# US 10,119,115 B2 · Methods and compositions for producing solvents > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,119,115 B2 - Document ID: US10119115B2 - Status: Granted - Kind: B2 - Application number: 14/796,253 - Filed: 2015-07-10 - Published: 2016-03-10 - Granted: 2018-11-06 - Earliest claimed: 2007-05-17 - Assignees: The Board of Trustees of the University of Illinois; Eastman Renewable Materials, LLC - Applicants: The Board of Trustees of the University of Illinois; Eastman Renewable Materials, LLC - Inventors: Hans P. Blaschek; Steven F. Stoddard; Zhen Shi - Web page: https://patentgrep.com/patent/US10119115B2 ## CPC - C12N 1/20: Bacteria; Culture media therefor - C12N 1/205: Bacterial isolates - C12N 9/0006: {acting on CH-OH groups as donors (1.1)} - C12N 9/001: {acting on the CH-CH group of donors (1.3)} - C12N 9/1217: {Phosphotransferases with a carboxyl group as acceptor (2.7.2)} - C12N 15/70: Vectors or expression systems specially adapted for E. coli - C12P 7/065: {with microorganisms other than yeasts} - C12P 7/16: Butanols - C12P 7/28: Acetone-containing products - C12R 2001/145: Clostridium - C12Y 101/01001: Alcohol dehydrogenase (1.1.1.1) - Y02E 50/10: Biofuels, e.g. bio-diesel - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS - C12Y: ENZYMES - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract Described herein are methods, compositions and synthetic biology approaches for solvent production, including but not limited to butanol production. Described herein are recombinant bacteria and yeast strains which may be used in production of a solvent, including but not limited to butanol, from lignocellulosic and other plant-based feedstocks. Described herein are methods of producing solvents, including but not limited to butanol, using bacteria and yeast strains. Described herein are methods of producing organisms that display highly efficient butanol production. ## Claims ### Claim 1 A recombinant solventogenic microorganism, wherein the microorganism is transformed with: (a) a nucleic acid molecule comprising at least one Clostridium beijerinckii Adh (alcohol dehydrogenase) polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 14, wherein the polynucleotide encodes a polypeptide having alcohol dehydrogenase activity; (b) a nucleic acid molecule comprising at least one Clostridium beijerinckii Bcd (butyryl-CoA dehydrogenase) polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 2, wherein the polynucleotide encodes a polypeptide having butyryl-CoA dehydrogenase activity; and (c) a nucleic acid molecule comprising at least one Clostridium beijerinckii Buk (butyrate kinase) polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 3, wherein the polynucleotide encodes a polypeptide having butyrate kinase activity; wherein the recombinant solventogenic microorganism is capable of (i) more efficient butanol production, (ii) faster butanol production, and/or (iii) increased butanol production relative to the same recombinant solventogenic microorganism that is not transformed with the nucleic acid molecules of (a), (b), and (c). ### Claim 2 (depends on 1) The recombinant solventogenic microorganism of claim 1, wherein the microorganism is further transformed with a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 4, 5, 6 or combinations thereof, and wherein the polynucleotide encodes a polypeptide having chemotaxis protein activity. ### Claim 3 (depends on 1) The recombinant solventogenic microorganism of claim 1, wherein expression of the Clostridium beijerinckii Adh polynucleotide is controlled by an inducible or a constitutive promoter. ### Claim 4 (depends on 1) The recombinant solventogenic microorganism of claim 1, which is a yeast, bacterium, or fungi. ### Claim 5 (depends on 4) The recombinant solventogenic microorganism of claim 4, wherein the bacterium is an Escherichia or a Clostridium. ### Claim 6 (depends on 4) The recombinant solventogenic microorganism of claim 4, wherein the bacterium is a Clostridium beijerinckii or Clostridium acetobutylicum. ### Claim 7 (depends on 4) The recombinant solventogenic microorganism of claim 4, wherein the bacterium is a recombinant Clostridium beijerinckii 8052. ### Claim 8 (depends on 1) The recombinant solventogenic microorganism of claim 1, which is cellulolytic. ### Claim 9 (depends on 1) The recombinant solventogenic microorganism of claim 1, wherein the Clostridium beijernickii Adh polynucleotide is a heterologous Adh polynucleotide from Clostridium beijernickii NCIMB 8052. ### Claim 10 (depends on 1) A method of producing butanol comprising culturing the recombinant solventogenic microorganism of claim 1, such that butanol is produced. ### Claim 11 (depends on 1) A method for making the recombinant solventogenic microorganism of claim 1, comprising transforming a solventogenic microorganism with: (a) a nucleic acid molecule comprising at least one Clostridium beijerinckii Adh polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 14, wherein the polynucleotide encodes a polypeptide having alcohol dehydrogenase activity, (b) a nucleic acid molecule comprising at least one Clostridium beijerinckii Bcd polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 2, wherein the polynucleotide encodes a polypeptide having butyryl-CoA dehydrogenase activity, and (c) a nucleic acid molecule comprising at least one Clostridium beijerinckii Buk polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 3, wherein the polynucleotide encodes a polypeptide having butyrate kinase activity; wherein the recombinant solventogenic microorganism is capable of (i) more efficient butanol production, (ii) faster butanol production, and/or (iii) increased butanol production relative to the microorganism prior to the transformation with the nucleic acid molecules of (a), (b), and (c). ### Claim 12 (depends on 11) The method claim 11, wherein, the recombinant solventogenic microorganism is Clostridium sp., Escherichia sp., Clostridium beijerinckii, Clostridium beijerinckii 8052, Clostridium beijernickii BA101, or Clostridium acetobutylicum. ### Claim 13 (depends on 11) The method of claim 11, wherein the Clostridium beijerinckii Adh polynucleotide is operably linked to a promoter. ### Claim 14 (depends on 1) The recombinant solventogenic microorganism of claim 1, wherein the nucleic acid molecule comprising at least one Clostridium beijerinckii Adh polynucleotide encodes an alcohol dehydrogenase protein comprising the amino acid sequence of SEQ ID NO: 15. ### Claim 15 (depends on 11) The method of claim 11, wherein the nucleic acid molecule comprising at least one Clostridium beijerinckii Adh polynucleotide encodes an alcohol dehydrogenase protein comprising the amino acid sequence of SEQ ID NO: 15. ### Claim 16 (depends on 1) The recombinant solventogenic microorganism of claim 1, which is non-cellulolytic.