulose.

[0108] Accordingly, lignocellulolytic cells containing a recombinant cellulosome consume less sugar for growth and enzyme production while maintaining a high rate of cellulose degradation. Methods of expressing recombinant cellulosomes in lignocellulolytic cells are well known in the art, and include expressing cellulosome components under the control of a cellulose gene promoter. In certain embodiments, lignocellulolytic cells are engineered such that the recombinant cellulosome is assembled on the surface of the cell by linking the scaffidin component of the cellulosome to a protein which binds to the surface of the lignocellulolytic cell.

Fermentative Microorganisms

[0109] Certain aspects of the present disclosure relate to a co-culture composition containing a population of fermentative microorganisms. As disclosed herein, the

fermentative microorganisms may be either aerobic microorganisms or anaerobic

microorganisms. In certain preferred embodiments, the fermentative microorganisms are anaerobic microorganisms.

[0110] Fermentative microorganisms of the present disclosure are capable of efficiently converting oligosaccharides (e.g., cellodextrins and xylobiose) to commodity chemicals under aerobic (i.e., ATP-rich) or anaerobic (i.e., ATP-scarce) conditions.

[0111] Fermentative microorganisms of the present disclosure preferentially utilize cellulosic oligosaccharides (e.g., cellodextrin and cellobiose) and hemicellulose

oligosaccharides (e.g., xylobiose) to produce commodity chemicals. Additionally, fermentative microorganisms of the present disclosure can also utilize hemicellulose oligosaccharides, such as xylose, to produce commodity chemicals.

[0112] In certain embodiments fermentative microorganisms of the present disclosure are co-cultured with lingocellulolytic cells of the present disclosure under aerobic conditions. In such embodiments, a local micro-aerobic or anaerobic environment may be created for the fermentative microorganisms to efficiently convert the oligosaccharides produce by the lingocellulolytic cells into commodity chemicals. A local micro-aerobic environment may be created in the co-culture by immobilizing the fermentative microorganisms. The

fermentative microorganisms may be immobilized utilizing cell immobilization technology to entrap the fermentative microorganisms inside a gel matrix to create a mass transfer barrier to oxygen so that the fermentative microorganisms will have a local micro-aerobic environment. In certain embodiments, the gel matrix is composed of gel beads, and the fermentative microorganisms are immobilized within the beads. Additionally, a local anaerobic environment may be created in the co-culture by co-immobilizing the lignocellulolytic cells and the fermentative microorganisms on gel beads. Co-immobilization may be achieved by immobilizing the lignocellulolytic cells in the outer layer of a gel bead and the fermentative microorganisms in the core of the gel bead.

[0113] Fermentative microorganisms of the present disclosure may be genetically modified by modifying one or more genes. Methods of generating one or more gene mutations are well known in the art and include, without limitation, any of the methods disclosed herein.

[0114] Fermentative microorganisms of the present disclosure include, without limitation, bacteria, fungi, and yeast.

[0115] Examples of suitable fermentative bacteria include, without limitation, E. coli, Bacillus subtilis, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum (Moorella thermoacetica), Thermoanaerobacterium saccharolyticum, Klebsiella oxytoca,

Carboxydocella sp., Corynebacterium glutamicum, Enterobacteriaceae, Erwinia

chrysanthemi, Lactobacillus sp., Pediococcus acidilactici, Rhodopseudomonas capsulata, Streptococcus lactis, Vibrio furnissii, Vibrio furnissii Ml, Caldicellulosiruptor

saccharolyticus, and Xanthomonas campestris. Additional examples of fermentative bacteria include, without limitation, those species assigned to the Escherichia, Enterobacter,

Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Synechococcus, Synechocystis, and Paracoccus taxonomical classes. Suitable fermentative bacteria may also include cyanobacteria. In certain preferred embodiments, the fermentative microorganism is Klebsiella oxytoca.

[0116] Examples of suitable fermentative fungi include, without limitation, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis, Fusarium

crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum,

Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium

sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Scytalidium thermophilum, Sporotrichum thermophile, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride. Additional examples of suitable fermentative fungi include, without limitation, those fungal species assigned to the Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, and Trichoderma taxonomical classes.

[0117] Examples of suitable fermentative yeast include, without limitation,

Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis,

Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus,

Saccharomyces pombe, Saccharomyces oviformis, Kluyveromyces lactis, Kluyveromyces fragilis, Kluyveromyces marxiamus, Pichia stipitis, Candida shehatae, Candida tropicalis, Yarrowia lipolytica, Brettanomyces custersii, and Zygosaccharomyces roux. Additional examples of suitable fermentative yeast include, without limitation, those yeast species assigned to the Candida, Hansenula, Issatchenkia, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia taxonomical classes.

Cellodextrin transporters

[0118] Fermentative microorganisms of the present disclosure may contain at least one cellodextrin transporter that allows the microorganisms to transport cellodextrins, such as cellobiose into the cells where the cellodextrins can be utilized by the microorganisms to produce commodity chemicals.

[0119] A cellodextrin transporter is any transmembrane protein that transports a cellodextrin molecule from outside of the cell to the inside of the cell and/or from inside of the cell to outside of the cell. Cellodextrin transporters have been described in US

2011/0020910, which is herein incorporated by reference in its entirety. Examples of suitable cellodextrin transporters include, without limitation, NCU00801, NCU00809, NCU8114, XP_001268541.1, and LAC2. [0120] Fermentative microorganisms of the present disclosure may contain an endogenous cellodextrin transporter or a recombinant cellodextrin transporter. In

embodiments where the fermentative microorganism contains an endogenous cellodextrin transporter, the microorganism may be engineered to contain at least one additional recombinant cellodextrin transporter. In embodiments where the fermentative microorganism does not contain an endogenous cellodextrin transporter, the microorganism is engineered to contain a recombinant cellodextrin transporter. Preferably, the cellodextrin transporter is a cellobiose transporter.

Intracellular β-glucosidases and cellobiose phosphorylases

[0121] Fermentative microorganisms of the present disclosure may also contain at least one intracellular β-glucosidase that allows the microorganisms to convert cellodextrins, such as cellobiose, to glucose, which can be utilized by the microorganisms to produce commodity chemicals.

[0122] Fermentative microorganisms of the present disclosure may contain any intracellular β-glucosidase of the present disclosure, including without limitation, those encoded by NCU00130, NCU05577, NCU07487, NCU08054, homologs thereof, orthologs thereof, and paralogs thereof.

[0123] Alternatively, fermentative microorganisms of the present disclosure may contain at least one intracellular cellobiose phosphorylase rather than an intracellular

β-glucosidase. The cellobiose phosphorylase also allows the microorganisms to convert cellodextrins, such as cellobiose, to glucose, which can be utilized by the microorganisms to produce commodity chemicals.

[0124] When fermentative microorganisms contain an intracellular β-glucosidase, the β-glucosidase hydrolyzes the cellodextrins to form glucose. The resulting glucose must be phosphorylated using ATP as a phosphate donor in order to be used by the microorganism. An alternative mechanism for utilizing cellodextrins may instead rely on cellobiose phosphorylases (EC2.4.1.20). These enzymes use phosphate to cleave the

beta-glucosidic linkage between glucose moieties in cellodextrins. The phosphorolysis reaction saves 1 ATP equivalent per cleavage reaction as compared to β-glucosidase. [0125] Fermentative microorganisms of the present disclosure may contain an

endogenous β-glucosidase or cellobiose phosphorylase, or a recombinant β-glucosidase or cellobiose phosphorylase. In embodiments where the fermentative microorganism contains an endogenous β-glucosidase or cellobiose phosphorylase, the microorganism may be engineered to contain at least one additional recombinant β-glucosidase or cellobiose phosphorylase. In embodiments where the fermentative microorganism does not contain an endogenous β-glucosidase or cellobiose phosphorylase, the microorganism is engineered to contain a recombinant β-glucosidase or cellobiose phosphorylase.

Monosaccharide, sugar alcohol, and sugar aldonic acid transporters

[0126] Fermentative microorganisms of the present disclosure may also have reduced expression of one or more monosaccharide, sugar alcohol, and sugar aldonic acid transporter genes, by containing a modification, such a mutation, in one or more monosaccharide, sugar alcohol, and sugar aldonic acid transporter genes. Mutations in one or more monosaccharide, sugar alcohol, and sugar aldonic acid transporter genes that reduce expression of the genes or that delete the genes result in fermentative microorganisms that preferentially transport oligosaccharides, such as cellobiose, into the cell for utilization. Advantageously, fermentative microorganisms with reduced expression of one or more monosaccharide, sugar alcohol, and sugar aldonic acid transporter genes do not compete with lignocellulolytic cells of the present disclosure for monosaccharides, such as glucose, in a mixed culture.

[0127] A monosaccharide transporter is any transmembrane protein that transports a monosaccharide molecule from outside of the cell to the inside of the cell and/or from inside of the cell to outside of the cell.

[0128] Examples of suitable monosaccharide transporter genes include, without limitation, hexose transporter genes, pentose transporter genes, glucose transporter genes, xylose transporter genes, galactose transporter genes, mannose transporter genes, arabinose transporter genes, fructose transporter genes, and combinations thereof.

[0129] Oligosaccharide transporters are also capable of transporting monosaccharides. Accordingly, in certain embodiments fermentative microorganisms of the present disclosure may have reduced expression of one or more oligosaccharide transporters genes, such as maltose transporter genes and lactose transporter genes. [0130] A sugar alcohol transporter is any transmembrane protein that transports a sugar alcohol molecule from outside of the cell to the inside of the cell and/or from inside of the cell to outside of the cell. Examples of suitable sugar alcohol transporter genes include, without limitation, sorbitol transporter genes.

[0131] A sugar aldonic acid transporter is any transmembrane protein that transports a sugar aldonic acid molecule from outside of the cell to the inside of the cell and/or from inside of the cell to outside of the cell. Examples of suitable sugar aldonic acid transporter genes include, without limitation, gluconic acid transporter genes.

