AU6875298A - Development of high-ethanol resistant (escherichia coli) - Google Patents

Description

WO98/45425 PCTIUS98/06405 -1 DEVELOPMENT OF HIGH-ETHANOL RESISTANT ESCHERICHIA COLI BACKGROUND OF THE INVENTION The fermentation of waste paper and other lignocellulosic products, such as crop residues, into 5 ethanol offers the opportunity to reduce environmental waste problems and reduce reliance on petroleum-based automotive fuels. Genetically engineered bacteria, such as Escherichia coli KO11 (U.S Patent No. 5,000,000) and Klebsiella oxytoca P2 (U.S Patent No. 5,424,202), have 10 been developed which convert both pentose and hexose sugars, produced by the hydrolysis of hemicellulose, into ethanol. Relative to yeast, such as Saccharomyces, which is currently used for commercial ethanol production from 15 cane syrup and from hydrolyzed corn starch, Escherichia coli KO11 is much less ethanol tolerant. Thus, even though bacteria have been developed that have the ability to convert the sugars from, for example, lignocellulose to ethanol, the problem remains that 20 ethanol tolerance in these bacteria limits both the rate of ethanol production and the final ethanol concentration which can be achieved in the fermentors. SUMMARY OF THE INVENTION This invention is based upon the discovery that 25 novel mutants of Escherichia coli KO11 exhibit the ability to grow and survive in ethanol concentrations beyond that in which the parent Escherichia coli KO11 can survive. The invention is also based upon the discovery of an improved ethanol selection process which WO98/45425 PCTIUS98/06405 -2 alternates between selection for ethanol resistance in liquid medium and selection for rapid growth on solid medium containing a high level of chloramphenicol. This selection process resulted in novel strains of 5 Escherichia coli KO11, as discussed above, which are useful for ethanol production. It was discovered that, by using this new approach, mutants producing 20% more ethanol and completing fermentation more rapidly than the parental E. coli KO11 10 strain, could be produced. Moreover, the mutants are capable of growth in up to 50 g/L ethanol while the parent is incapable of growth at 35 g/L ethanol. Finally, the mutants show dramatically enhanced survival exposure to 100 g/L ethanol as compared to the parent. 15 These characteristics of the mutants means that the expense of ethanol production from lignocellulosic hydrolysates will decrease by achieving higher ethanol concentrations in shorter times and reducing the costs of nutrients, capital equipment, product recovery and 20 waste disposal. In one embodiment, the invention comprises a method for the selection of ethanologenic microorganisms comprising contacting the microorganisms sequentially to a liquid medium and a solid medium, wherein said liquid 25 medium is used to select for ethanol tolerance and said solid medium is used to select for ethanologenic microorganisms having the ability to grow and produce ethanol. In a preferred embodiment, the invention comprises 30 liquid media containing increasing concentrations of ethanol and solid media containing antibiotics and a WO98/45425 PCT/US98/06405 -3 fermentable sugar for use in the above selection process. In another embodiment the invention comprises an ethanologenic microorganism having the ability to grow 5 in ethanol concentrations of greater than 35 g/L. In a preferred embodiment the ethanologenic microorganism is selected from the group consisting of Erwinia, Klebsiella, Xanthomonas, Zymomonas and Escherichia, specifically K. oxytoca and E. coli. In a more 10 preferred embodiment the E. coli bacterium is selected from the group comprising LY01, LY02 and LY03. In still another embodiment the invention comprises an ethanologenic mutant having the ability to produce at least 10% more ethanol than the parental bacteria, 15 preferably Escherichia coli KO11, under equivalent fermentation conditions. Furthermore, it has been discolvered that the inactivation of cyclic AMP receptor protein and/or active biosynthetic alanine racemase results in a 20 microorganism with improved ethanol tolerance. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred 25 embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of 30 the invention.

WO98/45425 PCT/US98/06405 -4 Figure 1A shows the ability of mutants to grow in increasing concentrations of ethanol relative to the parental Escherichia coli KO11. Figure 1B shows the ability of mutants to survive 5 in 10% ethanol (w/w) relative to the parental Escherichia coli KOll. Figure 2A shows the ability of mutants, relative to the parental Escherichia coli KOll, to grow in the presence of glucose and no ethanol. 10 Figure 2B shows the ability of the LYO1 mutants to grow in varying concentrations of ethanol and constant glucose relative to the parental Escherichia coli KO11 strain in 3.5% ethanol and constant glucose. Figure 2C shows the ability of mutants, relative to 15 the parental Escherichia coli KO11, to grow in the presence of xylose and no ethanol. Figure 2D shows the ability of the LYO1 mutants to grow in varying concentrations of ethanol and constant xylose relative to the parental Escherichia coli KO11 20 strain in 3.5% ethanol and constant xylose. Figure 3A shows the osmotic tolerance of mutants to increasing concentrations of glucose relative to the parental Escherichia coli KOll. Figure 3B shows the osmotic tolerance of mutants to 25 increasing concentrations of xylose relative to the parental Escherichia coli KO11. Figure 4A shows the ability of mutants to convert 14% glucose to ethanol relative to the parental Escherichia coli KOll. 30 Figure 4B shows the ability of mutants to convert 14% xylose to ethanol relative to the parental Escherichia coli KO11.

