# US 2014/0154760 A1 · 3-Hydroxypropionic Acid Production By Recombinant Yeasts > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0154760 A1 - Document ID: US20140154760A1 - Status: Published application - Kind: A1 - Application number: 14/089,219 - Filed: 2013-11-25 - Published: 2014-06-05 - Earliest claimed: 2012-11-30 - Assignees: Novozymes, Inc. - Applicants: Novozymes, Inc. - Inventors: Mariah Bindel - Web page: https://patentgrep.com/patent/US20140154760A1 ## CPC - C12P 7/42: Hydroxy-carboxylic acids - C12N 1/16: Yeasts; Culture media therefor - C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)} - C12N 15/52: Genes encoding for enzymes or proenzymes - C12Y 102/01009: Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (1.2.1.9) - Y02E 50/10: Biofuels, e.g. bio-diesel - Y02P 20/52: using catalysts, e.g. selective catalysts - 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)} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION - Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS ## Abstract Provided herein are recombinant yeast cells having an active 3-Hydroxypropionic Acid (3-HP) pathway and further comprising a heterologous polynucleotide encoding a non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN). Also described are methods of using the recombinant yeast cells to produce 3-HP and acrylic acid. ## Claims ### Claim 1 A recombinant yeast cell, comprising: (1) an active 3-HP pathway that proceeds through a β-alanine intermediate, and (2) a heterologous polynucleotide encoding a non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN), wherein the cell capable of producing 3-HP. ### Claim 2 (depends on 1) The recombinant yeast cell of claim 1, wherein the cell comprises a heterologous polynucleotide encoding an ADC. ### Claim 3 (depends on 2) The recombinant yeast cell of claim 2, wherein the ADC has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 17, 18, 133, 135, 137, or 139. ### Claim 4 (depends on 2) The recombinant yeast cell of claim 2, wherein the ADC has an amino acid sequence comprising or consisting of SEQ ID NO: 17, 18, 133, 135, 137, or 139. ### Claim 5 (depends on 1) The recombinant yeast cell of claim 1, wherein the non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 154, 155, 156, 157, 158, 159, 160, 161, 162, 189, 190, 192, or 194. ### Claim 6 (depends on 1) The recombinant yeast cell of claim 1, wherein the non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) has an amino acid sequence comprising or consisting of SEQ ID NO: 154, 155, 156, 157, 158, 159, 160, 161, 162, 189, 190, 192, or 194. ### Claim 7 (depends on 1) The recombinant yeast cell of any of claim 1, wherein the heterologous polynucleotide encoding the non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) comprises a promoter foreign to GAPN coding sequence. ### Claim 8 (depends on 1) The recombinant yeast cell of claim 1, wherein the cell is capable of producing a greater amount of 3-HP compared to the cell without the heterologous polynucleotide encoding the non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN), when cultivated under identical conditions. ### Claim 9 (depends on 8) The recombinant yeast cell of claim 8, wherein the cell is capable of producing at least 10% more 3-HP compared to the cell without the heterologous polynucleotide encoding the non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN), when cultivated under identical conditions. ### Claim 10 (depends on 1) The recombinant yeast cell of claim 1, wherein said cell further comprises a disruption to one or more endogenous genes encoding a PDC, ADH, GALE, CYB2A, CYB2B, GPD, GPP, ALD, or PCK. ### Claim 11 (depends on 10) The recombinant yeast cell of claim 10, wherein said cell further comprises a disruption to an endogenous gene encoding a PDC. ### Claim 12 (depends on 10) The recombinant yeast cell of claim 10, wherein said cell further comprises a disruption to an endogenous gene encoding a GPD. ### Claim 13 (depends on 1) The recombinant yeast cell of claim 1, wherein the yeast cell belongs to a genus selected from Issatchenkia, Candida, Kluyveromyces, Pichia, Schizosaccharomyces, Torulaspora, Zygosaccharomyces, and Saccharomyces. ### Claim 14 (depends on 13) The recombinant yeast cell of claim 13, wherein the yeast cell belongs to a clade selected from the I. orientalis/P. fermentans clade and the Saccharomyces clade. ### Claim 15 (depends on 13) The recombinant yeast cell of claim 13, wherein the yeast cell is selected from I. orientalis, C. lambica, and S. bulderi. ### Claim 16 (depends on 15) The recombinant yeast cell of claim 15, wherein the yeast cell is an I. orientalis CNB1 yeast cell. ### Claim 17 (depends on 16) The recombinant yeast cell of claim 16, wherein the yeast cell is unable to ferment pentose sugars. ### Claim 18 (depends on 1) The recombinant yeast cell of claim 1, wherein the cell is capable of growing at a pH of less than 4 in media containing 75 g/L or greater 3-HP. ### Claim 19 (depends on 1) The recombinant yeast cell of claim 1, wherein the cell is a 3-HP-resistant yeast cell. ### Claim 20 (depends on 1) The recombinant yeast cell of claim 1, wherein the cell has undergone mutation and/or selection, such that the mutated and/or selected cell possess a higher degree of resistance to 3-HP than a wild-type cell of the same species. ### Claim 21 (depends on 20) The recombinant yeast cell of claim 20, wherein the cell has undergone selection in the presence of lactic acid or 3-HP. ### Claim 22 (depends on 20) The recombinant yeast cell of claim 20, wherein the selection is chemostat selection. ### Claim 23 A recombinant yeast cell, comprising (1) an active 3-HP pathway, and (2) a heterologous polynucleotide encoding a non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN), wherein the GAPN has: (a) at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 192; or (b) at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 194; and wherein the cell is capable of producing 3-HP. ### Claim 24 (depends on 23) The recombinant yeast cell of claim 23, wherein the non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) has an amino acid sequence comprising or consisting of SEQ ID NO: 192 or 194. ### Claim 25 (depends on 1, 23) A method of producing 3-HP, comprising: (a) cultivating the recombinant yeast cell of claim 1 or claim 23 in a fermentable medium under suitable conditions to produce 3-HP; and (b) recovering the 3-HP. ### Claim 26 (depends on 25) The method of claim 25, wherein the fermentable medium comprises sucrose, glucose, and/or fructose. ### Claim 27 (depends on 25) The method of claim 25, wherein the yeast cell is an I. orientalis CNB1 yeast cell and the fermentable medium comprises less than 1% pentose sugars.