[0132] Expression of monosaccharide, sugar alcohol, and sugar aldonic acid transporter genes found in fermentative microorganisms can be reduced by any method known to those of skill in the art and disclosed herein, including without limitation, gene disruption, a deletion mutation, a knock-out mutation, a truncation mutation, a point mutation, a missense mutation, a substitution mutation, a frameshift mutation, an insertion mutation, a duplication mutation, an amplification mutation, a translocation mutation, or an inversion mutation, promoter modification, and RNAi. In certain preferred embodiments, one or more hexose transporter genes contain a mutation that results in a partial deletion or a complete deletion of the gene.

Oligosaccharide utilization polypeptides

[0133] Fermentative microorganisms of the present disclosure may further contain at least one recombinant oligosaccharide utilization polypeptide that allows the fermentative microorganisms to utilize oligosaccharides, such as the hemicellulosic xylobiose produced by lignocellulolytic cells of the present disclosure.

[0134] Suitable oligosaccharide utilization polypeptides include, without limitation, polypeptides necessary for fermentative microorganisms to utilize oligosaccharides.

Examples include, without limitation, cellodextrin utilization polypeptides, cellobiose utilization polypeptides, xylodextrin utilization polypeptides, xylobiose utilization polypeptides, mannobiose utilization polypeptides, galactobiose utilization polypeptides, maltose utilization polypeptides, lactose utilization polypeptides, and combinations thereof. [0135] In certain preferred embodiments, the at least one recombinant oligosaccharide utilization polypeptide is a cellodextrin transporter, a cellobiose transporter, a xylodextrin transporter, a xylobiose transporter, a mannobiose transporter, a galactobiose transporter, a maltose transporter, a lactose transporter, and combinations thereof.

Commodity chemical production

[0136] Fermentative microorganisms of the present disclosure can produce commodity chemicals from the oligosaccharides produced by lignocellulolytic cells of the present disclosure.

[0137] Commodity chemicals include, without limitation, any saleable or marketable chemical that can be produced either directly or as a by-product of the fermentative microorganisms of the present disclosure. Examples of commodity chemicals include, without limitation, biofuels, polymers, specialty chemicals, and pharmaceutical

intermediates. Biofuels include, without limitation, alcohols such as ethanol, propanol, isopropanol, acetone, butanol, isobutanol, 2-methyl-l-butanol, 3-methyl-l-butanol, phenylethanol, fatty alcohols, and isopentenol; aldehydes, such as acetylaldehyde, propionaldehyde, butryaldehyde, isobutyraldehyde, 2-methyl-l-butanal, 3-methyl-l-butanal, phenylacetaldehyde, and fatty aldehydes; hydrocarbons, such as alkanes, alkenes,

isoprenoids, fatty acids, wax esters, and ethyl esters; and inorganic fuels such as hydrogen. Polymers include, without limitation, 1,3-propandiol, 1,4-butanediol, polyhydroxyalkanoate, polyhydroxybutyrate, and isoprene. Specialty chemicals include, without limitation, carotenoids, such as lycopene, β-carotene, etc. Pharmaceutical intermediates include, without limitation, polyketides, statins, omega-3 fatty acids, isoprenoids, steroids, and erythromycin (antibiotic). Further examples of commodity chemicals include, without limitation, lactate, succinate, glutamate, citrate, malate, 3-hydroxypropionate, ascorbate, sorbitol, amino acids (leucine, valine, isoleucine, etc.), and hydroxybutyrate.

[0138] In some embodiments, fermentative microorganisms of the present disclosure naturally produce any of the precursors for the production of the desired commodity chemical. These genes encoding the desired enzymes may be heterologous to the

fermentative microorganism, or these genes may be endogenous to the fermentative microorganism but are operatively linked to heterologous promoters and/or control regions which result in higher expression of the gene(s) in the fermentative microorganism. For example, in certain embodiments, fermentative microorganisms of the present disclosure may be further modified to contain one or more modifications sufficient for the fermentative microorganisms to produce a commodity chemical from one or more oligosaccharides. The one or more modifications may be either a deletion of one or more genes, or the recombinant expression of one or more genes.

[0139] In other embodiments, fermentative microorganisms of the present disclosure do not naturally produce the desired commodity chemical, and thus contains heterologous polynucleotide constructs capable of expressing one or more genes necessary for producing the desired commodity chemical. In one non-limiting example, fermentative microorganisms of the present disclosure may be modified to produce isobutanol by recombinantly expressing an isobutanol metabolic pathway, and by deleting the genes coding for alcohol- aldehyde dehydrogenase, phosphotransacetylase, isocitrate dehydrogenase, fumarate reductase, and pyruvate formate lyase to divert intermediates into the isobutanol production pathway.

Fatty Acid-Producing and Hydrocarbon-Producing Microorganisms

[0140] Further aspects of the present disclosure relate to a co-culture composition that further contains a population of fatty acid-producing and/or hydrocarbon-producing microorganisms. Produced fatty acids include, without limitation, saturated fatty acids and unsaturated fatty acids having chain lengths that range from 6 to 50. Produced hydrocarbons include, without limitation, saturated hydrocarbons and unsaturated hydrocarbons having chain lengths that range from 6 1 100. Suitable fatty acid-producing and hydrocarbon- producing microorganisms include, without limitation, cyanobacteria, algae, yeast, fungi, and other bacteria. In certain embodiments, fatty acid-producing and hydrocarbon-producing microorganisms include, without limitation, Thalassiosira pseudonana, Cyclotella cryptica, Cylindrothecafusiformis, Mucor circinelloides, and Mortierella isabellina.

[0141] Fatty acid-producing and hydrocarbon-producing microorganisms of the present disclosure are capable of efficiently producing fatty acids and hydrocarbons from the oligosaccharides, such as cellodextrins and hemicellulose oligosaccharides, generated by lignocellulolytic cells of the present disclosure under either aerobic (i.e., ATP-rich) conditions or anaerobic (i.e., ATP-scarce) conditions. Methods of Producing and Co-Culturing Microorganisms of the Present Disclosure

[0142] Other aspects of the present disclosure relate to the production of genetically modified lignocellulolytic cells and fermentative organisms, and to co-culture compositions containing populations of such microorganisms.

[0143] Methods of producing and culturing microorganisms of the present disclosure may include the introduction or transfer of expression vectors containing recombinant

polynucleotides into the host cell. Such methods for transferring expression vectors into microorganisms are well known to those of ordinary skill in the art. For example, one method for transforming E. coli with an expression vector involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate. Other salts, e.g., calcium phosphate, may also be used following a similar procedure. In addition, electroporation (i.e., the application of current to increase the permeability of cells to nucleic acid sequences) may be used to transfect the host cell. Also, microinjection of the nucleic acid sequences provides the ability to transfect host cells. Other means, such as lipid complexes, liposomes, and dendrimers, may also be employed. Those of ordinary skill in the art can transfect a host cell with a desired sequence using these or other methods.

[0144] The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host, or a transposon may be used.

[0145] The vectors preferably contain one or more selectable markers which permit easy selection of transformed hosts. A selectable marker is a gene the product of which provides, for example, biocide or viral resistance, resistance to heavy metals, prototrophy to

auxotrophs, and the like. Selection of bacterial microorganisms may be based upon antimicrobial resistance that has been conferred by genes such as the amp, gpt, neo, and hyg genes. [0146] Suitable markers for yeast microorganisms are, for example, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal microorganism include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyl transferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in Aspergillus are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus. Preferred for use in Trichoderma are bar and amdS.

[0147] The vectors preferably contain an element(s) that permits integration of the vector into the microorganism' s genome or autonomous replication of the vector in the

microorganism independent of the genome.

[0148] For integration into the host genome, the vector may rely on the gene's sequence or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host. The additional nucleotide sequences enable the vector to be integrated into the host genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of

homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host by non-homologous recombination.

[0149] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host in question. The origin of replication may be any plasmid replicator mediating autonomous replication which functions in a cell. The term "origin of replication" or "plasmid replicator" is defined herein as a sequence that enables a plasmid or vector to replicate in vivo. Examples of origins of replication for use in yeast are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. Examples of origins of replication useful in a filamentous fungus are AMA1 and ANSI (Gems et al., 1991; Cullen et al., 1987; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

[0150] For other microorganisms, transformation procedures may be found, for example, in Jeremiah D. Read, et al., Applied and Environmental Microbiology, Aug. 2007, p. 5088- 5096, for Kluyvewmyces; in Osvaldo Delgado, et al., FEMS Microbiology Letters 132, 1995, 23-26, for Zymomonas; in U.S. Pat, No. 7,50L275 for Pichia stipitis and in WO

2008/040387 for Clostridium.

[0151] More than one copy of a gene may be inserted into the microorganism to increase production of the gene product. An increase in the copy number of the gene can be obtained by integrating at least one additional copy of the gene into the host genome or by including an amplifiable selectable marker gene with the nucleotide sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the gene, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0152] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present disclosure are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0153] The microorganism is transformed with at least one expression vector. When only a single expression vector is used (without the addition of an intermediate), the vector will contain all of the nucleic acid sequences necessary.

[0154] Once the microorganism has been transformed with the expression vector, the microorganism is allowed to grow. Methods of the present disclosure may include culturing the microorganism such that recombinant nucleic acids in the microorganism are expressed. For microorganisms, this process entails culturing the microorganisms in a suitable medium. Typically cells are grown at 35°C in appropriate media. Preferred growth media in the present disclosure include media that is compatible to both the lignocellulolytic microorganisms and the fermentative microorganisms, for example, common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth Yeast medium (YM) broth, or Vogel's minimal medium. Other defined or synthetic growth media may also be used and the appropriate medium for the co-culture of the particular

lignocellulolytic microorganisms and fermentative microorganisms will be known by someone skilled in the art of microbiology or fermentation science. Temperature ranges, pH ranges, salt concentrations, and other conditions suitable for co-culture growth are known in the art (see, e.g., Bailey and Ollis 1986).

[0155] According to some aspects of the present disclosure, the co-culture media contains lignocellulosic biomass that is processed by the lignocellulolytic microorganisms to produce a carbon source for both the lignocellulolytic microorganisms themselves and the

fermentative microorganisms. Such a "carbon source" generally refers to a substrate or compound suitable to be used as a source of carbon for cell growth. Carbon sources can be in various forms, including, without, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, etc. These include, for example, various monosaccharides such as glucose, oligosaccharides such as cellodextrins, polysaccharides, a biomass polymer such as cellulose or hemicellulose, xylose, arabinose, disaccharides, such as sucrose, saturated or unsaturated fatty acids, succinate, lactate, acetate, ethanol, etc., or mixtures thereof.