WO98/45425 PCTIUS98/06405 -5 Figure 5 shows a map of pLOI1531 and subclones. The map is of insert DNA from KO11. The vector is not shown. Figure 6 is a map of pLOI1534 and subclones. 5 DETAILED DESCRIPTION OF THE INVENTION As described above, the invention relates to an ethanologenic mutant having improved ethanol tolerance. In one embodiment the mutant can produce at least 10% more ethanol than the parental bacteria, (e.g. 10 Escherichia coli KO11) when grown under equivalent conditions. In another embodiment the mutant can grow in ethanol concentrations which exceed those of the parental microorganism. A microorganism (e.g., an ethanologenic 15 microorganism), or "mutant" of the subject invention can be produced by the process of (1) contacting the parental microorganism (e.g., an ethanologenic microorganism) with a first liquid medium comprising an aqueous solution comprising ethanol, selecting one or 20 more microorganisms that survive; (2) contacting one or more microorganisms obtained from the preceding step with a solid growth medium for a sufficient period of time to permit growth. This process can be repeated, such as two, three, four or more times to further 25 improve ethanol tolerance. With each repeating step, the concentration of ethanol is incrementally increased. Thus, the microorganism(s) obtained from step (2) can be contacted (step (3)) with a second liquid medium comprising an aqueous solution comprising an amount of 30 ethanol greater than present in said first liquid WO98/45425 PCT/US98/06405 -6 medium, selecting one or more microorganisms that survive; and (4) contacting one or more microorganisms obtained from the preceding step with a solid growth medium for a sufficient period of time to permit growth. 5 The microorganisms selected in such a manner have improved ethanol tolerance to the parental microorganisms. The liquid and solid mediums can contain additional components, as necessary or desirable. For example, the 10 solid medium can include nutrients, such as sources of carbon, sulfur and nitrogen suitable for growth of the parental microorganism. Examples of suitable growth medium include Luria broth and Basal Salts Media. Generally, the solid medium will contain a sugar, such 15 as xylose and/or glucose. This is desirable to ensure that the microorganisms selected form this step can produce ethanol from the sugar source in good to excellent yields (such as possessing the same or better ethanol production ability as the parent microorganism). 20 The medium can also include an antibiotic, e.g. chloramphenicol, tetracycline, or ampicillin and a fermentable sugar. The liquid media include an aqueous solution of ethanol. In addition, the media can, optionally, 25 contain nutrients as well, including a suitable carbon, sulfur and nitrogen source. The liquid medium can also contain a buffer to control the pH of the medium. As in the solid media, the liquid media preferably contain sugar, such as xylose and/or glucose. The first liquid 30 medium, second liquid medium or both generally contain at least about 3.5% (by weight) ethanol. The second and subsequent liquid media contain incrementally greater WO98/45425 PCT/US98/06405 -7 concentrations of ethanol. For example, the second liquid medium can contain at least about 4% (by weight) ethanol. A third liquid medium (when present) can contain at least about 4.5% (by weight) ethanol. 5 The microorganisms which can be subjected to the above process can be prokaryotic or eukaryotic and include bacteria, yeasts and fungi. The process is particularly suited for ethanologenic microorganisms (such as ethanologenic bacterium) which comprise one or 10 more enzymes which convert a sugar (such as a pentose (e.g., xylose) or a hexose (e.g., glucose)) to ethanol. Examples of suitable ethanologenic microorganisms comprise one or more nucleic acid molecules which encode alcohol dehydrogenase and pyruvate decarboxylase. 15 Ethanologenic bacteria comprising one or more nucleic acid molecules which encode alcohol dehydrogenase and pyruvate decarboxylase (as isolated from, for example, Zymomonas species, such as Zymomonas mobilis) are known. Also microorganisms that possess 20 xylulokinase, transaldolase, transketolase and xylose isomerase are known (such as those expressed by enteric bacteria, such as Escherichia coli). Many micro organisms have the ability to convert both xylose and glucose to ethanol. Such organisms possess both sets of 25 enzymes. These organisms have been manufactured by recombinant DNA technology by inserting the nucleic acid molecules which encode one set (or operon) of these enzymes into a host cell which, preferably, expresses the second set (or operon) of these enzymes. Table 1 30 summarizes several enzymes which possess the ability to convert both pentose and hexose to ethanol in good to high yields.

