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  Cloning and sequencing of pyruvate decarboxylase (pdc) genes from bacteria and uses thereforUSPTO Application #: 20080009609Title: Cloning and sequencing of pyruvate decarboxylase (pdc) genes from bacteria and uses therefor Abstract: The invention provides isolated nucleic acids molecules which encode pyruvate decarboxylase enzymes having improved decarboxylase activity, substrate affinity, thermostability, and activity at different pH. The nucleic acids of the invention also have a codon usage which allows for high expression in a variety of host cells. Accordingly, the invention provides recombinant expression vectors containing such nucleic acid molecules, recombinant host cells comprising the expression vectors, host cells further comprising other ethanologenic enzymes, and methods for producing useful substances, e.g., acetaldehyde and ethanol, using such host cells. (end of abstract)
Agent: Edwards Angell Palmer & Dodge LLP - Boston, MA, US Inventors: Julie A. Maupin-Furlow, Lee Ann Talarico, Krishnan Chandra Raj, Lonnie O. Ingram USPTO Applicaton #: 20080009609 - Class: 530387900 (USPTO) Related Patent Categories: Chemistry: Natural Resins Or Derivatives; Peptides Or Proteins; Lignins Or Reaction Products Thereof, Proteins, I.e., More Than 100 Amino Acid Residues, Blood Proteins Or Globulins, E.g., Proteoglycans, Platelet Factor 4, Thyroglobulin, Thyroxine, Etc., Globulins, Immunoglobulin, Antibody, Or Fragment Thereof, Other Than Immunoglobulin Antibody, Or Fragment Thereof That Is Conjugated Or Absorbed, Binds Specifically-identified Amino Acid Sequence The Patent Description & Claims data below is from USPTO Patent Application 20080009609. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED INFORMATION [0001] This application claims priority to U.S. provisional application No. 60/288,638, entitled "High-Level Production of Active Sarcina ventriculi Pyruvate Decarboxylase in Recombinant Bacillus megaterium"; U.S. provisional application No. 60/288,671, entitled "Cloning, Expression, and Characterization of Pyruvate Decarboxylase from the Acid-Tolerant, Anaerobic Gram-Positive Bacterium Sarcina ventriculi Goodsir"; U.S. provisional application No. 60/288,698, entitled "Acetobacter pasteurianus Pyruvate Decarboxylase: Biochemical, Genetic, and Physiological Properties"; U.S. provisional application No. 60/288,622, entitled "Biochemical and Biophysical Characterization of Pyruvate Decarboxylase from the Acetic Acid Bacterium Acetobacter pasteurianus"; and U.S. provisional application No. 60/288,699, entitled "Pyruvate Decarboxylase: A Key Enzyme for the Oxidative Metabolism of Lactic Acid by Acetobacter pasteurianus"; all of which were filed on May 4, 2001 and are incorporated herein in their entireties by this reference. The contents of all patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties. BACKGROUND OF THE INVENTION [0003] Many environmental and societal benefits would result from the replacement of petroleum-based automotive fuels with renewable fuels obtained from plant materials (Lynd et al., (1991) Science 251:1318-1323; Olson et al., (1996) Enzyme Microb. Technol. 18:1-17; Wyman et al., (1995) Amer. Chem. Soc. Symp. 618:272-290). Each year, the United States burns over 120 billion gallons of automotive fuel, roughly equivalent to the total amount of imported petroleum. The development of ethanol as a renewable alternative fuel has the potential to eliminate United States dependence on imported oil, improve the environment, and provide new employment (Sheehan, (1994) ACS Symposium Series No. 566, ACS Press, pp 1-53). [0004] In theory, the solution to the problem of imported oil for automotive fuel appears quite simple. Rather than using petroleum, a finite resource, ethanol, a renewable resource, can be produced efficiently by the fermentation of plant material. Indeed, Brazil has demonstrated the feasibility of producing ethanol and the use of ethanol as a primary automotive fuel for more than 20 years. Similarly, the United States produces over 1.2 billion gallons of fuel ethanol each year. Currently, fuel ethanol is produced from corn starch or cane syrup utilizing either Saccharomyces cerevisiae or Zymomonas mobilis (Z. mobilis). However, neither of these sugar sources can supply the volumes needed to realize a replacement of petroleum-based automotive fuels. In addition, both cane sugar and corn starch are relatively expensive starting materials, which have competing uses as food products. [0005] Moreover, these sugar substrates represent only a fraction of the total carbohydrates in plants. Indeed, the majority of the carbohydrates in plants are in the form of lignocellulose, a complex structural polymer containing cellulose, hemicellulose, pectin, and lignin. Lignocellulose is found in, for example, the stems, leaves, hulls, husks, and cobs of plants. Hydrolysis of these polymers releases a mixture of neutral sugars including glucose, xylose, mannose, galactose, and arabinose. No known natural organism can rapidly and efficiently metabolize all of these sugars into ethanol. [0006] Nonetheless, in an effort to exploit this substrate source, the Gulf Oil Company developed a method for the production of ethanol from cellulose using a yeast-based process termed simultaneous saccharification and fermentation (SSF) (Gauss et al. (1976) U.S. Pat. No. 3,990,944). Fungal cellulase preparations and yeasts were added to a slurry of the cellulosic substrate in a single vessel. Ethanol was produced concurrently during cellulose hydrolysis. However, Gulf's SSF process has some shortcomings. For example, the cell cycle time for yeast is relatively long (24-36 hours) and they are unable to ferment complex sugars. Further, fungal cellulases have to be added which have been considered, thus far, to be too expensive for use in large scale bioethanol processes (Himmel et al., (1997) Amer. Chem. Soc. pp. 2-45; Ingram et al., (1987) Appl. Environ. Microbiol. 53:2420-2425; Okamoto et al., (1994) Appl. Microbiol. Biotechnol. 42:563-568; Philippidis, G., (1994) Amer. Chem. Soc. pp. 188-217; Saito et al., (1990) J. Ferment. Bioeng. 69:282-286; Sheehan, J., (1994) Amer. Chem. Soc. pp 1-52; Su et al., (1993) Biotechnol. Lett. 15:979-984). [0007] Moreover, producing ethanol using other organisms is difficult because pyruvate decarboxylase (PDC), a key enzyme for fermenting ethanol, is common only to plants, yeast, and fungi; and is rarely found in bacteria and is absent in animals (9, 25). SUMMARY OF THE INVENTION [0008] The development of inexpensive enzymatic methods for ethanol fermentation has great potential for improving the efficiency of substrate utilization and the economics of the fermentation process. Accordingly, developing enzymes and, advantageously, biocatalysts that produce such enzymes which can be used for the efficient depolymerization of complex sugars and subsequent rapid fermentation of the sugar into alcohol, would be of great benefit. [0009] Certain microbes, such as Gram-negative and Gram-positive bacteria produce a number of fermentation enzymes, which are capable of catalyzing, for example, the depolymerization of cellulose and hemicellulose to produce fermentable sugars, conversion of a sugar into pyruvate, the substrate pyruvate into acetaldehyde, and finally, the substrate acetaldehyde into ethanol. However, such organisms rarely produce all of the necessarily enzymes at the most desirable levels. [0010] Accordingly, the invention provides genes encoding pyruvate decarboxylases which can be expressed at high levels in a range of organisms. Thus, when expressed in an organism, or cultured with an organism, that produces the remaining key enzymes needed for ethanol fermentation, superior levels of ethanol production can be achieved. These enzymes, for example pyruvate decarboxylase (PDC), alone or in combination with alcohol dehydrogenase (ADH), can be used as a crude extract having a desired mixture of activity or, can be used as a purified composition. [0011] Moreover, a biocatalyst, advantageously a recombinant bacterium, more advantageously a ethanologenic bacterium, can be engineered to express one or more of these enzymatic activities in particular amounts sufficient for fermenting a sugar(s). Such a biocatalyst is suitable for the efficient degradation of complex sugars and subsequent fermentation into alcohol by a process known as simultaneous saccharification and fermentation (SSF). [0012] The present