[0156] Lignocellulosic biomass is composed of cellulose, hemicellulose, and lignin. In some embodiments, the carbon source is a biomass polymer such as cellulose or

hemicellulose. A "biomass polymer" as described herein is any polymer contained in biological material. The biological material may be living or dead. A biomass polymer includes, for example, cellulose, xylan, xylose, hemicellulose, lignin, mannan, and other materials commonly found in biomass. Non-limiting examples of lignocellulosic biomass include agricultural crops, agricultural residues, livestock solid waste, industrial solid waste, human sewage, yard waste, wood and forestry waste, Miscanthus, energy grass, elephant grass, switchgrass, cord grass, rye grass, reed canary grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, oat spelt, sorghum, rice hulls, sugarcane bagasse, corn fiber, barley, oats, flax, wheat, linseed, citrus pulp, cottonseed, groundnut, rapeseed, sunflower, peas, lupines, palm kernel, coconut, konjac, locust bean gum, gum guar, soy beans, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine, conifer softwood, eucalyptus, birchwood, willow, aspen, poplar wood, hybrid poplar, energy cane, short-rotation woody crop, crop residue, yard waste, and sludge from paper manufacture.

[0157] In addition to an appropriate carbon source, co-culture media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the co-cultures and promotion of the enzymatic pathways necessary for the production of hydrolytic enzymes in the lignocellulolytic microorganisms, and for the fermentation of various sugars and the production of commodity chemicals in the fermentative microorganisms.

Methods for the Production of Commodity Chemicals

[0158] Further aspects of the present disclosure relate to methods for the production of a commodity chemical from lignocellulosic biomass. In one aspect, the present disclosure provides a method for the production of a commodity chemical from lignocellulosic biomass, by providing a fermentation broth containing any of the co-culture compositions of the present disclosure, where the co-culture composition contains a first population of lignocellulolytic cells of the present disclosure, where the cells have reduced expression of one or more β-glucosidase genes, and a second population of fermentative microorganisms; contacting lignocellulosic biomass with the fermentation broth; and incubating the fermentation broth and lignocellulosic biomass under conditions sufficient for the co-culture composition to produce a commodity chemical from the lignocellulosic biomass. In certain embodiments, the lignocellulolytic cells contain a mutation in the one or more β-glucosidase genes, where the mutation reduces expression of the one or more β-glucosidase genes.

[0159] Incubation conditions sufficient for the co-culture composition to produce a commodity chemical are known in the art and include any suitable co-culturing and fermentation conditions disclosed herein. In some embodiments of the methods for producing commodity chemicals, the co-culture composition is incubated under aerobic conditions. In other embodiments, oxygen is supplied to the fermentation broth to achieve the aerobic conditions. In still other embodiments, the fermentative microorganisms are incubated under a local anaerobic or micro-aerobic environment. In certain preferred embodiments, the local anaerobic or micro-aerobic environment is produced by immobilizing the fermentative microorganisms. Preferably, the fermentative microorganisms are immobilized within gel beads. [0160] In some embodiments of the methods for producing commodity chemicals, the lignocellulolytic cells produce glucose and cellodextrin from the lignocellulosic biomass. Preferably, the cellodextrin is cellobiose. In other embodiments, the lignocellulolytic cells utilize the produced glucose as a carbon source. In still other embodiments, the fermentative microorganisms utilize the cellodextrin to produce the commodity chemical. In yet other embodiments, the lignocellulolytic cells further produce at least one hemicellulose oligosaccharide from the lignocellulosic biomass. In some embodiments, the at least one hemicellulose oligosaccharide is xylobiose. In other embodiments, the fermentative microorganisms further utilize the xylobiose to produce the commodity chemical.

[0161] In certain embodiments of the methods for producing commodity chemicals, the commodity chemical is extracted from the fermentation broth by gas stripping. In certain preferred embodiments, air or oxygen is utilized to gas strip the commodity chemical.

Advantageously, air or oxygen supplied to the fermentation broth to generate an aerobic environment may also be used to strip out volatile commodity chemicals, such as ethanol and isobutanol, which are produced by the fermentative microorganisms. In further

embodiments, the commodity chemical is a biofuel selected from alcohol, ethanol, propanol, isopropanol, acetone, butanol, isobutanol, 2-methyl-l-butanol, 3-methyl-l-butanol, phenylethanol, a fatty alcohol, isopentenol, an aldehyde, acetylaldehyde, propionaldehyde, butryaldehyde, isobutyraldehyde, 2-methyl-l-butanal, 3-methyl-l-butanal,

[0162] It is to be understood that, while the present disclosure has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

[0163] The following examples are offered to illustrate provided embodiments and are not intended to limit the scope of the present disclosure. EXAMPLES

Introduction

Background

[0164] Lignocellulosic biomass is a sustainable source for organic fuels, chemicals, and materials that is available at low cost and in large abundance. The central obstacle impeding the widespread utilization of lignocellulosic biomass is the absence of a low-cost processing technology. Production of biofuels from cellulosic biomass featuring enzymatic hydrolysis involves six key steps: (1) pretreatment; (2) cellulase production; (3) enzymatic hydrolysis; (4) hexose fermentation; (5) pentose fermentation; and (6) product recovery (Column 1 of Figure 4).

[0165] One of the strategies to lower the overall process cost involves process consolidation. Columns 1 to 4 of Figure 4 present the evolution of biomass processing configurations featuring enzymatic hydrolysis over the past thirty years. When pretreatment, cellulose hydrolysis, hexose fermentation, pentose fermentation, and product recovery take place in different reactors; the process is called Separate Hydrolysis and Fermentation (SHF). Simultaneous saccharification and fermentation (SSF) consolidates hexose fermentation and enzymatic hydrolysis into one reactor. Simultaneous saccharification and co-fermentation (SSCF) consolidates enzymatic hydrolysis, hexose fermentation, and pentose fermentation into one step by engineering the fermentative microorganism to co-ferment both pentose and hexose sugars. Consolidated bioprocessing (CBP) features the combination of cellulase production, cellulose hydrolysis, hexose fermentation, and pentose fermentation in a single process.

[0166] Disclosed herein, are two novel configurations for biofuel and chemical production from lignocellulosic biomass that overcome the challenges of SSF, SSCF, and CBP. One configuration is an integrated bioprocessing and separation (IBS) process that can consolidate cellulase production, enzymatic hydrolysis, pentose fermentation, hexose fermentation, and product recovery into one single step (Column 5 of Figure 4). A second configuration is a one-step bioconversion and separation (OBS) process that integrates lignocellulosic pretreatment into the IBS process (Column 6 of Figure 4). In OBS, lignocellulolytic microorganisms are engineered to efficiently degrade lignin by producing lignin-solubilizing enzymes. The IBS and OBS configurations have higher levels of consolidation than CBP. Thus, IBS and OBS have the potential to achieve even greater reduction in processing costs.

[0167] As shown in Column 5 of Figure 4, the novel IBS process combines the same four steps that CBP consolidates, but in under aerobic rather than anaerobic conditions. In addition, the product separation step can also be combined with the four steps of CBP. The IBS configuration can be achieved by a co-culture composed of a lignocellulolytic microorganism, such as a lignocellulolytic fungus, and a fermentative microorganism, such as a bacterium or yeast. The lignocellulolytic fungus produces hydrolysis enzymes under ATP- plentiful conditions in an aerobic environment. The hydrolysis enzymes produced by the fungus hydrolyze the cellulose and hemicellulose of lignocellulosic biomass resulting in hydrolysis products. The fermentative microorganism then consumes the hydrolysis products for biofuel and chemical production to realize the benefits of simultaneous saccharification and fermentation.

[0168] In the IBS process, the lignocellulolytic microorganism is engineered to divert most of the hydrolysis product to the fermentative microorganism for biofuel and chemical production, while maintaining a robust co-culture. However, most fermentative

microorganisms do not produce reduced biofuel products at high yields under aerobic conditions. Accordingly, the fermentative microorganism is immobilized utilizing cell immobilization technology to entrap the fermentative microorganism inside a gel matrix to create a mass transfer barrier to oxygen so that the fermentative microorganism will have a local micro-aerobic and/or anaerobic environment for biofuel and chemical production. It has been previously shown that oxygen transfer to cells entrapped inside gel beads is very poor. Oxygen can penetrate only a very narrow outer shell of beads. Thus, a locally micro- aerobic environment can be maintained for most of the cells entrapped inside the beads by optimizing the cell-immobilization conditions. Additionally, a complete anaerobic

environment in an aerobic culture can be achieved by co -immobilizing the aerobic lignocellulolytic microorganism and a strict anaerobic fermentative microorganism. Co- immobilization is achieved by immobilizing the aerobic microorganism in the outer layer of a gel bead, and the anaerobic microorganism in the core of the gel bead. An added advantage of such a configuration is that the air supplied to the system for aeration can also strip out volatile biofuel products, such as ethanol and isobutanol, produced by the fermentative organism. Thus, the fermentation product will not accumulate to high concentrations that are inhibitory to both the lignocellulolytic microorganism and to the fermentative

microorganism.

[0169] In the OBS process, the lignocellulosic pretreatment step is further consolidated into the IBS process to achieve by utilizing or engineering a lignocellulolytic microorganism to produce lignin-solubilizing enzymes efficiently so that they can hydrolyze cellulose and hemicellulose in the presence of lignin. Advantageously, lignin degradation is an aerobic process that is fully compatible with the IBS process.

[0170] One challenge of the IBS and OBS configurations is maintaining a proper population of lignocellulolytic and fermentative microorganisms such that cellulose and hemicellulose hydrolysis proceeds at a reasonable rate and that the majority of carbon flows to biofuel and chemical production. It has been previously shown that the co-culture of a free aerobic amylase-producing fungus, Aspergillus awamori, and a facultative anaerobe, Saccharomyces cerevisiae, immobilized inside of gel beads, and the co-immobilization of the aerobic A. awamori and a strict anaerobe, Zymomonas mobilis, inside of gel beads were used for direct ethanol production from starch. However, the fungus and the fermentative microorganism competed for the same end hydrolysis product, glucose, for growth in both cases. The amount of supplied oxygen was the only means of controlling the relative population sizes of the two microorganisms. As a result, the aerobic fungi outgrew the bacteria or yeast due to locality advantages of gaining better access to oxygen and the produced glucose by residing in a free solution or on the outer shell of the immobilized beads. The yields of ethanol from starch were very low due to the excessive consumption of the carbon source for fungal biomass production.