WO 98/45425 PCT/US98/06405 -8 TABLE 1 Bacteria (Plasmid) Characteristics Accession Number (Deposit Date) K. oxytoca M5A1(pLOI555) Cmr, petB ATCC 68564 K. oxytoca M5A1 S1 Cmr,Ipeta K. oxytoca M5A1 S2 Cmr,Ipeta K. oxytoca M5A1 S3 Cmr,Ipeta K. oxytoca M5A1 P1 Cmr,Ipeta K. oxytoca M5A1 P2 Cmr,Ipeta K. oxytoca M5A1 B1 Cmr,Ipeta E. coli K011 frd, Cmr, Ipeta E. coli (pLOI510) pet e ATCC 68484 (11/28/90) E. coli (pLOI308-10) petc ATCC 67983 (5/15/89) E. coli C4 (pLOI292) pet e ATCC 68237 (2/23/90) E. coli TC4 (pLOI308-11) pet e ATCC 68238 (2/23/90) E. coli TC4 (pLOI297) pet e ATCC 68239 (2/23/90) E. coli TC4 (pLOI295) pet e ATCC 68240 (2/23/90) a Ipet refers to the integration of Z. mobilis pdc and adhB genes into the chromosome. b pet refers to the presence of Z. mobilis pdc and adhB genes in plasmid pLOI555. c pet refers to the presence of Z. mobilis pdc and adhB genes in the indicated plasmid. Cmr is the an E. coli shuttle vector carrying the cat gene. A more detailed description of these and other related recombinant organisms, as well as the techniques and materials used in their preparation can be found in, for example, United States Patent Nos. 5,028,539 to 5 Ingram et al., 5,000,000 to Ingram et al. 5,424,202 to Ingram et al., 5,487,989 to Fowler et al., 5,482,846 to WO98/45425 PCT/US98/06405 -9 Ingram et al., 5,554,520 to Fowler et al., 5,514,583 to Picataggio, et al., copending applications having U.S.S.N 08/363,868 filed on December 27, 1994, U.S.S.N. 08/475,925 filed on June 7, 1995 and U.S.S.N. 08/218,914 5 filed on March 28, 1994 and standard texts such as, Ausubel et al., Current Protocols in Molecular Biology, Wiley-Interscience, New York (1988) (hereinafter "Ausubel et al."), Sambrook et al., Molecular Cloning: A Laboratory Manual, Second and Third Edition, Cold 10 Spring Harbor Laboratory Press (1989 and 1992) (hereinafter "Sambrook et al.") and Bergey's Manual of Systematic Bacteriology, William & Wilkins Co., Baltimore (1984) (hereinafter "Bergey's Manual") the teachings of all of which are hereby incorporated by 15 reference in their entirety. Additional microorganisms having improved ethanologenic activity are described in copending application U.S.S.N. 08/834,901, to Ingram et al., filed April 7, 1997, which is also incorporated herein by 20 reference. Ethanologenic microorganisms, or host cells which can be employed for the insertion of enzymes which convert one or more sugars to ethanol, can be selected from bacteria, yeasts, fungi, or other cells. Suitable 25 bacteria include Erwinia, Klebsiella, Xanthomonas, Zymomonas (such as Zymomonas mobilis) and Escherichia. Preferred species include K. oxytoca and E. coli. Also envisioned are gram-positive bacteria, such as members of the genera Bacillus, for example, B. pumilus, B. 30 subtilis and B. coagulans, members of the genera Clostridium, for example, Cl. acetobutylicum, Cl. aerotolerans, Cl. thermocellum, Cl.

WO98/45425 PCTIUS98/06405 -10 thermohydrosulfuricum and C1. thermosaccharolyticum, members of the genera Cellulomanas like C. uda and Butyrivibrio fibrisolvens. Acceptable yeasts, for example, are of the species of Cryptococcus like Cr. 5 albidus, Monilia, and Pichia stipitis and Pullularia pullulans. The above microorganisms can be subjected to the selection process of the claimed invention as they occur in nature or after isolation or genetic manipulation, as 10 in mutation or genetic engineering. For example, soil or fecal samples containing microorganisms can be subjected to the described process. In such an embodiment, the ethanologenic properties of the mutated microorganism can be introduced or further improved by 15 inserting one or more enzymes which convert a sugar to ethanol, as described in the above patents. Alternatively, an isolated ethanologenic microorganism with good to excellent ethanol producing properties (such as one or more of the above recombinant 20 microorganisms) are subjected to the above process. The temperature and pressure of the process are generally not critical but should be selected to ensure viability and growth of the microorganisms. Suitable temperatures can be between about 200 and about 60'C. 25 Pressure will generally be atmospheric. The pH should also be selected towards the ability of the microorganism to remain viable and grow. For example, the pH may generally be selected to be between about 4.5 and 8.0. The tolerance of a microorganism (or members 30 of the species) can be determined readily and frequently are related to the conditions of the microorganism's native environment. Guidance for selecting optimal WO98/45425 PCT/US98/06405 -11 conditions for growth can be obtained for example in Bergey's Manual of Bacteriology. The retention time of each step of the process is also not generally critical. The time in which the 5 microorganisms are subjected to the ethanol-containing liquid medium is generally selected such that some, but not all, of the population has died. For example, the step can last between about 8 hours to two weeks. Frequently, several days can be sufficient. Where the 10 step lasts for several days, it may be desirable to exchange or add fresh liquid medium with the same or greater concentration of ethanol to the microorganisms. Likewise, the time for permitting growth of the microorganism on the solid medium is not generally 15 critical and is dependent upon the microorganism. Slow growing microorganisms will require longer retention times than fast growing microorganisms, as generally known in the art. The time is generally long enough to differentiate colonies which are growing better than 20 other colonies. Frequently, several days are sufficient for a fast growing bacteria. As set forth above, the mutants produced by the claimed process have greater ethanol tolerance than the parental microorganisms. In mutating ethanologenic 25 microorganisms, the microorganisms can have improved ability to produce ethanol, as well. Once the mutants have been isolated, the genetic basis for the mutation can be identified. For example, the chromosome and/or the RNA transcripts produced by the mutant microorganism 30 can be compared to the parent microorganism. This can be done through hybridization technology, as described generally in Sambrook, et al. and Ausubel, et al.

