US2023028039A1PendingUtilityA1

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

70
Assignee: LYGOS INCPriority: Apr 13, 2018Filed: Jun 10, 2022Published: Jan 26, 2023
Est. expiryApr 13, 2038(~11.8 yrs left)· nominal 20-yr term from priority
Inventors:Jeffrey A. DietrichMario OuelletAndrew Conley
C12N 15/81C12N 1/16C12Y 101/02004C12Y 101/01027C12N 1/14C12N 1/20C12N 9/0006C12N 15/815C12P 7/56C12R 2001/84C12N 15/905C12N 15/52
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Claims

Abstract

Methods and materials related to producing D-lactic acid are disclosed. Specifically, isolated synthetic or natural nucleic acids, synthetic or natural polypeptides, host cells, and methods and materials for producing D-lactic acid by direct fermentation from carbon sources are disclosed, along with methods of preparing D-lactic acid polymers.

Claims

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

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