[0171] In order to overcome this challenge, the lignocellulolytic and fermentative microorganisms are both genetically engineered to control the cell population and carbon flow. Since the hydrolysis products of cellulose and hemicellulose are composed of both oligosaccharides and monosaccharides, the lignocellulolytic microorganism are engineered to divert most of the produced oligosaccharides to the fermentative microorganism for cell growth and biofuel production, and to divert most of the produces monosaccharides to the lignocellulolytic microorganism for cell growth and cellulase production (Fig. 5). By engineering the cellulase system of the lignocellulolytic microorganism to produce oligosaccharides as the main hydrolysis products and the monosaccharides as the minor hydrolysis products, greater yields of biofuels and chemicals will be produced. Such a lignocellulolytic microorganism can be engineered by disabling the microorganism' s ability to produce extracellular oligosaccharide hydrolases, such as β-glucosidases and xylosidases, so that oligosaccharides are produced as the main products of cellulose and hemicellulose hydrolysis, and monosaccharides are produced as the minor hydrolysis product; and by disabling the microorganism's ability to produce intracellular oligosaccharide hydrolases, such as β-glucosidases, to prevent the microorganisms from utilizing the produced oligosaccharides as a carbon source. Additionally, the fermentative microorganism is engineered to reduce or disable the ability of the fermentative microorganism to utilize the produced monosaccharides as a carbon source so that the majority of the monosaccharides will be utilized by the lignocellulolytic microorganism for growth and cellulase production. This is accomplished by genetically modifying the fermentative microorganism to disable expression of monosaccharide transporters in the fermentative microorganism.

[0172] The carbon flow toward the lignocellulolytic and fermentative microorganisms is also dependent on their respective sugar uptake kinetics. How fast the lignocellulolytic microorganism can utilize cellobiose and glucose is dependent on the genetics of the microorganism (e.g., the number of β-glucosidase genes that are disrupted), while the rate of sugar utilization by the fermentative microorganism depends not only on their genetics but also on the immobilization conditions. Thus, the biofuel and chemical product yields from sugars are related to oxygen supply and the immobilization conditions.

Overview

[0173] Accordingly, the Examples described herein describe the development of an IBS configuration for biofuel production from lignocellulosic biomass that lowers the overall processing cost. The IBS system is developed using Neurospora crassa as the aerobic lignocellulolytic microorganism and Klebsiella oxytoca as the anaerobic fermentative microorganism. The IBS system is developed to convert cellulose contained in

lignocellulosic biomass to isobutanol.

[0174] N. crassa was chosen as the lignocellulolytic microorganism for the co-culture for the following reasons. (1) N. crassa produces a wide variety of cellulases and hemicellulases, and is one of the most efficient cellulase and hemicellulase producers. Unlike other cellulase and hemicellulose producers, such as Trichoderma reesei and Aspergillus niger, N. crassa also produces a wide variety of oxidases and laccases that are involved in phenol degradation and potentially in lignin modification. Thus, N. crassa is also a good starting microorganism for OBS systems. (2) N. crassa is a genetically tractable microorganism, as its genome has been sequenced and tools for its genetic manipulation are readily available. (3) Hydrolysis is the only oligosaccharide utilization pathway that N. crassa possesses. For example, cellobiose must be cleaved into glucose by β-glucosidase (BGL) before being metabolized. Hence, knocking out multiple bgl genes will be decrease cellobiose utilization. (4) Some of the N. crassa bgl genes are redundant. Thus, knocking out multiple bgl genes will not necessarily lead to reduced cellulase production and sluggish growth. In the process of cellulose hydrolysis by N. crassa, endoglucanases and exoglucanases hydrolyze insoluble cellulose to soluble cello-oligosaccharides, with cellobiose as the main product and glucose monomers as the by-products. Extracellular BGL further hydrolyzes cellobiose to the monosaccharide glucose. N. crassa then takes up and utilizes the produced glucose.

Cellobiose may also be transported intracellularly and cleaved to glucose by intracellular BGL. As N. crassa does not possess pathways other than cellobiose hydrolysis, such as cellobiose phosphorylation, to utilize cellobiose, when multiple bgl genes are knocked out in N. crassa, it will be unable to utilize cellobiose by hydrolyzing cellobiose to glucose either intracellularly or extracellularly. Thus, N. crassa containing multiple bgl gene deletions will rely on glucose rather than cellobiose as the carbon source.

[0175] K. oxytoca was chosen as the fermentative microorganism for isobutanol production in the co-culture system for the following reasons. (1) It has been demonstrated that K. oxytoca is capable of efficiently metabolizing not only a wide variety of

monosaccharides (e.g., glucose, xylose, and arabinose) from lignocellulosic biomass, but also oligosaccharides such cellobiose, cellotriose, and xylobiose. (2) Monosaccharides and oligosaccharides are transported via different transporters proteins. Accordingly, it is possible to slow monosaccharide utilization in K. oxytoca by deleting genes related to monosaccharide transportation without affecting oligosaccharide transportation and utilization. (3) K. oxytoca can utilize monosaccharides and oligosaccharides at a pH as low as 5.2. This pH is fully compatible with the requirements of the lignocellulolytic

microorganism, which has an optimal pH of 5 to 6 in co-culture. [0176] Conversion of cellulose to isobutanol was chosen as biofuel product for the following reasons. (1) A two-step non-native isobutanol production pathway has been engineered in E. coli. Using metabolic engineering principles, this pathway has been optimized to yield titers of more than 20 g/L. The isobutanol success achieved in E. coli can be easily transferred to K. oxytoca. (2) The yield of isobutanol production from glucose by E. coli can reach maximal production under micro-aerobic conditions, which can be locally achieved with K. oxytoca by immobilizing the cell within gel beads under a fully aerobic environment. (3) Isobutanol is a very volatile chemical. Thus, in-situ removal of isobutanol from the co-culture system can be achieved by using gas stripping, which has been previously shown to be very efficient in increasing overall productivity.

Example 1: N. crassa mutant strains containing multiple bgl gene knockouts

Construction of multiple β-glucosidase gene deletion N. crassa strains

[0177] There are seven β-glucosidase (BGL) gene transcripts in the N. crassa genome (bgll, bgl2, bgl3, bgl4, bgl5, bgl6, and bgll). The bgll gene has accession number

NCU00130. The bgll gene has accession number NCU04952. The bgl3 gene has accession number NCU05577. The bg¼ gene has accession number NCU07487. The bgl5 gene has accession number NCU08054. The bgl6 gene has accession number NCU08755. The bgll gene has accession number NCU03641. Based on sequence analysis, it is thought that the bgll, bgl3, bgl4, and bgl5 genes code for intracellular BGLs, while the bgll, bgl6, and bgll genes code for extracellular BGLs. All seven of the N. crassa bgl genes have been knocked out and the single knockout strains are publicly available from the Fungal Genetics Stock Center (Kansas City, MO).

[0178] In the present study, seven mutant N. crassa strains were engineered with each strain containing deletions (knockouts) of six bgl genes (strains F1A-F7A), and one mutant strain containing knockouts of all seven bgl genes (strain G27A). The mutant strains were engineered by utilizing standard genetic crossing protocols to generate multiple knockout strains from the single knockout strains (Fig. 6). Double bgl knockout strains were constructed following a standard mating protocol. Gene knockout strains were grown on solid Vogel's minimal medium at 25°C for seven days. Conidia were then harvested and added to another gene knockout strain carrying an opposite mating type grown on the synthetic cross medium. Within a month, thousands of ascospores resulting from a compatible mating were recovered.

[0179] Asexual conidia were excluded from ascospores by treatment at 60°C. Double knockout strains were selected using a PCR-genotyping method as follows. Each single knockout mutant strain, obtained from the Fungal Genetics Stock Center (Kansas City, MO), contained a hygromycin resistance gene (hph^r) inserted within a specific β-glucosidase gene. Primers were designed based on the hph^r open reading frame and the flanking sequence of the knockout loci. The double knockout strains produced two PCR products corresponding to the two replaced genes. The double knockout strains were crossed, following the same crossing procedures, to generate quadruple knockout strains. The quadruple knockout strains were then crossed with a double knockout strain, following the same crossing procedures, to generate strains with 6 bgl gene deletions. A 6 bgl gene knockout strain was crossed with a single knockout strain, following the same crossing procedures, to generate the G27A knockout strain.

[0180] The bgl gene knockout strains were confirmed by genotyping (Fig. 6).

Additionally, the BGL activity of strains F1A, F4A, F3A, F5A, F7A, and G27A grown on either cellobiose or Avicel^® was also measured.

Cellobiose production

[0181] N. crassa strain F5A, which contains knockouts of all bgl genes coding for extracellular BGLs and three of the four bgl genes coding for intracellular BGLs, was utilized to produce cellobiose. The F5A strain was cultured with 20g/L of Avicel^® for five days, and oligosaccharide and monosaccharide production was measured using an HPLC column.

[0182] Strain F5A produced about 8.0 g/L of cellobiose after the five-day culture with Avicel^® (Fig. 7). However, no glucose was detected. About 0.4 g/L of Avicel^® was left in the culture. Accordingly, the cellobiose yield from Avicel^® was about 40%. The overall yield of oligosaccharide production may be even higher, as the HPLC column used to measure oligosaccharide production was not able to detect oligosaccharides with chain lengths greater than two (i.e., cellobiose). Other six-knockout strains and the seven-knockout strain G27A also produced cellobiose, but not with as high a yield as strain F5A. Neither cellobiose nor glucose production was detected in the culture when the wild type N. crassa strain was cultured with 20g/L of Avicel . It was also found that the F5A strain converted Avicel faster than the wild type strain.