WO98/45425 PCT/US98/06405 -12 Once the genetic basis of the improved mutant has been identified, the mutation can be repeated or an equivalent produced through genetic engineering. For example, where the mutation is based upon the 5 inactivation of a gene or genes, then a recombinantly produced equivalent can be made by deleting the gene, deleting the regulatory sequences of the gene or targeting a site-specific mutation to shift the reading frame or remove an active site of the gene, as described 10 in Ausubel, et al. and Sambrook, et al. In any event, the result is the inability of the microorganism to express an active gene product. Where the mutation is based upon the increased expression of a gene, then recombinantly produced equivalent can be made by 15 substituting the native promoter with a stronger promoter of the gene, adding an enhancer or introducing more copies of the gene. In yet another embodiment, where the mutation is based upon an activity caused by a mutation in the coding region of a gene, a recombinantly 20 produced equivalent can be prepared by introducing the mutated sequence under the control of a promoter region recognized by the host cell. In the examples below, it has been discovered that mutants which do not express active cyclic AMP receptor 25 protein, active biosynthetic alanine racemase or both have increased ethanol tolerance. Thus the invention includes microorganisms of increased ethanol tolerance wherein the microorganism does not express active cyclic AMP receptor protein, active biosynthetic alanine 30 racemase or both. This can be achieved, for example, by such molecular biology techniques as site-specific mutagenesis and knocking out the gene, also known as WO98/45425 PCT/US98/06405 -13 "knock outs." This can be accomplished, for example, by homologous recombination, described in Ausubel, et al. and Sambrook, et al. Microorganisms which do not express active cyclic 5 AMP receptor protein and/or active biosynthetic alanine racemase can be particularly suitable host cells for expressing enzymes (e.g., recombinantly) which convert a sugar to ethanol. Suitable microorganisms for use in the invention are as discussed above. Preferred 10 microorganisms are bacteria, particularly gram-negative bacteria (e.g., enteric bacteria) such as Escherichia coli, Erwinia chrysanthia and Klebsiella oxytoca. Particularly preferred microorganisms include those described in the U.S. Patents and applications to 15 Ingram, et al. and Picataggio, et al., above. The invention further relates to methods of using the ethanol tolerant microorganisms described herein. Microorganisms which have increased ethanol tolerance can be ethanologenic or not ethanologenic. 20 Ethanologenic microorganisms can be used in methods of producing ethanol, employing processes generally known in the art. Examples of suitable ethanol-producing processes include those described in the above patents and application to Ingram, et al. and Picataggio, et 25 al., which have been incorporated by reference. An improved process for producing ethanol is described in copending application U.S.S.N. 08/833,435, by Ingram, et al., filed April 7, 1997, which is also incorporated herein by reference. These processes are particularly 30 useful for the production of ethanol from lignocellulosic waste. Other processes for the use of ethanologenic microorganisms include the fermentation of WO 98/45425 PCT/US98/06405 -14 sugar containing materials to foods and beverages. For example, ethanologenic microorganisms are employed in the manufacture of soy sauce, sake, beer and wine. As such, this invention can be employed to further improve 5 the activities and ethanol tolerance of the microorganisms employed in these processes. Microorganisms of the invention which are not ethanologenic or slightly ethanologenic are particularly useful as host cells for inserting nucleic acid 10 molecules which encode one or more enzymes which catalyze one or more reactions in the glycolytic pathway, or other pathway which converts sugar to ethanol. That is, such microorganisms are particularly useful in the production of ethanologenic microorganisms 15 through recombinant DNA technology. The invention will now be illustrated by one or more non-limiting examples. EXEMPLIFICATION Methods and Materials 20 The methods and materials described below were used in carrying out the work described in the examples which follow. For convenience and ease of understanding, the methods and materials section is divided into sub headings as follows. 25 Bacterial Strains and Media E. coli KO11 was used in these studies. This is an ethanol producing derivative of E. coli B in which the Zymomonas mobilis genes for ethanol production (pdc, adhB) have been integrated into the chromosome 30 immediately upstream from chloramphenicol acyl WO98/45425 PCT/US98/06405 -15 transferase (cat) (U.S. Patent No. 5,424,202). In this strain, resistance to chloramphenicol (600 mg liter') was used to select for high level expression of pdc and adhB. Cultures were grown in modified Luria broth 5 containing per liter: 5 g NaCl, 5 g Yeast Extract, 10 g Tryptone, 40 or 600 mg chloramphenicol, and 20-140 g of fermentable carbohydrate. Stock cultures of alcohol-resistant mutants were maintained on solid medium containing glucose (20 g 10 liter-), chloramphenicol (600 mg liter'), isopropanol (10 g liter'), and agar (15 g liter-) . Ethanol was added to the broth on a weight basis to prepare a stock solution (100 g kg

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') which was diluted as necessary. Broth containing ethanol was filter sterilized using 15 Nalgene 50 mm, 0.45Am bottle top filters (SFCA). E. coli DH5a was used as a host for the construction of pUC18-based plasmids. This strain was grown in modified Luria broth without added carbohydrate. Ampicillin (50 mg liter

