US2022315956A1PendingUtilityA1

Recombinant host cells and methods for the production of l-lactic acid

73
Assignee: LYGOS INCPriority: Apr 13, 2018Filed: Feb 18, 2022Published: Oct 6, 2022
Est. expiryApr 13, 2038(~11.7 yrs left)· nominal 20-yr term from priority
Inventors:Jeffrey A. DietrichMario OuelletAndrew Conley
C12N 9/0006C12N 15/905C12Y 101/02004C12N 1/20C12P 7/56C12N 15/81C12Y 101/01027C12N 15/52C12N 1/16C12R 2001/84C12N 1/14C12N 15/815
73
PatentIndex Score
0
Cited by
0
References
0
Claims

Abstract

Provided herein are recombinant cells comprising a heterologous L-lactate dehydrogenase, and processes of preparing L-lactic acid employing such cells.

Claims

exact text as granted — not AI-modified
1 . A recombinant cell comprising:
 a heterologous nucleic acid encoding L-lactate dehydrogenase having at least 60% amino acid identity with SEQ ID NO: 1;   wherein the heterologous nucleic acid is expressed in a sufficient amount to produce L-lactic acid.   
     
     
         2 . The recombinant cell of  claim 1 , wherein the L-lactate dehydrogenase has at least 65% amino acid identity with SEQ ID NO: 1. 
     
     
         3 . The recombinant cell of  claim 1  or  claim 2 , wherein the L-lactate dehydrogenase has at least 70% amino acid identity with SEQ ID NO: 1. 
     
     
         4 . The recombinant cell of any one of  claims 1  to  3 , wherein the L-lactate dehydrogenase has at least 75% amino acid identity with SEQ ID NO: 1. 
     
     
         5 . The recombinant cell of any one of  claims 1  to  4 , wherein the L-lactate dehydrogenase has at least 80% amino acid identity with SEQ ID NO: 1. 
     
     
         6 . The recombinant cell of any one of  claims 1  to  5 , wherein the L-lactate dehydrogenase has at least 85% amino acid identity with SEQ ID NO: 1. 
     
     
         7 . The recombinant cell of any one of  claims 1  to  6 , wherein the L-lactate dehydrogenase has at least 90% amino acid identity with SEQ ID NO: 1. 
     
     
         8 . The recombinant cell of any one of  claims 1  to  7 , wherein the L-lactate dehydrogenase has at least 95% amino acid identity with SEQ ID NO: 1. 
     
     
         9 . The recombinant cell of any one of  claims 1  to  8 , wherein the L-lactate dehydrogenase has greater than 95% amino acid identity with SEQ ID NO: 1. 
     
     
         10 . The recombinant cell of any one of  claims 1  to  9 , wherein the L-lactate dehydrogenase is SEQ ID NO: 1. 
     
     
         11 . The recombinant cell of any one of  claims 1  to  10 , wherein the cell is of the genera  Candida  and does not express a Crabtree-negative phenotype. 
     
     
         12 . The recombinant cell of any one of  claims 1  to  10 , wherein the cell is not of the genera  Candida  and expresses a Crabtree-negative phenotype. 
     
     
         13 . The recombinant cell of any one of  claims 1  to  10 , wherein, the cell is a yeast cell. 
     
     
         14 . The recombinant cell of  claim 13 , wherein the yeast cell does not have a deletion or disruption of a native L- or D-lactate:ferricytochrome c oxidoreductase gene. 
     
     
         15 . The recombinant cell of  claim 13  or  claim 14 , which is selected from  Pichia kudriavzevii  and  Saccharomyces cerevisiae.    
     
     
         16 . The recombinant cell of any one of  claims 1  to  10 , wherein the cell is a prokaryotic cell. 
     
     
         17 . The recombinant cell of  claim 16 , which is selected from  Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis , and  Lactococcus lactis.    
     