[0183] As shown in Figure 8, the gene expression level of the major endoglucanases gh5- 1 and gh7-l in most of the six bgl gene knocked out strains F1-F7 were found to be many times higher than that of the wild type strain. Accordingly, deleting multiple bgl genes appears to have a positive effect on endoglucanase production. Without wishing to be bound by theory, it is believed that multiple bgl gene deletion has a similar effect on exoglucanases.

[0184] Without wishing to be bound by theory, it is believed that since the multiple bgl gene knockout strains are defective in β-glucosidase activity, the strains cannot get enough glucose through cellobiose hydrolysis. As a result, it must produce more endoglucanases and exoglucanases in order to get more of the cellulose hydrolysis by-product glucose as a carbon source. It was observed that cellobiose concentration accumulated at a high concentration, which can be inhibitory for cellulase production and activity. Without wishing to be bound by theory, it also believed that the multiple bgl gene knockout strains will utilize Avicel^® even faster if the inhibitory effect of cellobiose is removed by a fermentative microorganism. It is further believed that the cellobiose yield from Avicel^® would also be higher if the cellobiose is removed instantaneously by a fermentative microorganism, as the cellobiose would be exposed to oligosaccharide hydrolases for a shorter period of time.

Example 2: Engineering N. crassa strains for IBS

[0185] This Example describes the development of N. crassa mutant strains containing further deletions of oligosaccharide hydrolase genes, such as the two β-galactosidase genes NCU05956 and NCU00810 and the β-mannosidase gene NUC00130, to increase conversion of cellulose to cellobiose and cellodextrins. The two β-galactosidase genes and the

β-mannosidase gene are knocked out in a N. crass strain that contains deletions of the six bgl genes.

[0186] The multiple knockout strains are generated using a standard gene knockout method that has the following features: (1) a high rate of homologous integration using a mus52 deletion host; (2) a recyclable marker for unrestricted rounds of knockouts; and (3) a double selective marker system. To construct the multiple knockout strain, the F5AAmus52 strain, which has the tmus52 gene deleted, and recombinase Cre under the inducible xynl promoter at the pyr4 site, are used. The knock- in vector has a double marker cassette, which is composed of a herbicide bialaphos resistant gene (bar) that renders resistance to bialaphos, and the thymidine kinase gene, which renders sensitivity to fluorodeoxyuridine (FUDR), flanked by two LoxP sites. The target gene is then first replaced with the marker cassette sequence through homologous recombination. Screening is easily done by screening colonies on plates containing ignite, the active component of bialaphos. Subsequently, the marker sequence is excised from the genome by employing the Cre/loxP recombination system by growing the strain in plates containing xylan as the inducer. The transformants that have been excised of the marker cassette are selected using plates containing FUDR. The process is repeated to produce desired rounds of knocking out. After finishing all the rounds of gene deletion, the strain is re-transformed to change back to original pyr4 and tmus53 genes, respectively, to regain the original phenotype.

[0187] Generated knockout strains are characterized for growth on Vogel's medium with glucose, cellobiose, or Avicel^® as the carbon source. Both the linear growth in solid medium and the rates of carbon source utilization in liquid culture are characterized. To characterize linear growth, growth tubes are filled with 1.5% agar medium. The strain is inoculated at one end of the tube, and the position of the advancing mycelial front (leading hyphae) is marked at different time intervals. When the growth experiments are completed, distance is plotted against time to obtain the linear growth rate. Tubes are run in duplicate to ensure accuracy.

[0188] The strains are characterized for their rate of glucose, cellobiose, and Avicel^® utilization in liquid culture. Conidia are collected from 10 to 14-day- old slants and inoculated into flasks containing liquid Vogel's medium and 2% carbon source. The culture is carried out at 200 rpm and at 25°C with the lights on. Samples are taken at various time intervals to monitor sugar concentrations and cell mass. After one week, the residual Avicel^® and mycelia concentrations are measured.

Example 3: Engineering a K. oxytoca strain for isobutanol production in the IBS configuration

[0189] This Example describes the construction of a recombinant K. oxytoca strain that is suitable for isobutanol production in the IBS configuration by knocking out the transporters that are responsible for monosaccharide transportation to decrease monosaccharide utilization, by knocking by-product production pathways; and by recombinantly expressing an isobutanol production pathway.

Monosaccharide transporter deletion

[0190] The native K. oxytoca utilizes both oligosaccharides and monosaccharides. In the IBS co-culture system, glucose is preferentially utilized by the lignocellulolytic

microorganism for growth and cellulase production. Thus, it is necessary to slow the ability of K. oxytoca to utilize glucose by knocking out the transporter proteins that are responsible for glucose transportation, while maintaining those responsible for cellobiose transportation so that the rate of glucose utilization is greatly reduced.

[0191] To construct a K. oxytoca strain that is slower in glucose utilization, the genes coding for the glucose transporter (ptsG) and the mannose transporter (ptsLPM) are deleted. The gene deletion system developed by Datsenko and Wanner is utilized to construct the multiple knockout strain using recyclable markers. The target genes are replaced with a kanamycin cassette flanked by FLP recognition target sites by homologous recombination. The kanamycin resistance gene is then excised by using a helper plasmid encoding the FLP recombinase. The process is repeated to produce the desired number of rounds of knocking out.

[0192] To confirm the slower glucose utilization phenotype, the mutant strain is grown in synthetic medium using glucose or cellobiose as the carbon source. Batch studies are conducted in 500-mL Erlenmeyer flasks with 100 mL of liquid loading. Measurements accompanying batch studies include glucose and cellobiose concentration (HPLC) and cell concentration (optical density).

Isobutanol production pathway

[0193] The K. oxytoca mutant strain is further modified using a metabolic engineering strategy, which has proven successful in diverting more carbon flow toward isobutanol production in E. coli, to engineer the K. oxytoca strain for isobutanol production. The genes coding for alcohol- aldehyde dehydrogenase (adhE), phosphotransacetylase (pta), isocitrate dehydrogenase (IdhA), fumarate reductase (frdAB, fnr), and pyruvate formate lyase (pfl) are deleted. In addition, K. oxytoca has a very strong 2,3-butanediol production pathway, which represents another competing branch for pyruvate. It has been previously shown that knocking out budAB genes to eliminate 2,3-butanediol production was found successful in diverting more carbon flow toward ethanol production in an acidic environment for a recombinant ethanologen K oxytoca P2. Accordingly, the budAB genes are also deleted.

[0194] The plasmid carrying the isobutanol pathway has an isopropyl- -D- thiogalactoside (IPTG) inducible promoter PlacOl, which is well-characterized and very user-friendly. However, IPTG is too expensive for industrial use. Thus, the IPTG inducible promoter is replaced with a constitutive promoter, such as the E. coli promoters for rrnB and gapA, or the phage lambda promoters PL and PR. To improve genetic stability and avoid the utilization of antibiotic addition to maintain the plasmids, the DNA fragment containing the isobutanol biosynthetic pathway is inserted into the K. oxytoca chromosome at the pfl site using a standard protocol. To optimize isobutanol production, the pathway is fine-tuned by comparing polycistronic and monocistronic expression cassettes of each gene and modulating their expression by using promoters and ribosomal binding sites (RBS) of varying strengths.

Example4: Isobutanol production

[0195] To co-culture the engineered K. oxytoca mutant strain, produced in Example 3 above and the engineered N. crassa mutant strain, produced in Examples 1 and 2 above, in the same reactor, a defined medium which is suitable for culturing both strains is used. The minimal medium requirements for both strains including amino acids, vitamins, and nitrogen source are optimized. Each strain is grown on the defined medium alone or together to ensure proper growth. The effect of pH, temperature, and K+, P04 ^", and corn steep liquor on cell growth is evaluated.

[0196] Isobutanol production is first optimized using the immobilized K. oxytoca constructed in Example 3 above using the defined medium. Calcium alginate is used as the immobilization carrier. The K. oxytoca cells are grown until to exponential phase. Cells are then harvested and mixed with sodium alginate. The cell culture slurry is converted to droplet form while it is dripped into a calcium chloride bath using a syringe. Once the slurry is added to the bath, beads of calcium alginate with entrapped cells are formed. The bead size can be controlled by the size of syringe used. The matrix density and cell density is controlled by the concentrations of sodium alginate, calcium chloride, and cells. [0197] Isobutanol production is also optimized using immobilized K. oxytoca in the optimized medium using cellobiose and glucose as the carbon source in a Bioflo 110 reactor with a working volume of 300 mL. Sterile air is bubbled through the reactor. The air carrying isobutanol vapor is then condensed in a cooler and a condenser. The cooler is held at about - 2°C. Ethylene glycol is circulated through the condenser using an apparatus describe by Baez et al. The dissolved oxygen content, isobutanol, sugar concentration, glucose, and ethanol concentrations in fermentation broth and the isobutanol concentration in the adsorption reservoir are measured during the process of fermentation. The isobutanol production is optimized by varying the aeration rate (air flow rate, stirring speed), gel bead size, and gel matrix density.

[0198] The IBS configuration is conducted in the same reactor system using the same conditions described above except that 40 g/L of Avicel^® is utilized as the carbon source. The sterile medium is inoculated with 1 mL of 10⁶ /mL conidia of the N. crassa mutant strain harvested from a 7 day culturing slant and with the immobilized K. oxytoca at a concentration of 10⁶ /mL. Fermentation samples are collected to determinate growth, isobutanol production, and organic acid and glucose concentrations at various time intervals. The isobutanol production is optimized by varying the initial cellulose concentration, relative ratio of the initial concentration of the N. crassa and K. oxytoca strains, aeration rate, pH, and temperature.

Example 5: Construction of an IBS Configuration

Introduction

[0199] The following Example describes the construction of a microbial consortium (e.g., co-culture) for developing an IBS configuration, and test the IBS configuration for cellulosic biofuel production with the purpose of lowering the overall processing cost. The IBS system is developed using Neurospora crassa and K. oxytoca as the model microorganisms, and focusing on converting cellulose contained in cellulosic biomass to ethanol as the model system.