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') was used for 20 selection as appropriate. Enrichment and Selection of E. coli KO11 Mutants KO11 cultures were transferred daily by diluting 1:20 to 1:200 into 10 ml of fresh broth containing ethanol and glucose (50 g liter') in 18x150 mm culture 25 tubes. Tubes were incubated for 24 h at 35 0 C without agitation. As cultures increased in density during subsequent transfers, ethanol concentrations were progressively increased to select for resistant mutants. Twice weekly, cultures were diluted and spread on solid 30 medium to enrich for ethanol-resistant mutants which grew rapidly and retained high level expression of the WO98/45425 PCT/US98/06405 -16 Z. mobilis genes. Colonies on these plates were scraped into fresh broth and diluted. The dilutions were then used as inocula in ethanol-containing broth. KO11 was initially transferred into 3.5% ethanol. After 5 days 5 of sequential transfer, the ethanol concentration in the broth was increased to 4.0%; after 13 days, the ethanol concentration was increased to 4.5%; after 14 days, the ethanol concentration was increased to 5.0%. Dilutions into higher concentrations of ethanol did not appear to 10 yield mutants with further increases in ethanol resistance which were also capable of rapid growth. Cultures were diluted and spread on solid medium containing chloramphenicol to allow the isolation of mutants. Large raised colonies were individually tested 15 for ethanol resistance in comparison to the parent, KO11. Colonies were transferred to 3 ml of broth and the resulting suspension diluted 60-fold into 13x100 culture tubes containing 0%, 4.5%, and 5% ethanol. After incubation for 24 h at 35 0 C, cell growth was 20 measured as O.D.

5 5 0 nm• Mutants were maintained on plates containing 1% isopropanol and stored at -75 0 C in 40% glycerol. Cell Survival in 10% (w/w) Ethanol Survival of mutant strains was compared to KO11 25 after dilution into 10% ethanol. Cell suspensions (approximately 0.05 O.D. at 550 nm) were prepared in Luria broth containing glucose (50 g liter

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') by transferring cells from overnight plates. These were preheated to 35 0 C and diluted at time zero with an equal 30 volume of preheated broth containing 20% ethanol. Dilution into broth lacking ethanol served as a control.

WO98/45425 PCT/US98/06405 -17 Serial dilutions were spread on solid medium at time zero (no ethanol only), after 0.5 min, and after 5 min. After overnight incubation at 30 0 C, colonies from appropriate dilutions were counted to determine relative 5 survival as colony forming units (CFU). Fermentation Experiments Inocula were grown for 16 h (Beall, D.S. et al., "Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli." 10 Biotechnol. Bioeng. 38:296-303 (1991)) (30 0 C) without agitation in Luria broth containing glucose or xylose (50 g/L). Cells were harvested by centrifugation (6000 x g, 5 min, ambient temperature) and added to initiate fermentation to provide 1.0 OD at 550 nm (approximately 15 330 mg liter', dry cell weight). Batch fermentations were carried out at 35 0 C (100 rpm) in modified 500-ml Fleaker M beakers containing 350 ml of Luria broth supplemented with glucose or xylose (140 g liter-'). Sugar solutions were sterilized by autoclaving 20 separately. Automatic addition of 2 N KOH was used to prevent acidification above pH 6. Samples were removed to measure cell mass and ethanol. Base consumption and pH were also recorded. Genetic Methods and Construction of a Genomic Library 25 Chromosomal DNA was isolated from KO11 essentially as described by Cutting, S.M. and Vander Horn, P.B., Genetic analysis, p. 37-74. In C.R. Harwood and S.M. Cutting (ed.), Molecular biological methods for Bacillus. John Wiley & sons Ltd., Chichester, England 30 (1990). A genomic library of the parental strain, KO11, WO98/45425 PCT/US98/06405 -18 was constructed by ligating Sau3AI partial digestion products (4-9 kbp fragments) into the BamHI site of pUC18 followed by transformation into DHS. The resulting library consisted of approximately 8,000 5 clones. After pooling these colonies, plasmids were isolated to produce a library. Standard procedures were used for the construction, isolation, transformation, and analysis of plasmids (Sambrook et al., 1989). Isolation of Plasmids Containing Native DNA Fragments 10 from KO11 Which Decrease Ethanol Tolerance by Reverse Complementation D-cycloserine was used to selectively kill cells capable of growing in 3.5% ethanol (w/w) while allowing non-growing cells to survive. At this concentration of 15 ethanol, mutants continue to grow while the parent remains viable without increasing in cell number. LY02 and LYO3 were transformed with the KO11 plasmid library and allowed to grow overnight into colonies. Recombinant colonies of each mutant were harvested by 20 scraping into Luria broth containing glucose (50 g liter') and 3.5% ethanol, inoculated to provide 0.1 0.D. at 550 nm, and incubated at 35 0 C. After 1.5 hr, D-cycloserine (100 mg liter