     
         18 . The recombinant cell of any one of  claims 1  to  17 , wherein:
 the L-lactate dehydrogenase comprises the residues GXGXXG, wherein X refers to any amino acid; and 
 1-3 amino acid residues located 20-22 residues downstream from the GXGXXG sequence is mutated to a neutral amino acid. 
 
     
     
         19 . The recombinant cell of any one of  claims 1  to  17 , wherein:
 the L-lactate dehydrogenase comprises the residues GXGXXG, wherein X refers to any amino acid; and 
 a negatively charged amino acid located 18-20 residues downstream from the GXGXXG sequence is mutated to a neutral amino acid. 
 
     
     
         20 . The recombinant cell of any one of  claims 1  to  19 , further comprising one or more heterologous nucleic acids encoding one or more proteins selected from organic acid transporters and redox cofactor biogenesis proteins. 
     
     
         21 . The recombinant cell of  claim 20 , wherein one heterologous nucleic acid encodes an organic acid transporter having at least 60% amino acid identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. 
     
     
         22 . The recombinant cell of any one of  claims 1  to  21 , further comprising a genetic disruption of one or more genes encoding a pyruvate decarboxylase, a protein subunit of the pyruvate dehydrogenase complex, glycerol-3-phosphate dehydrogenase, NAD(P)H dehydrogenase, or combinations thereof. 
     
     
         23 . The recombinant cell of  claim 22 , wherein one genetic disruption is in a gene encoding a pyruvate decarboxylase having at least 60% amino acid identity with a sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. 
     
     
         24 . The recombinant cell of  claim 22  or  claim 23 , wherein one genetic disruption is in a gene encoding a glycerol-3-phosphate dehydrogenase having at least 60% amino acid identity with SEQ ID NO: 10. 
     
     
         25 . The recombinant cell of any one of  claims 22  to  24 , wherein one genetic disruption is in a gene encoding an NAD(P)H dehydrogenase having at least 60% amino acid identity with SEQ ID NO: 11. 
     
     
         26 . A method of producing L-lactic acid comprising:
 culturing the recombinant cell of any one of  claims 1  to  25  under fermentation conditions suitable to produce L-lactic acid, or a salt thereof.   
     
     
         27 . The method of  claim 26 , wherein the culturing is performed at an oxygen transfer rate greater than 10 mmol/L/hr. 
     
     
         28 . The method of  claim 26  or  claim 27 , wherein the culturing is performed at a temperature of 25° C. to 45° C. 
     
     
         29 . The method of any one of  claims 26  to  28 , wherein the culturing is performed at a pH of less than 5. 
     
     
         30 . The method of any one of  claims 26  to  29 , wherein the culturing is performed by providing at least 100 g/L glucose to the recombinant cells to yield at least 25% L-lactic acid. 
     
     
         31 . The method of any one of  claims 26  to  30 , wherein the recombinant cell produces less than 5 g/L of ethanol, 5 g/L of pyruvate, 5 g/L of acetate, 5 g/L of glycerol, or any combination thereof. 
     
     
         32 . The method of any one of  claims 26  to  31 , wherein the L-lactic acid is produced at enantiomeric purity of >99.5%. 
     
     
         33 . The method of any one of  claims 26  to  32 , further comprising isolating the L-lactic acid, or salt thereof. 
     
     
         34 . A method for producing a lactic acid polymer, comprising:
 culturing the recombinant cell of any one of  claims 1  to  25  under fermentation conditions suitable to produce L-lactic acid, or a salt thereof;   isolating the L-lactic acid or salt thereof;   optionally converting the L-lactic acid or salt thereof to an L-lactic acid derivative; and   producing a lactic acid polymer using the isolated L-lactic acid, the salt thereof, or the L-lactic acid derivative.

Join the waitlist — get patent alerts

Track US2022315956A1 — get alerts on status changes and closely related new filings.

We store only your email — no account needed. See our privacy policy.