[0200] N. crassa was chosen as the lignocellulolytic fungus in the mixed culture for several reasons. N. crassa is a fast growing fungus. (2) N. crassa does not produce any known toxins during culture. (3) N. crassa produces a wide variety of cellulases and hemicellulases and is one of the most efficient cellulase and hemicellulase producers. (4) N. crassa is a genetically tractable microorganism, as its genome has been sequenced and tools for its genetic manipulation are readily available. (5) N. crassa possesses a known sexual cycle, it is possible to combine multiple mutations by genetic crossing and facilitate strain improvement stemming from recombinant DNA methods. (6) N. crassa does not possess oligomer utilization pathways other than hydrolysis. For example, cellobiose must be cleaved into glucose by BGL before being metabolized. Hence, knocking out multiple copies of bgl will be directly linked to slower cellobiose utilization.

[0201] K. oxytoca was chosen as the fermentative microorganism for biofuel production in the co-culture system for the following reasons. (1) K. oxytoca is genetically tractable microorganism and its whole genome sequence is available and tools for its genetic manipulation are readily available. (2) It has been demonstrated to be capable of metabolizing not only a wide variety of monomer sugars (glucose, xylose, and arabinose) from cellulosic biomass, but also sugar oligomers such cellobiose, cellotriose, and xylobiose, very efficiently. Monomers and oligomers are transported via different transporters. It is possible to slow its monomer sugar utilization by knocking out genes related to monomer transportation without affecting oligomer transportation and utilization. (3) K. oxytoca P2 (engineered for ethanol production) can efficiently ferment monomer and oligomer sugars at a pH 5.2-7.0 at a yield of more than 90%. This pH is fully compatible with the requirements of the N. crassa cellulase production, which has an optimal pH of 5 to 7, in the co-culture.

[0202] Conversion of cellulose to ethanol was chosen as the model system mainly due to ethanol is a volatile biofuel and the bacterium strain K. oxytoca P2 which has been

engineered for ethanol production is readily accessible for further genetic modification.

However, the same principle is applicable for other volatile biofuels production such as isobutanol, butanol etc.

[0203] Cellulolytic fungi have a remarkable potential for cellulase production and direct enzymatic hydrolysis. They have remained the main workhorse for cellulase production in the industry. However, their application in direct enzymatic hydrolysis is limited. One of the major factors that limit their application is the over consumption of the sugars released for their own. The strain engineering strategy described herein provides a new approach to prevent excessive sugar utilization by the fungi during hydrolysis, hence enabling the direct utilization of the lignocellulolytic fungi in enzymatic hydrolysis process and fermentation. [0204] The proposed IBS configuration distinguishes from consolidated bioprocessing in the following aspects: a. Yield optimization by expert strain co-culture and targeted bioengineering

[0205] In the CBP enabling microorganism development, it is very hard to engineer strains to degrade cellulose at rate matching that of a native cellulose degrader or produce biofuels at a yield matching that of a native biofuel producer. The genetic engineering efforts affect the strain performance substantially in both cases. In the IBS configuration, a cellulase production expert strain and a biofuel production expert strain are utilized in the consortium. As detailed below, the genetic modification of the fungus not only did not compromise the rate of cellulose utilization, on the contrary, it enhanced the rate of cellulose degradation. The genetic modification of the bacterium did not affect the oligomer utilization and the ethanol yield from the sugars. Hence, without wishing to be bound by theory it is believed that the IBS configuration yields a robust cellulose degradation rate and high yield of biofuel production hat are close to those achieved by the native microorganism. b. The IBS configuration is more consolidated than CBP and is an inhibition free system, hence higher productivity is possible

[0206] In the conventional SHF, the cellulose hydrolysis products, such as cellobiose or glucose, are inhibitors for the cellulase activity and cellulase production. The fermentation products, such as ethanol, are inhibitory to the fermentative microorganism at high concentrations. The SSF and CBP configuration combines the hydrolysis and fermentation in a single step, and can efficiently alleviate the inhibition of the sugars on cellulase. Similar to the cases of SSF and CBP, the hydrolysis product oligomers and monomers are instantly removed by the fungus and fermentative microorganism so that they will not inhibit the cellulase and hemicellulose enzymes in the IBS configuration. Moreover, the product ethanol is removed by in-situ stripping by the aeration in the IBS configuration. The inhibition of the product on the cellulase enzyme and the microorganisms may also be alleviated as well. Hence, higher process efficiency and productivity are possible. c. Less product tolerant strains are needed

[0207] The IBS configuration also features in-situ product removal, which leads to lower product concentration in the fermentation broth. The low product concentration imposes less product tolerance requirements on the strains. One of the strain development goals for CBP or SSF is to increase the strain's product inhibition tolerance. However, increasing the strain's product tolerance generally leads to lower product yield or decreased productivity. Advantageously, in the IBS configuration, strains are not required to exhibit high product tolerance, and hence their biofuel production yield, capacity, and productivity are not compromised as a side-effect. d. Potential to incorporate pretreatment to achieve one step conversion

[0208] If the fungus is engineered to produce lignin solubilizing enzymes to solubilize lignin, which is an aerobic process that is completely compatible with the IBS configuration, then pretreatment can be further consolidated into the IBS configuration to achieve a one-step conversion in which all the individual unit operations can be achieved in one reactor.

Materials and methods

Glucose and cellobiose utilization

[0209] The wild type N. crassa strain and the F5A-bgl5 strain were cultured on Vogel's slants or flasks containing 2% sucrose to develop conidia. Mycelia-free conidia were harvested after 10 days of culture, and were inoculated into 50 ml of Vogel's medium containing 20g/L glucose, 20g/L cellobiose, or 20g/L Avicel^® at a final concentration of 10⁶ conidia/ml. In the medium containing cellobiose or Avicel^®, a small amount of glucose (0.6g/L) was supplied to facilitate the germination of the spores. The fermentation was carried out in a rotary shaker at 200 rpm and at 27°C for 120 hours with the light on. Samples were taken at various intervals to analyze the concentrations of glucose and cellobiose in the broth by HPLC. After 4 days of culture, the residues in the bottles containing Avicel^® were filtered and dried at 72°C for 2 days. The nitrogen content of the residual was measured by a LECO TruSpec CHN elemental determinator (St. Joseph, MO, USA). The mycelial dry weight was calculated indirectly based on the nitrogen content. The amount of residual Avicel was calculated as the weight difference between the residual sample and mycelial dry weight. [0210] The cellobiose yield was expressed as the amount of cellobiose produced (g) divided by the amount of cellulose consumed (g). The cell yield was calculated by the amount of mycelial dry weight (g) divided by the amount of cellulose consumed (g).

Quantification of endoglucanase and exoglucanase gene transcripts by RT-PCR

[0211] Ten-day-old conidia were inoculated into Vogel's medium containing 20 g/L glucose as a carbon source. Mycelia were collected after 15 hours of culture and were rinsed three times in Vogel's medium to remove all residual glucose. The resulting mycelia were weighed to determine wet cell mass, transferred to 200 ml of Vogel's medium containing 20 g/L Avicel^® (Sigma, St. Louis, MO) as the sole carbon source, and cultured at 27°C at 220 rpm. Samples were taken at 4 hours to quantify the transcription levels of the selected genes.

[0212] The mycelia harvested from the cell broth were immediately frozen in liquid nitrogen and stored at -80°C. The frozen samples were ground in a mortar and pestle containing liquid nitrogen, and RNA was extracted using Trizol (Invitrogen, Foster City, CA) according to the manufacturer's protocol. The extracted total RNA was purified using the RNeasy Mini Protocol (Qiagen, Valencia, CA). The integrity of total RNA was examined by electrophoresis and visual inspection on 1% agarose gel, and the purity was determined by calculating the ratio of absorbance at 260 nm and 280 nm (acceptable range: 1.8-2.2).

[0213] For cDNA synthesis, 1 μg of purified total RNA was used as template. Reverse transcription was performed using an AffinityScript qPCR cDNA synthesis kit (Agilent, Santa Clara, CA) with oligo dT primers following the manufacturer's instructions. Real-time quantitative PCR was performed in an Applied Biosystems StepOne Plus system (Applied Biosystems, Foster City, CA). The PCR conditions were optimized to ensure that all selected genes had high amplification efficiencies. The slopes of the standard curves for all four genes ranged from -3.22 to -3.29, which confirmed that the amplification efficiency was between 99.2% and 101.3%. The comparative CT method was used to analyze all qPCR data, and β- actin was used as the internal standard. The relative expression was obtained by comparing the transcription levels to that of the wild type at the same time point. All of the primers used in the qPCR were designed with the aid of software Primer3Plus and were synthesized by Invitrogen (Foster City, CA). The primers are listed in Table 1. Table 1

Glucose and cellobiose utilization by K. oxytoca

[0214] Anaerobic fermentation was carried out in 200 mL serum bottles with a working volume of 100 mL and a N₂ gas phase. Luria Bertani (LB) broth with glucose or cellobiose as the carbon source was used as the culturing medium. The pH was adjusted to 7.0. The batch experiments were initiated by inoculating 1% of the wild type strain K oxytoca P2 and the mutant strain P2AptsG AmanXYZ were inoculated to the serum bottles containing inoculums and incubating on a rotary shaker at 200 rpm and at 30°C. Samples were taken at various time intervals. The concentration of glucose, cellobiose, and ethanol were analyzed by HPLC.

Results

N. crassa strain containing multiple bgl deletions

[0215] As described above, there are seven gene transcripts named β-glucosidase (bgl) in the N. crassa genome. Using the technique described in Example 1, we successfully constructed seven sextuple (X) bgl gene deletion strains (N. crassa F1-F7) and one septuple bgl deletion strain (N. crassa G27A) by genetic crossing starting from the single bgl deletion strains. [0216] As described in Example 1 above, the sextuple bgl deletion strain F5A-bgl5 has all three bgl genes coding for extracellular BGL and three out of four bgl genes coding for intracellular BGL deleted. The F5A-bgl5 strain performed best in terms of cellulose degradation and cellobiose production. As shown in Figure 9, the F5-bgl5 strain utilized glucose at the same rate as that of wild type N. crassa. However, the strain did not utilize cellobiose at all, while wild type N. crassa utilized 20g/L cellobiose within two days (Fig. 9).