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') was added and the incubation continued for 4 hours. After 4 h, CFU/ml had 25 dropped by over 95%. Clones harboring putative genes for decreased ethanol tolerance were isolated from these plates. DNA Sequencing and Sequence Analysis The QIAprep spin plasmid kit (Qiagen, Chatsworth, 30 CA) was used for plasmid purification. Dideoxy WO98/45425 PCT/US98/06405 -19 sequencing was performed using fluorescent primers [forward, 5'CACGACGTTGTAAAACGAC-3' (SEQ ID NO:1); reverse, 5'-ATAACAATTTCACACAGGA-3' (SEQ ID NO:2)] (LI-COR, Lincoln, NE). Extension reactions were 5 performed with a Perkin Elmer GeneAmp PCR System 9600 (Norwalk, CT) using an Excel Sequencing Kit-LC (Epicentre Technologies, Madison, WI) (30 cycles; denaturation for 30 sec at 95 0 C, annealing for 30 sec at 600C, and extension for 1 min at 700C). Extension 10 products were separated and read with a LI-COR DNA Sequencer model 4000L. Sequences were analyzed using the Wisconsin Genetics Computer Group (GCG) software package and the National Center for Biotechnology Information BLAST 15 network service. Analytical Procedures Cell density was measured using a Bausch & Lomb Spectronic 70 spectrophotometer and converted to dry cell weight based on a standard curve for this organism. 20 Ethanol was measured by gas chromatography with n-propanol as an internal standard (Beall et al., 1991) using a Varian Star 3400 CX gas chromatograph. EXAMPLE 1 - Isolation of Ethanol-tolerant Mutants of E. Coli KOll 25 Using the materials and procedures outlined above, a total of 135 colonies were initially tested for growth in 4.5% (w/w) ethanol, a concentration at which the parental KO11 failed to grow. Forty three colonies were WO98/45425 PCT/US98/06405 -20 turbid after 24 h incubation at 35 0 C and were saved for further testing. Stability of the ethanol resistance trait was examined by retesting clones which had been stored 5 frozen at -75 0 C and clones which had been maintained on solid medium lacking ethanol. EXAMPLE 2 - Testing Mutants of E. Coli KO11 for Ethanol Tolerance As shown in Figure 1A, after 24 h, the mutants were 10 much more resistant to ethanol than the parent KO11. Figure 2B, Figure 2D, and Table 1A and 1B compare initial growth upon dilution into media containing various concentrations of ethanol. In the absence of ethanol, KO11 appeared to grow slightly better than the 15 mutants both with glucose and with xylose as the fermentable sugar as shown in Figures 2A and 2C. However, KO11 was unable to grow in 3.5%(w/w) ethanol while all mutants grew in ethanol concentrations of up to 5% (w/w). Ethanol tolerance was also compared using 20 other fermentable sugars which may be of interest for fuel ethanol production: lactose, arabinose, and mannose, galactose, sucrose and raffinose. KO11 growth was consistently above that of the mutants in the absence of ethanol. However, KO11 failed to grow in 25 ethanol concentrations above 3%(w/w) while the mutants grew in 5% ethanol with all sugars tested. Next, cell survival during exposure to 10% ethanol was examined (Figure 1B). All mutants tested were more resistant than KO11. This difference was particularly 30 dramatic for 0.5 min exposure where 63-84% of the WO98/45425 PCTIUS98/06405 -21 mutants retained colony forming ability while less than 10% of KO11 survived. The stability of the ethanol-tolerance trait was also examined. LYO1, LYO2, LYO3, and LYO4 were 5 transferred daily on solid medium lacking alcohol for 30 days. Cells were then used to inoculate broth cultures containing ethanol as shown in Figures 2C and 2D. The resulting growth curves were identical to those initially determined. 10 The retention of osmotic tolerance to sugars is essential for the utility of ethanol-tolerant mutants of KO11. As illustrated in Figures 3A and 3B, tolerance to xylose and glucose are similar on a molar basis. With both sugars, KO11 appeared more resistant to osmotic 15 stress than the ethanol-resistant mutants, although this difference was not dramatic. No differences were observed in growth at 48 0 C, the maximum temperature for growth. Both KO11 and the mutants grew slowly at this temperature.

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WO 98/45425 PCT/US98/06405 -25 0 a) +1 + ( H-IH +1 ~ C +1+1-H+1 +1+1+1++1+1+1+ 0 m(0m HH 0rC4- ~-1r- OtN- 0CrLn .1w N-A o fLn -0 0 nC 0)0 0 H) C qr 0 - - I _ _ Ln'_ -v _ __ri-1N _q C I4-4 4 ) +1 1+1 +- +1 +1+1 +1 +1I+1 +1+1 +1 +1 +1+1 0 -H 0) 0 0 W- N 0 H LA)LnO'C m N ()0 -10 o U 0 r M 0J 03Nw( OQ I 0 co N w0 D N 0 00 N' W0 o 414 0)4k c C'4 ('mN4 0 q vr-a N -1 0) E4-) 0 rJ (-iCN m 0C40 PQ 0 n 1,11 NH .. 0 0 HHHH1 H 4) H iHHH r-Om liH Q) H- +1 +1 +1+1 Q)1 00 +-1-1+1+1 +14+1 +1 +1 +1 +1 +1+1 w H H U) E . . . . HN(r-'4 L 0) TE m ~O0 Ln )w o) i kO-Hmn 1 ~Hr- r- ~O00 0)0 U H 0 (f1- m *l *Hi - H +1 +t +1 +1 4-4) Q H H+1 :3 -H1 CQ) m 1P 1P 144O 40 -HO P 4 kD cl 0))4-d -4 0) >1 a) 0 4 4-4) a 04J4 H-i H~0 (TI HD ('4P1 J-) 0C 00 4c l 4 4 WO98/45425 PCT/US98/06405 -26 EXAMPLE 3 - Fermentation of Sugars to Ethanol Twenty of the most promising mutants were screened for their ability to produce ethanol from 14% glucose (Figure 4A and Table 2) and 14% xylose (Figure 4B and 5 Table 2) in pH-controlled fermenters. All were superior to the parent strain KO11 in their ability to produce ethanol more rapidly and in their ability to achieve higher final ethanol concentrations. All mutants were also superior to the parent strain KO11 in their ability 10 to achieve higher yields. Mutants LYO1, LYO2, LYO3, and LYO4 were examined in more detail to establish the variability in testing. With all mutants, the cell mass produced during the fermentation of glucose or xylose was consistently higher than that produced by KO11. Base 15 consumed for the neutralization of small amounts of organic acids and dissolved CO 2 was higher for glucose than for xylose due to the higher rate for fermentation. Roughly equivalent amounts of KOH were required to maintain pH 6.0 for both KO11 and the mutants. Addition 20 of base resulted in a small dilution of product. Ethanol values in Table 2 are corrected for this dilution (ethanol produced = measured ethanol *(1000+ 1/2 mM KOH/1000)) to allow the estimation of ethanol yield. After 96 h, the ethanol yield for KO11 with 14% 25 sugar was 74% and 80% of the theoretical maximum (0.51 g ethanol per gram of pentose or hexose) for glucose and xylose, respectively. Strain LYO1 was among the best of the mutants tested. This strain achieved 85% of the maximum theoretical yield from glucose and xylose after 30 72 h and reached a final ethanol concentration of almost 60 g liter

