# US 10,696,988 B2 · Methods and systems for 1-butanol production > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,696,988 B2 - Document ID: US10696988B2 - Status: Granted - Kind: B2 - Application number: 15/552,647 - Filed: 2016-02-23 - Published: 2018-02-08 - Granted: 2020-06-30 - Earliest claimed: 2015-02-23 - Assignees: Biocheminsights, Inc. - Applicants: Biocheminsights, Inc. - Inventors: David R. Dodds; William B. Armiger; Mattheos Koffas - Web page: https://patentgrep.com/patent/US10696988B2 ## CPC - C12P 7/16: Butanols - C12M 45/07: {by electrical or electromagnetic forces} - C12N 9/0006: {acting on CH-OH groups as donors (1.1)} - C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)} - C12N 9/001: {acting on the CH-CH group of donors (1.3)} - C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)} - C12N 9/88: Lyases (4.) - C12N 9/90: Isomerases (5.) - C12N 15/52: Genes encoding for enzymes or proenzymes - C12Y 101/01001: Alcohol dehydrogenase (1.1.1.1) - C12Y 101/01027: L-Lactate dehydrogenase (1.1.1.27) - C12Y 101/01157: 3-Hydroxybutyryl-CoA dehydrogenase (1.1.1.157) - C12Y 102/0101: Acetaldehyde dehydrogenase (acetylating) (1.2.1.10) - C12Y 102/01046: Formaldehyde dehydrogenase (1.2.1.46) - C12Y 102/01057: Butanal dehydrogenase (1.2.1.57) - C12Y 102/04001: Pyruvate dehydrogenase (acetyl-transferring) (1.2.4.1) - C12Y 103/01006: Fumarate reductase (NADH) (1.3.1.6) - C12Y 103/01038: Trans-2-enoyl-CoA reductase (NADPH) (1.3.1.38) - C12Y 203/01008: Phosphate acetyltransferase (2.3.1.8) - C12Y 203/01009: Acetyl-CoA C-acetyltransferase (2.3.1.9) - C12Y 203/01054: Formate C-acetyltransferase (2.3.1.54), i.e. pyruvate formate-lyase or PFL - C12Y 401/02043: 3-Hexulose-6-phosphate synthase (4.1.2.43) - C12Y 503/01027: 6-Phospho-3-hexuloisomerase (5.3.1.27) - C25B 3/07: Oxygen containing compounds - C25B 3/25: Reduction - C25B 9/19: with diaphragms - C25B 15/02: Process control or regulation - C25B 15/08: Supplying or removing reactants or electrolytes; Regeneration of electrolytes - Y02E 50/10: Biofuels, e.g. bio-diesel - 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)} - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - C25B: ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR (anodic or cathodic protection C23F13/00; single-crystal growth C30B) - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract A combination of an electrochemical device for delivering reducing equivalents to a cell, and engineered metabolic pathways within the cell capable of utilizing the electrochemically provided reducing equivalents is disclosed. Such a combination allows the production of commodity chemicals by fermentation to proceed with increased carbon efficiency. ## Claims ### Claim 1 A system for 1-butanol production, comprising: an electrochemical bioreactor module for providing reducing equivalents; a first engineered pathway for producing 1-butanol from acetyl-CoA, comprising acetyl-CoA acetyltransferase (AtoB, EC 2.3.1.9), 3-hydroxybutyryl-CoA dehydrogenase (Hbd, EC 1.1.1.157), 3-hydroxybutyryl-CoA dehydratase (Crt, EC 4.2.1.5), trans-enoyl-CoA reductase (Ter, EC 1.3.1.38) and aldehyde/alcohol dehydrogenase (AdhE2, EC 1.2.157/EC 1.1.1.1); and a second engineered pathway for converting pyruvate into formate without producing CO2, and converting the formate to fructose-6-phosphate; wherein the second pathway comprises pyruvate:formate lyase (Pfl, EC 2.3.1.54), formaldehyde dehydrogenase (Fld, EC 1.2.1.46), hexulose-6-phosphate synthase (HPS, EC 4.1.2.43), and 6-phospho-3-hexuloisomerase (HPI, EC 5.3.1.27); wherein the reducing equivalents are provided to one or more redox enzymes in the first and/or second engineered pathways; and wherein optionally the first and second engineered pathways are present in an engineered cell selected from a bacterium or a fungus. ### Claim 2 (depends on 1) The system of claim 1 wherein in the engineered cell, the endogenous pyruvate dehydrogenase (Pdh, EC 1.2.4.1) has been disabled, deleted or otherwise rendered non-functional. ### Claim 3 (depends on 1) The system of claim 1 wherein in the engineered cell, the endogenous fumarate reductase (FrdBC, EC 1.3.1.6), lactate dehydrogenase (Ldh, EC 1.1.1.27), acetaldehyde dehydrogenase (AdhE, EC 1.2.1.10) and/or acetyl-CoA-phosphate acetyltransferase (Pta, EC 2.3.1.8) have been disabled, deleted or otherwise rendered non-functional. ### Claim 4 (depends on 1) The system of claim 1 wherein the one or more redox enzymes are selected from formaldehyde dehydrogenase (Fld, EC 1.2.1.46), 3-hydroxybutyryl-CoA dehydrogenase (Hbd, EC 1.1.1.157), trans-enoyl-CoA reductase (Ter, EC 1.3.1.38) and aldehyde/alcohol dehydrogenase (AdhE2, EC 1.2.157/EC 1.1.1.1). ### Claim 5 (depends on 1) The system of claim 1 wherein the reducing equivalents are transported by an electron transfer mediator (ETM). ### Claim 6 (depends on 5) The system of claim 5 wherein the ETM is selected from one or more of Neutral Red, Methylene Blue, Methyl Viologen, quinone, NAD+ and NADP+. ### Claim 7 (depends on 1) The system of claim 1 wherein the bacterium is selected from E. coli, Bacillus subtilis, Clostridium acetylbutylicum or another Clostridium species. ### Claim 8 (depends on 1) The system of claim 1 wherein the fungus is selected from Saccharomyces cerevisiae, a Pichia species or an Aspergillus species. ### Claim 9 (depends on 1) A method for producing 1-butanol, comprising providing the system of claim 1 in the presence of a carbon source. ### Claim 10 (depends on 9) The method of claim 9, wherein the carbon source is glucose.