[0217] As described in Example 1 above, the F5A-bgl5 strain also utilized cellulose faster than wild type N. crassa. Starting from 20g/L Avicel^®, the F5A-bgl5 strain consumed about 11.6 g/L Avicel^® in 3 days, and produced about 6.7 g/L cellobiose (Fig. 7). Even in the presence of high concentrations of cellobiose, which is a strong inhibitor for

cellobiohydrolases, the F5A-bgl5 strain utilized cellulose faster than wild type N. crassa and produced only about half the amount of cell mass as the wild type strain.

[0218] The characterization of gene expression levels of the major cellulase genes in the F5-bgl5 strain after 4 hours induction on Avicel^® revealed that major endoglucanase and exoglucanase genes were expresses at a higher level than those of wild type N. crassa (Fig. 10).

[0219] Multiple bgl gene deletions did not affect cellulase induction. Instead, the deletions appear to have a positive effect. Without wishing to be bound by theory, we believe that the reason why the F5-bgl5 strain produced higher levels of endoglucanases and exoglucanases is that the F5-bgl5 strain cannot get enough glucose through cellobiose hydrolysis because it is defective in β-glucosidase activity. As a result, it is believed that the strain must produce more endoglucanases and exoglucanases in order to get more of the cellulose hydrolysis by-product glucose as a carbon source. Accordingly and without wishing to be bound by theory, we believe that the F5-bgl5 strain can utilize Avicel^® even faster when cellobiose is removed by a fermentative microorganism.

[0220] The cellobiose yield from consumed cellulose is about 50% under the condition that cellobiose was accumulating at a high concentration, which is highly inhibitory to the cellobiohydrolases. In order to determine the ratio of cellobiose to glucose produced by the cellulases produced by the F5-bgl5 strain under condition where cellobiose inhibition is not induced, we collected the supernatant of 12 hour culture broth of wild type N. crassa and the

F5-bgl5 strain when the cellobiose was not detectable, concentrated each broth sample, and mixed it with cell-free Avicel . After 72 hours of hydrolysis, the concentration of cellobiose and glucose in the broth was measured. The ratio of cellobiose to glucose in the wild type broth was about 1: 3, which indicates that glucose was the major hydrolysis product produced. However, the ratio of cellobiose to glucose produced by the F5-bgl5 broth was about 11: 1, which indicates that the cellobiose is the major hydrolysis product. These results show that deleting multiple bgl genes in N. crassa efficiently diverts most of the carbon toward oligomer sugar production.

[0221] The added advantage of the mutant F5A-bgl5 strain is that it showed a distinct, fragmented mycelial morphology, which resulted in a much lower viscosity phenotype.

Without wishing to be bound by theory, it is believed that the inherent low viscosity of the F5-bgl5 strain can ease several of the problems related to filamentous fungi fermentation.

Development of a mus51 deletion strain

[0222] The F5-bgl5 strain was constructed by crossing the each single bgl deletion strain, which had the functional bgl gene replaced with a hygromycin resistance marker. Hence, the F5-bgl5 strain contains six copies of the hygromycin resistance gene in the chromosome, which limits the utilization of this marker for future gene deletion work. In order to facilitate future gene deletions, a self-excising marker employing the -rec/six site-specific

recombination system established previously for Aspergillus fumigatus was adapted and validated in N. crassa. The system features repetitive gene deletion using a bar resistance marker, and one transformation per gene deletion. Using this system, we successfully deleted the mus-51 gene in the F5-bgl5 strain created by means of genetic crossing, recycled the marker cassette and subsequently deleted a transcriptional regulator cre-1 (the global regulator of carbon catabolite repression), generating the N. crassa strain F5-bgl5-Amus51Acrel. The results of this experiment show that the -rec/six site-specific recombination system can be used to generate further gene deletions in the F5-bgl5 strain.

K. oxytoca P2 strain containing two hexose transporter gene deletions

[0223] We deleted the genes encoding the glucose transporter (ptsG) and the mannose transporter (manXYZ) from the K. oxytoca P2 genome, using a standard gene deletion system developed by Datsenko and Wanner (Datsenko and Wanner (2000), Proc Natl Acad Sci U S A 97, 6640-6645). As shown in Figures 11 and 12, the double knock-out strain (AptsG AmanXYZ) showed much slower glucose utilization than wild type K. oxytoca. Starting from 20g/L glucose, wild type K. oxytoca consumed all the glucose in 24 hours, while the AptsG AmanXYZ strain left about 14 g/L glucose after 24 hours. However, the rate of cellobiose utilization for both wild type K. oxytoca and the P2AptsG AmanXYZ strain were almost the same.

[0224] As shown in Figure 11 and 12, the deletion of ptsG and manXYZ slowed glucose utilization substantially. However, the rate of cellobiose utilization was about the same.

[0225] We further deleted the genes encoding the galactose transporter (galP) and galactose transporter (mglABC), or the maltose transporter (malEFG) from the K. oxytoca P2 genome. These deletions were made in the K. oxytoca P2 strain containing deletions of the ptsG glucose transporter gene and the manXYZ mannose transporter gene. As shown in Figure 13, the double knock-out strain (AptsG AmanXYZ), quadruple knockout strains (AptsGAmanXYZAgalPAmglABC, and AptsG AmanXYZ AgalPAmalEFG) showed much slower glucose utilization than wild type K oxytoca P2 (p2). Starting from 20g/L glucose, wild type K oxytoca P2 consumed all the glucose in 24 hours, while the mutants strain left about 14 g/L glucose after 24 hours.

Cell immobilization leads to higher ethanol yield

[0226] Using the ethanologen K. oxytoca P2 for ethanol production, immobilization of the cells inside of gel beads produced a higher concentration of ethanol, as compared to free cells under aerobic conditions. The yield achieved by immobilized cells using calcium alginate and κ -carrageenan was about 80% and 87.5% of the theoretical yield, respectively, while the free cells only achieved 60% of the theoretical yields. The results demonstrate that cell immobilization can generate a locally micro-aerobic environment that results in high ethanol yields.

Co-culture ofN. crassa F5-bgl5 and K. oxytoca PlAptsG AmanXYZ

[0227] The N. crassa F5-bgl5 strain was cultured on Vogel's slants or flasks containing 2% sucrose to develop conidia. Mycelia-free conidia were harvested after 10 days of culture and were inoculated into 50 ml of Vogel's medium containing 20 g/L Avicel^® at a final concentration of 10⁶ conidia/ ml. In the medium containing cellobiose or Avicel^®, a small amount of glucose (0.6g/L) was supplied to facilitate the germination of the spores. The fermentation was carried out in a rotary shaker at200 rpm and at 27°C. The K. oxytoca VlAptsG AmanXYZ strain was added to the flask at a final OD of 0.1.

[0228] Samples were taken at various time intervals to analyze the concentration of glucose and cellobiose in the broth. After 4 days of culture, the residue in the bottles containing Avicel^® were filtered and dried at 72°C for 2 days. For the F5-bgl5 strain without the addition of K. oxytoca, the nitrogen content of the residue was measured by a LECO TruSpec CHN elemental determinator (St. Joseph, MO, USA). The mycelial dry weight was calculated indirectly based on the nitrogen content. The amount of residual Avicel^® was calculated as the weight difference between the residual sample and mycelial dry weight. For the strains that produced no cellobiose, the cellobiose conversion was calculated based on the weight difference, based on a cell yield of 0.47g cell / g cellulose consumed for both the N. crassa and the K. oxytoca.

[0229] As shown in Figure 14 and Table 2, the N. crassa F5-bgl5 and K. oxytoca

VlAptsGAmanXYZ co-culture had higher cellulose conversion than the pure culture of N. crassa alone, which demonstrates that the co-culture has a higher rate of cellulose utilization since the cellobiose as the inhibitor was removed (consumed) by the K. oxytoca bacteria.

Table 2

[0230] However, we did not detect any ethanol in the fermentation broth. Without wishing to be bound by theory, we believe that ethanol was not produced because the ethanol was consumed by N. crassa F5-bgl5strain, which has the ethanol utilization pathway.

Without wishing to be bound by theory, we also believe that deleting the two alcohol dehydrogenase genes in the N. crassa F5-bgl5strain (adhl: NCU01754 and adh3:

NCU02476) will solve this problem. [0231] The N. crassa F5-bgl5 strain, Adhl knockout strain (FGSC 12735), and Adh3 knockout strain (FGSC 12920) were cultured on Vogel's slants or flasks containing 2% sucrose to develop conidia. Mycelia-free conidia were harvested after 10 days of culture and were inoculated into 50 ml of Vogel's medium containing 29g/L ethanol at a final concentration of 10⁶ conidia/ ml. In the medium containing cellobiose or Avicel^®, a small amount of glucose (0.6g/L) was supplied to facilitate the germination of the spores. The fermentation was carried out in a rotary shaker at 200 rpm and at 27°C. Flasks with about 29g/l ethanol and without strain addition were used as the control. Samples were taken at various time intervals to analyze ethanol concentration.

[0232] As shown in Figure 15, the ethanol concentration decreased in the control flasks due to evaporation. The ethanol concentration detected in the flasks in which the Adhl knockout strain was cultured had an ethanol profile similar to that of the control, which indicated that the ADH1 knockout strain does not consume ethanol. The Adh3 knockout strain consumed ethanol, however the consumption was much slower than that of the F5-bgl5 strain.

Claims

CLAIMS We claim:

1. A co-culture composition comprising: a first population of mutant lignocellulolytic cells, wherein the mutant cells comprise a mutation in one or more β-glucosidase genes; and a second population of fermentative microorganisms.

2. The co-culture composition of claim 1, wherein the mutant cells are aerobic mutant cells.

3. The co-culture composition of claim 1, wherein the mutant cells are anaerobic mutant cells.

4. The co-culture composition of any one of claims 1-3, wherein the fermentative microorganisms are anaerobic fermentative microorganisms.

5. The co-culture composition of any one of claims 1-3, wherein the fermentative microorganisms are aerobic fermentative microorganisms.

6. The co-culture composition of any one of claims 1-5, wherein the mutant cells comprise a mutation in two or more, three or more, four or more, five or more, six or more, or seven β-glucosidase genes.

7. The co-culture composition of any one of claims 1-6, wherein the β- glucosidase genes encode extracellular β-glucosidases.