-

(7.5% ethanol by volume).

WO 98/45425 PCT/US98/06405 -27 TABLE 2 SPECIFIC GROWTH RATE (A) Organism KO11 LY01 LYO2 LYO3 Glucose 0% EtOH 2.50 1.96 2.01 1.83 3.5% EtOH 0.00 0.20 0.19 0.19 4.0% EtOH nda 0.13 0.12 0.12 4.5% EtOH nd 0.08 0.08 0.08 5.0% EtOH nd 0.06 0.06 0.06 Xylose 0% EtOH 1.40 0.99 1.00 0.98 3.5% EtOH 0.00 0.16 0.15 0.15 4.0% EtOH nd 0.10 0.09 0.09 4.5% EtOH nd 0.06 0.05 0.05 5.0% EtOH nd 0.04 0.05 0.04 EXAMPLE 4 - Isolation of Plasmids Containing Native Genes from KO11 Which Decrease Ethanol Tolerance After D-cycloserine enrichment for survivors which fail to grow at 3.5% ethanol, a total of 32 recombinants 5 of LYO2 and 40 recombinants of LYO3 were screened in triplicate for growth in Luria broth containing 5% glucose and 4% ethanol. All grew to a lower density after 24 h than the original mutant. Six replicates of the most promising 19 clones were compared 10 simultaneously. Four clones which uniformly resulted in a decrease in ethanol tolerance as compared to LYO2(pUC18) were selected for further study.

WO98/45425 PCT/US98/06405 -28 EXAMPLE 5 - Identification of Genes Which Reduce Ethanol Tolerance Sequence analysis of the ends of the inserts in the four selected plasmids revealed that they consisted of 5 two pairs of siblings. The entire E. coli genome is now available in the GenBank database. The cloned fragments were readily identified by using the terminal sequences and BLAST network server. Plasmid pLOIl531 contains a 3.6 kbp fragment of 10 KO11 DNA within the 67.4 min-76.0 min region of the E. coli chromosome (GenBank Accession Number U18997). Subclones were prepared and sequenced to confirm gene arrangement in KO11 and to identify the genes which are responsible for a decrease in ethanol tolerance (Figure 15 5). Only 2 open reading frames were present: crp and a large open reading frame of unidentified function (ORF 0696). A comparison of the effects of pUC18, pLOI1531 and deleted clones (pLOIl532 and pLOI1533) on LYO3 indicated that the crp gene (cyclic AMP receptor 20 protein) was responsible for the decrease in ethanol tolerance. pLOI1534 contains a 7.39 kbp fragment of KO11 DNA within the 89.2 min -92.8 min segment (Accession Number U00006 of the E. coli chromosome (Figure 6). Deleted 25 derivatives of this clone were also sequenced to confirm gene arrangement and revealed the presence of 3 complete and 2 partial open reading frames: dnaB', alr, tyrB, napA(hobH), and uvrA'. A comparison of deleted derivatives identified the alr gene (biosynthetic 30 alanine racemase) as being responsible for decreasing the ethanol tolerance of LYO2.

WO 98/45425 PCT/US98/06405 -29 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: University of Florida Research Foundation Incorporated (ii) TITLE OF INVENTION: Development of High-Ethanol Resistant Escherichia Coli (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Hamilton, Brook, Smith & Reynolds, P.C. (B) STREET: Two Militia Drive (C) CITY: Lexington (D) STATE: MA (E) COUNTRY: USA (F) ZIP: 02173-4799 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: Windows 95 (D) SOFTWARE: FastSEQ for Windows Version 2.0b (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/834,900 (B) FILING DATE: 07-APR-1997 WO 98/45425 PCT/US98/06405 -30 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Elmore, Carolyn S (B) REGISTRATION NUMBER: 37,567 (C) REFERENCE/DOCKET NUMBER: UF97-02.PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 781-861-6240 (B) TELEFAX: 781-861-9540 (C) TELEX: (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CACGACGTTG TAAAACGAC 19 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ATAACAATTT CACACAGGA 19 WO98/45425 PCT/US98/06405 -31 EQUIVALENTS While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the 5 art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, 10 many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the claims.

Claims (29)

1. An ethanologenic microorganism comprising one or more nucleic acid molecules which encode alcohol 5 dehydrogenase and pyruvate decarboxylase; wherein said bacterium does not express active cyclic AMP receptor protein, active biosynthetic alanine racemase or both.

2. The ethanologenic microorganism according to Claim 10 1, wherein said microorganism is a bacterium selected from the group consisting of Erwinia, Klebsiella, Xanthomonas and Escherichia.

