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United States Patent (19) 11) Patent Number: 5,482,846 Ingram Et Al

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United States Patent (19) 11) Patent Number: 5,482,846 Ingram Et Al US005482846A United States Patent (19) 11) Patent Number: 5,482,846 Ingram et al. 45) Date of Patent: Jan. 9, 1996 54 ETHANOL PRODUCTION IN Bioengineering 34:1295-1304. GRAM-POSITIVE MICROBES Mistry, F. R., C. L. Cooney (1989) “Production of Ethanol by Clostridium thermosaccharolyticum. II. A Quantitative (75) Inventors: Lonnie O’Neal Ingram; Maria D. F. Model Describing Product Distrubutions' Biotechnology Barbosa-Alleyne, both of Gainesville, and Bioengineering 34:1305–1320. Fla. Neale, A. D. et al. (1987) “Nucleotide sequence of the pyruvate decarboxylase gene from Zymomonas mobilis' 73) Assignee: University of Florida, Gainesville, Fla. Nucleic Acids Research 15(4): 1753-1761. O'Hara, M.B., J. H. Hageman (1990) "Energy and Calcium (21) Appl. No.: 220,072 Ion Dependence of Proteolysis during Sporulation of Bacil lus subtilis Cells' Journal of Bacteriology 172(8): (22 Filed: Mar. 30, 1994 4161-4170. Sarthy, A. V. et al. (1987) “Expression of the Escherichia Related U.S. Application Data coli Xylose Isomerase Gene in Saccharomyces cerevisiae" 63 Continuation-in-part of Ser. No. 26,051, Mar. 5, 1993, which Applied and Environmental Microbiology is a continuation-in-part of Ser. No. 946,290, Sep. 17, 1992, 53(9):1996-2000. which is a continuation-in-part of Ser. No. 846,344, Mar. 6, Tolan, J. S., R. K. Finn (1987) "Fermentation of D-Xylose 1992, Pat. No. 5,424,202, which is a continuation-in-part of and L-Aravinose to Ethanol by Erwina chrysanthemi” Ser. No. 670,821, Mar. 18, 1991, abandoned, and a continu ation-in-part of Ser. No. 624,227, Dec. 7, 1990, abandoned, Applied and Environmental Microbiology 53(9): each is a continuation-in-part of Ser. No. 352,062, May 15, 2033-2038. 1989, Pat. No. 5,000,000, which is a continuation-in-part of Tolan, J. S., K. Finn (1987) “Fermentation of D-Xylose to Ser. No. 239,099, Aug. 31, 1988, abandoned. Ethanol by Genetically Modified Klebsiella planticola” (51) Int. Cl." .................... C12N 1/21; C12P 7/10 Applied and Environmental Microbiology 53(9): 2039-2044. 52 U.S. Cl. ............. 435/161; 435/163; 435/252.31 Wood, B.E., L. O. Ingram (1992) "Ethanol Production from 58) Field of Search ............................ 435/252.3, 252.31, Cellobiose, Amorphous Cellulose, and Crystalling Cellulose 435/161, 163,320.1 by Recombinant Klebsiella oxytoca Containing Chromo somally Integrated Zymomonas mobilis Genes for Ethanol (56) References Cited Production and Plasimds Expressing Thermostable Cellu U.S. PATENT DOCUMENTS lase Genes from Clostridium thermocellum' Applied and Environmental Microbiology 58(7): 2103-2110. 4,493,893 1/1985 Mielenz et al. ...................... 435/1723 Barbosa et al., Current Microbiol. 28:279-282 (1994). OTHER PUBLICATIONS Brock et al., Biology of Microorganisms, 4th Edition, Prentice-Hall, Inc., Englewood Cliffs, N.J., 1984, pp. Beall, D. S., L. O. Ingram (1993) "Genetic Engineering of 803-805. Soft-rot Bacteria for Ethanol Production from Lignocellu Sneath et al., Eds., Bergey's Manual of Systematic Biology, lose' J. Ind. Microbiol. 11:151-155. vol. 2, Williams & Wilkins, Baltimore, Md., 1986, pp. Bräu, B., H. Sahm (1986) “Cloning and expression of the xxi-xxiii. structural gene for pyruvate decarboxylase of Zymomonas Hashiba et al., Biosci. Biotech. Biochem. 56: 190-194 mobilis in Escherichia coli'' Arch. Microbiol 144:296-301. (1992). Bringer-Meyer, S. et al. (1986) “Pyruvate decarboxylase Danilevich et al., Molecular Biology 28: 158-166 (1994). from Zymomonas mobilis. Isolation and partial character Coleman et al., J. Bacteriol. 169: 4302-4307 (1987). ization' Arch Microbiol 146:105-110. Gold et al., J. Ind. Microbiol. 10: 45-54 (1992). Conway, T. et al. (1987) “Cloning and Sequencing of the Panbangred et al., Appl. Microbiol. Biotechnol. 22:259-264 Alcohol Dehydrogenase II Gene from Zymomonas mobilis' (1985). Journal of Bacteriology 169(6): 2591-2597. Lawford et al., Appl. Biochem. Biotechnol. 28/29: 221-236 Gong, C.-S. et al. (1981) “Production of Ethanol from (1991). D-Xylose by Using D-Xylose Isomerase and Yeasts' Applied and Environmental Microbiology 41(2): 430-436. Primary Examiner-Robert A. Wax Ingram, L. O., T. Conway (1988) "Expression of Different Assistant Examiner-Eric Grimes Levels of Ethanologenic Enzymes from Zymomonas mobilis Attorney, Agent, or Firm-Saliwanchik & Saliwanchik in Recombinant Strains of Escherichia coli'' Applied and (57) ABSTRACT Environmental Microbiology 54(2): 397–404. Ingram, L. O. et al. (1987) "Genetic Engineering of Ethanol The subject invention concerns the transformation of Gram Production in Escherichia coli'' Applied and Environmental positive bacteria with heterologous genes which confer upon Microbiology 53(10): 2420-2425. these microbes the ability to produce ethanol as a fermen Koide, Y. et al. (1986) "Cloning and Sequencing of the tation product. Specifically exemplified is the transformation Major Intracellular Serine Protease Gene of Bacillus subti of bacteria with genes, obtainable from Zymomonas mobilis, lis' Journal of Bacteriology 167(1): 110-116. which encode pyruvate decarboxylase and alcohol dehydro Mistry, F. R., C. L. Cooney (1989) “Production of Ethanol genase. by Clostridium thermosaccharolyticum. I. Effect of Cell Recycle and Environmental Parameters' Biotechnology and 2 Claims, 2 Drawing Sheets U.S. Patent Jan. 9, 1996 Sheet 1 of 2 5,482,846 U.S. Patent Jan. 9, 1996 Sheet 2 of 2 5,482,846 5,482,846 1. 