invention is based, at least in part, on the discovery of key enzyme-encoding genes of ethanol fermentation in bacteria. In particular, the identification of the pdc gene of Zymobacter palmae, Acetobacter pasteurianus, and Sarcina ventriculi has been achieved. These genes have been determined to encode pyruvate decarboxylase enzymes having superior pyruvate decarboxylase activity, substrate affinity, for, e.g., pyruvate, as well as thermostability, and superior activity at different pH. Still further, the pdc genes of the invention have a codon usage that affords for their high expression in a range of organisms. [0013] Accordingly, in one aspect, the invention provides isolated nucleic acid molecules encoding pyruvate decarboxylase polypeptides (PDC) or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of PDC-encoding nucleic acids. [0014] In one embodiment, an pyruvate decarboxylase (pdc) nucleic acid molecule of the invention is at least about 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more identical to the nucleotide sequence (e.g., when compared to the overall length of the nucleotide sequence) shown in SEQ ID NO:1, 3, 5, or a complement thereof. [0015] In a particular embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:1, 3, 5, or a complement thereof. [0016] In another embodiment, a pdc nucleic acid molecule includes a nucleic acid sequence encoding a polypeptide having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, 4, or 6. In a particular embodiment, a pdc nucleic acid molecule includes a nucleotide sequence encoding a (PDC) polypeptide having at least about 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more identity (e.g., when compared to the overall length of the amino acid sequence) to the amino acid sequence shown in SEQ ID NO:2, 4, or 6. [0017] In one particular embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of the pyruvate decarboxylase enzyme of Zymobacter palmae having the amino acid sequence of SEQ ID NO: 2. [0018] In another particular embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of the pyruvate decarboxylase enzyme of Acetobacter pasteurianus having the amino acid sequence of SEQ ID NO: 4. [0019] In yet another particular embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of the pyruvate decarboxylase enzyme of Sarcina ventriculi having the amino acid sequence of SEQ ID NO: 6. [0020] In another particular embodiment, the nucleic acid molecule is at least about 1600 nucleotides in length and encodes a polypeptide having pyruvate decarboxylase activity (as described herein). [0021] In a more particular embodiment, the invention provides a plasmid, pJAM3440, encoding a pdc gene derived from Zymobacter palmae represented by a deposit with the American Type Culture Collection designated as deposit number ATCC ______. In a related embodiment, the invention provides a plasmid, pJAM304, encoding apdc gene derived from Acetobacter pasteurianus represented by a deposit with the American Type Culture Collection designated as deposit number ATCC ______. In another related embodiment, the invention provides a plasmid, pJAM419, encoding apdc gene derived from Sarcina ventriculi represented by a deposit with the American Type Culture Collection designated as deposit number ATCC ______. [0022] Another embodiment of the invention features nucleic acid molecules, advantageously pyruvate decarboxylase nucleic acid molecules, which specifically detect pyruvate decarboxylase nucleic acid molecules (i.e., pdc gene(s)) relative to nucleic acid molecules encoding non-pyruvate decarboxylase (PDC) polypeptides. For example, in one embodiment, such a nucleic acid molecule is at least 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 500-1000, 1000-1500, 1500-1500 or more nucleotides in length and/or hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, 3, or 5, or a complement thereof. It should be understood that the nucleic acid molecule can be of a length within a range having one of the numbers listed above as a lower limit and another number as the upper limit for the number of nucleotides in length, e.g., molecules that are 60-80, 300-1000, or 150-400 nucleotides in length. Continue reading... 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