3. The ethanologenic bacterium according to Claim 2, wherein said bacterium is Klebsiella oxytoca. 15

4. The ethanologenic bacteria according to Claim 2, wherein said bacterium is Escherichia coli.

5. The ethanologenic bacteria according to Claim 2 wherein said bacterium comprises a heterologous nucleic acid molecule which encodes Zymomonas 20 alcohol dehydrogenase and pyruvate decarboxylase.

6. The ethanologenic bacterium according to Claim 5 wherein said Zymomonas alcohol dehydrogenase and pyruvate decarboxylase are encoded by a nucleic acid molecule isolated from Zymomonas mobilis. WO 98/45425 PCT/US98/06405 -33

7. An ethanologenic Escherichia coli which comprises a heterologous nucleic acid molecule isolated from Zymomonas mobilis which encodes alcohol dehydrogenase and pyruvate decarboxylase, wherein 5 said bacterium does not express active cyclic AMP receptor protein, active biosynthetic alanine racemase or both.

8. An ethanologenic microorganism produced by the process comprising the steps of: 10 (a) contacting an ethanologenic microorganism with a first liquid medium comprising an aqueous solution comprising ethanol, selecting one or more microorganisms that survive; (b) contacting one or more microorganisms obtained 15 from step (a) with a solid growth medium for a sufficient period of time to permit growth; (c) contacting one or more microorganisms obtained from step (b) with a second liquid medium comprising an aqueous solution comprising an 20 amount of ethanol greater than present in said first liquid medium, selecting one or more microorganisms that survive; and (d) contacting one or more microorganisms obtained from step (c) with a solid growth medium for a 25 sufficient period of time to permit growth.

9. The ethanologenic microorganism according to Claim 8 wherein said microorganism is a bacterium. WO98/45425 PCT/US98/06405 -34

10. The ethanologenic microorganism according to Claim 9, wherein said microorganism is selected from the group consisting of Erwinia, Klebsiella, Xanthomonas and Escherichia. 5

11. The ethanologenic microorganism according to Claim 10, wherein said microorganism is Klebsiella oxytoca.

12. The ethanologenic microorganism according to Claim 10, wherein said microorganism is Escherichia coli. 10

13. The ethanologenic microorganism according to Claim 10, wherein said microorganism comprises a heterologous nucleic acid molecule which encodes Zymomonas alcohol dehydrogenase and pyruvate decarboxylase. 15

14. The ethanologenic microorganism according to Claim 13, wherein said Zymomonas alcohol dehydrogenase and pyruvate decarboxylase are encoded by a nucleic acid molecule isolated from Zymomonas mobilis.

15. A method for producing an ethanologenic 20 microorganism mutant comprising: (a) contacting an ethanologenic microorganism with a first liquid medium comprising an aqueous solution comprising ethanol, selecting one or more microorganisms that survive; 25 (b) contacting one or more microorganisms obtained from step (a) with a solid growth medium for a sufficient period of time to permit growth; WO98/45425 PCTIUS98/06405 -35 (c) contacting one or more microorganisms obtained from step (b) with a second liquid medium comprising an aqueous solution comprising an amount of ethanol greater than present in said 5 first liquid medium, selecting one or more microorganisms that survive; and (d) contacting one or more microorganisms obtained from step (c) with a solid growth medium for a sufficient period of time to permit growth. 10

16. The method according to Claim 15 wherein said microorganism is a bacterium.

17. The method according to Claim 16, wherein said microorganism is selected from the group consisting of Erwinia, Klebsiella, Xanthomonas and 15 Escherichia.

18. The method according to Claim 17, wherein said microorganism is Klebsiella oxytoca.

19. The method according to Claim 17, wherein said microorganism is Escherichia coli.

20 20. The method according to Claim 17, wherein said microorganism comprises a heterologous nucleic acid molecule which encodes Zymomonas alcohol dehydrogenase and pyruvate decarboxylase.

21. The method according to Claim 20 wherein said 25 Zymomonas alcohol dehydrogenase and pyruvate decarboxylase are encoded by a nucleic acid molecule isolated from Zymomonas mobilis. WO 98/45425 PCT/US98/06405 -36

22. The method according to Claim 15, wherein said solid medium contains an antibiotic and a fermentable sugar.

23. The method according to Claim 22, wherein said 5 antibiotic is selected from the group consisting of chloramphenicol, tetracycline and ampicillin.

24. The method according to Claim 15, wherein said first liquid medium, said second liquid medium or both further comprise a sugar. 10

25. The method according to Claim 24, wherein said first liquid medium, said second liquid medium or both comprise at least about 3.5% (by weight) ethanol.

26. The method according to Claim 25, wherein said 15 second liquid medium comprises at least about 4% (by weight) ethanol.

27. The method according to Claim 26, further comprising steps (e) contacting one or more microorganism obtained 20 from step (d) with a third liquid medium comprising an aqueous solution comprising an amount of ethanol greater than present in said second liquid medium, selecting one or more microorganisms that survive; and 25 (f) contacting one or more microorganisms obtained from step (e) with a solid growth medium for a sufficient period of time to permit growth. WO98/45425 PCT/US98/06405 -37

28. The method according to Claim 15, wherein said ethanologenic microorganism mutant can grow in ethanol concentrations of greater than 35 g L - .

29. The method according to Claim 15, wherein said 5 ethanologenic microorganism mutant can produce at least 10% more ethanol than Escherichia coli KO11 under equivalent fermentation conditions.