2 ETHANOL PRODUCTION IN different enzymatic steps are required for alcoholic fermen GRAM-POSITIVE MICROBES tation. Pyruvate decarboxylase cleaves pyruvate into acetal dehyde and carbon dioxide. Alcohol dehydrogenase serves This research was supported in part by Grant Nos. to regenerate NAD" by transferring hydrogen equivalents 92-37308-7471 and 583620-2-112 from the Department of 5 from NADH to acetaldehyde, thereby producing ethanol. Agriculture and Grant No. FG05-86ER13574 from the Divi The reactions for the regeneration of NAD" by alcoholic sion of Energy Biosciences in the Department of Energy. fermentation are: CROSS-REFERENCE TO RELATED APPLICATIONS 2 Pyruvate-2 Acetaldehyde-H2 CO2 Acetaldehyde -2 NADH-92 Ethanol-2 NAD This is a continuation-in-part of application Ser. No. 10 08/026,051, filed Mar. 5, 1993; which is a continuation-in The net reaction for alcoholic fermentation is: part of U.S. Ser. No. 07/946,290, filed Sep. 17, 1992; which is a continuation-in-part of U.S. Ser. No. 07/846,344, filed 2 Pyruvate-2 NADH-2 Ethanol-2 CO+2 NAD" Mar. 6, 1992 (now U.S. Pat. No. 5,424,202); which is a continuation-in-part of U.S. Ser. No. 07/670,821, filed Mar. 15 Pentose sugars, which can also be converted to ethanol, 18, 1991, now abandoned, and U.S. Pat. No. 07/624,227, are abundant in nature as a major component of lignocellu filed Dec. 7, 1990, now abandoned; both of which are losic biomass. One such pentose sugar is xylose, which is continuations-in-part of application Ser. No. 07/352,062, second only to glucose in natural abundance. Thus, as with fled May 15, 1989 (now U.S. Pat. No. 5,000,000), itself a hexose Sugars, pentose sugars such as Xylose can be con 20 verted into pyruvate by modified glycolytic pathways. The continuation-in-part of application Ser. No. 07/239,099, pyruvate can then be redirected to ethanol. The net reaction filed Aug. 31, 1988 (now abandoned). The respective con for a pentose sugar is typically: three pentose sugars yield tents of these patent documents are hereby incorporated by five ethanol and five carbon dioxide molecules. Because of reference. the abundance of pentose sugars, the fermentation of xylose BACKGROUND OF THE INVENTION 25 and other hemicellulose constituents is an attractive option for the development of an economically viable process to During glycolysis, cells convert simple sugars, such as produce ethanol from biomass. However, no naturally occur glucose, into pyruvic acid, with a net production of ATP and ring microorganisms have been found which rapidly and NADH. In the absence of a functioning electron transport efficiently ferment pentoses to high levels of ethanol. Yeasts system for oxidative phosphorylation, at least 95% of the 30 such as Pachysolen tannophilus, Candida shehatae, and pyruvic acid is consumed in short pathways which regen Pichia stipitis have been investigated as candidates for erate NAD", an obligate requirement for continued glyco xylose fermentation. Efficient fermentation by these pen lysis and ATP production. The waste products of these tose-fermenting yeasts has proven difficult due to a require NAD" regeneration systems are commonly referred to as ment for oxygen during ethanol production, acetate toxicity, fermentation products. 35 and the production of xylitol as a by-product. Other In most animals and plants as well as bacteria, yeast, and approaches to xylose fermentation include the conversion of fungi, glucose is degraded initially by an anaerobic pathway xylose to xylulose using xylose isomerase prior to fermen prior to either oxidative or fermentative metabolism. The tation by Saccharomyces cerevisiae (Gong et at, 1981) and most common such pathway, termed glycolysis, refers to the the development of genetically engineered strains of S. series of enzymatic steps whereby the six-carbon glucose cerevisiae which express xylose isomerase (Sarthy et at, molecule is broken down, via multiple intermediates, into 1987). The thermophilic bacterium, Clostridium thermosac two molecules of the three carbon compound, pyruvate. charotyticum, represent an alternative and promising During this process, two molecules of NAD" are reduced to approach to xylose fermentation (Mistry and Cooney, 1989 form NADH. The net reaction in this transformation of p. 1295); Mistry and Cooney, 1989 p. 1305). High volu glucose into pyruvate is: 45 metric productivities have been achieved in continuous glucose-1-2 P-2 ADP+2 NAD'- 2 pyruvate-2 ATP+2 NADH+2 culture although final ethanol concentrations remained low.
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