8. The co-culture composition of any one of claims 1-6, wherein the β- glucosidase genes encode intracellular β-glucosidases.

9. The co-culture composition of any one of claims 1-8, wherein the β- glucosidase genes are selected from the group consisting of NCU00130, NCU04952, NCU05577, NCU07487, NCU08054, NCU08755, NCU03641, homologs thereof, orthologs thereof, and paralogs thereof.

10. The co-culture composition of any one of claims 1-9, wherein the mutant cells further comprise a mutation in one or more additional cellulose oligosachharide hydrolase genes.

11. The co-culture composition of claim 10, wherein the one or more additional cellulose oligosachharide hydrolase genes are one or more additional hexose hydrolase genes selected from the group consisting of the β-galactosidase gene NCU05956, the β-galactosidase gene NCU05956, the β-mannosidase gene NUC00130, homologs thereof, orthologs thereof, and paralogs thereof.

12. The co-culture composition of any one of claims 1-11, wherein the mutant cells further comprise a mutation in one or more transcription factors.

13. The co-culture composition of any one of claims 1-12, wherein the mutant cells further comprise a mutation in at least one hemicellulose oligosaccharide hydrolase gene.

14. The co-culture composition of claim 13, wherein the at least one hemicellulose oligosaccharide hydrolase gene is a xylosidase gene.

15. The co-culture composition of any one of claims 1-14, wherein the mutant cells further comprise a mutation in at least one alcohol dehydrogenase gene.

16. The co-culture composition of claim 15, wherein the at least one alcohol dehydrogenase gene is the adhl gene or the adh3 gene.

17. The co-culture composition of any one of claims 1-16, wherein the gene mutation comprises a partial deletion or a complete deletion of the gene.

18. The co-culture composition of any one of claims 1-17, wherein the mutant cells produce one or more of cellulases, hemicellulases, lignin-solubilizing enzymes, or combinations thereof.

19. The co-culture composition of any one of claims 1-18, wherein the mutant cells have higher cellulase activity as compared to a corresponding cell that does not comprise a mutation in one or more β-glucosidase genes and/or hemicellulose

oligosaccharide hydrolase genes.

20. The co-culture composition of claim 18 or claim 19, wherein the one or more hemicellulases are recombinantly expressed in the mutant cells.

21. The co-culture composition of any one of claims 18-20, wherein the one or more lignin-solubilizing enzymes are recombinantly expressed in the mutant cells.

22. The co-culture composition of any one of claims 1-21, wherein the mutant cells further comprise a recombinant cellulosome.

23. The co-culture composition of claim 22, wherein the cellulosome is assembled on the surface of the mutant cells.

24. The co-culture composition of any one of claims 1-23, wherein the mutant cells produce cellodextrin.

25. The co-culture composition of claim 24, wherein the cellodextrin is cellobiose.

26. The co-culture composition of any one of claims 1-25, wherein the mutant cells are fungal cells.

27. The co-culture composition of any one of claims 1-26, wherein the fungal cells are filamentous fungal cells.

28. The co-culture composition of any one of claims 1-26, wherein the fungal cells are Neurospora crassa.

29. The co-culture composition of any one of claims 1-28, wherein the fermentative microorganisms comprise at least one cellodextrin transporter.

30. The co-culture composition of claim 29, wherein the cellodextrin transporter is a cellobiose transporter.

31. The co-culture composition of claim 29 or claim 30, wherein the cellodextrin transporter is endogenous to the fermentative microorganisms.

32. The co-culture composition of claim 29 or claim 30, wherein the cellodextrin transporter is a recombinant cellodextrin transporter.

33. The co-culture composition of any one of claims 1-32, wherein the fermentative microorganisms further comprise at least one intracellular β-glucosidase or cellobiose phosphorylase.

34. The co-culture composition of claim 33, wherein the intracellular β- glucosidase or cellobiose phosphorylase is endogenous to the fermentative microorganisms.

35. The co-culture composition of claim 33, wherein the intracellular β- galactosidase or cellobiose phosphorylase is a recombinant intracellular β-galactosidase or cellobiose phosphorylase.

36. The co-culture composition of any one of claims 1-35, wherein the fermentative microorganisms further comprise a mutation in one or more monosaccharide sugar transporter genes.

37. The co-culture composition of claim 36, wherein the one or more monosaccharide sugar transporter genes are selected from the group consisting of hexose transporter genes, pentose transporter genes, and combinations thereof.

38. The co-culture composition of claim 36, wherein the one or more monosaccharide sugar transporter genes are selected from the group consisting of glucose transporter genes, xylose transporter genes, galactose transporter genes, mannose transporter genes, arabinose transporter genes, fructose transporter genes, maltose transporter genes, lactose transporter genes, and combinations thereof.

39. The co-culture composition of any one of claims 1-35, wherein the fermentative microorganisms further comprise a mutation in one or more sugar alcohol transporter genes or one or more sugar aldonic acid transporter genes.

40. The co-culture composition of 39, wherein the one or more sugar alcohol transporter genes are sorbitol transporter genes and the one or more sugar aldonic acid transporters genes are gluconic acid transporter genes.

41. The co-culture composition of any one of claims 36-40, wherein the gene mutation comprises a partial deletion or a complete deletion of the gene.

42. The co-culture composition of any one of claims 1-41, wherein the fermentative microorganisms further comprise at least one recombinant oligosaccharide utilization polypeptide.

43. The co-culture composition of claim 42, wherein the at least one recombinant oligosaccharide utilization polypeptide is selected from the group consisting of a cellodextrin utilization polypeptide, a cellobiose utilization polypeptide, a xylodextrin utilization polypeptide, a xylobiose utilization polypeptide, a mannobiose utilization polypeptide, a galactobiose utilization polypeptide, a maltose utilization polypeptide, a lactose utilization polypeptide, and combinations thereof.

44. The co-culture composition of claim 42, wherein the at least one recombinant oligosaccharide utilization polypeptide is selected from the group consisting of a cellodextrin transporter, a cellobiose transporter, a xylodextrin transporter, a xylobiose transporter, a mannobiose transporter, a galactobiose transporter, a maltose transporter, a lactose transporter, and combinations thereof.

45. The co-culture composition of any one of claims 1-44, wherein the fermentative microorganisms further comprise one or more modifications sufficient for the fermentative microorganisms to produce a commodity chemical from one or more oligosaccharides.

46. The co-culture composition of claim 45, wherein the one or more modifications comprise a deletion in one or more genes.

47. The co-culture composition of claim 45 or claim 46, wherein the one or more modifications further comprise the recombinant expression of one or more genes.

48. The co-culture composition of any one of claims 45-47, wherein the commodity chemical is a biofuel selected from the group consisting of an alcohol, ethanol, propanol, isopropanol, acetone, butanol, isobutanol, 2-methyl-l-butanol, 3-methyl-l-butanol, phenylethanol, a fatty alcohol, isopentenol, an aldehyde, acetylaldehyde, propionaldehyde, butryaldehyde, isobutyraldehyde, 2-methyl-l-butanal, 3-methyl-l-butanal,

49. The co-culture composition of any one of claims 1-48, wherein the fermentative microorganisms are bacteria.

50. The co-culture composition of claim 49, wherein the bacteria are Klebsiella oxytoca.

51. The co-culture composition of any one of claims 1-48, wherein the fermentative microorganisms are fungi or yeast.

52. The co-culture composition of any one of claims 1-51, wherein the co- culture composition further comprises a third population of aerobic fatty acid-producing and/or hydrocarbon-producing microorganisms.

53. A method for the production of a commodity chemical from

lignocellulosic biomass, comprising:

(a) providing a fermentation broth comprising the co-culture composition of any one of claims 1-52;

(b) contacting lignocellulosic biomass with the fermentation broth; and

(c) incubating the fermentation broth and lignocellulosic biomass under conditions sufficient for the co-culture composition to produce a commodity chemical from the lignocellulosic biomass.

54. The method of claim 53, wherein the co-culture composition is incubated under aerobic conditions.

55. The method of claim 54, wherein air or oxygen is supplied to the fermentation broth to achieve the aerobic conditions.

56. The method of any one of claims 53-55, wherein the fermentative microorganisms are incubated under a local anaerobic or micro-aerobic environment.

57. The method of claim 56, wherein the local anaerobic or micro-aerobic environment is produced by immobilizing the fermentative microorganisms.

58. The method of claim 57, wherein the fermentative microorganisms are immobilized within gel beads.

59. The method of claim 57, wherein the fermentative microorganisms are co- immobilized with the mutant lignocellulolytic cells within gel beads.

60. The method of any one of claims 53-59, wherein the mutant

lignocellulolytic cells produce glucose and cellodextrin from the lignocellulosic biomass.

61. The method of claim 60, wherein the cellodextrin is cellobiose.

62. The method of claim 60, wherein the mutant lignocellulolytic cells utilize the produced glucose as a carbon source.

63. The method of any one of claims 60-62, wherein the fermentative microorganisms utilize the cellodextrin to produce the commodity chemical.

64. The method of any one of claims 53-63, wherein the mutant

lignocellulolytic cells further produce at least one hemicellulose oligosaccharide from the lignocellulosic biomass.

65. The method of claim 64, wherein the at least one hemicellulose oligosaccharide is xylobiose.

66. The method of claim 64 or claim 65, wherein the fermentative microorganisms further utilize the hemicellulose oligosaccharide to produce the commodity chemical.

67. The method of any one of claims 53-66, wherein the commodity chemical is extracted from the fermentation broth by gas stripping.

68. The method of claim 67, wherein air or oxygen is utilized to gas strip the commodity chemical.

69. The method of any one of claims 53-68, wherein the commodity chemical is a biofuel selected from the group consisting of an alcohol, ethanol, propanol, isopropanol, acetone, butanol, isobutanol, 2-methyl-l-butanol, 3-methyl-l-butanol, phenylethanol, a fatty alcohol, isopentenol, an aldehyde, acetylaldehyde, propionaldehyde, butryaldehyde, isobutyraldehyde, 2-methyl-l-butanal, 3-methyl-l-butanal, phenylacetaldehyde, a fatty aldehyde, a hydrocarbon, an alkane, an alkene, an isoprenoids, a fatty acid, a wax ester, an ethyl ester, hydrogen, and combinations thereof.