US2010137551A1PendingUtilityA1

Methods and materials for the production of D-lactic acid in yeast

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Assignee: RAJGARHIA VINEETPriority: May 30, 2002Filed: Jan 12, 2009Published: Jun 3, 2010
Est. expiryMay 30, 2022(expired)· nominal 20-yr term from priority
Inventors:Vineet RajgarhiaCatherine Asleson DundonStacey OlsonPirkko SuominenBen Hause
C12N 15/815C12Q 1/02G01N 2333/39Y02E50/10C12N 9/0006C12N 1/16C12P 7/56C12P 7/625Y02P20/52
66
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Claims

Abstract

The present invention relates to biocatalysts that are cells, optimally of the Crabtree-negative phenotype, comprising expression vectors encoding genes heterologous to the cell that enable increased production of organic products. More specifically, the invention relates to genetically modified Kluyveromyces cells, methods for making the Kluyveromyces cells, and their use in production of organic products, particularly D-lactic acid.

Claims

exact text as granted — not AI-modified
1 . A recombinant yeast cell of a species that does not naturally accumulate pyruvate, comprising at least one exogenous D-lactate dehydrogenase gene integrated into its genome, wherein the D-lactate dehydrogenase gene is operatively linked to functional promoter and terminator sequences. 
     
     
         2 . The recombinant yeast cell of  claim 1 , which is of the genera  Candida  or  Kluyveromyces.    
     
     
         3 . The recombinant yeast cell of  claim 2 , wherein the cell is of the species  C. sonorensis  or  K. marxianus.    
     
     
         4 . The recombinant yeast cell of  claim 1 , wherein the promoter sequence is at least 90% homologous to a promoter that is native to the yeast species. 
     
     
         5 . The recombinant yeast cell of  claim 4 , wherein the terminator sequence is at least 90% homologous to a terminator that is native to the yeast species. 
     
     
         6 . The recombinant yeast cell of  claim 4 , wherein the yeast species contains a native PDC gene having a native PDC promoter, and the promoter sequence is at least 90% homologous to the native PDC promoter. 
     
     
         7 . The recombinant yeast cell of  claim 6 , wherein the native PDC gene has a native PDC terminator, and the terminator sequence is at least 90% homologous to the native PDC terminator. 
     
     
         8 . The recombinant yeast cell of  claim 7 , wherein the native PDC gene is deleted. 
     
     
         9 . The recombinant yeast cell of  claim 1 , wherein the yeast species contains a native PDC gene, and the native PDC gene is deleted. 
     
     
         10 . The recombinant yeast cell of  claim 1 , wherein the nucleotide sequence encodes a D-lactate dehydrogenase protein from  Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus bulgaricus, Lactobacillus delbrueckii, lactobacillus plantarum  and  Lactobacillus pentosus.    
     
     
         11 . The recombinant yeast cell of  claim 10 , wherein the promoter is from a yeast species from the genera  Kluyveromyces  or  Saccharomyces.    
     
     
         12 . The recombinant yeast cell of  claim 11 , wherein the promoter is from  Kluyveromyces marxianus.    
     
     
         13 . The recombinant yeast cell of  claim 11 , wherein the promoter is from  Saccharomyces cerevisiae.    
     
     
         14 . The recombinant yeast cell of  claim 1  which exhibits reduced PDC activity. 
     
     
         15 . The recombinant yeast cell of  claim 8  wherein the D-LDH gene is integrated at the locus of the deleted PDC gene. 
     
     
         16 . A method for fermenting a carbohydrate to lactic acid comprising culturing the cell of  claim 1  under fermentation conditions in a medium containing a carbohydrate that is fermentable by the cell. 
     
     
         17 . A method for fermenting a carbohydrate to lactic acid comprising culturing the cell of  claim 8  under fermentation conditions in a medium containing a carbohydrate that is fermentable by the cell. 
     
     
         18 . A method for fermenting a carbohydrate to lactic acid comprising culturing the cell of  claim 9  under fermentation conditions in a medium containing a carbohydrate that is fermentable by the cell. 
     
     
         19 . A method for fermenting a carbohydrate to lactic acid comprising culturing the cell of  claim 14  under fermentation conditions in a medium containing a carbohydrate that is fermentable by the cell. 
     
     
         20 . A method according to the method of  claims 16 ,  17 ,  18 , or  19 , further comprising the step of converting at least a portion of the lactic acid to lactide. 
     
     
         21 . The method of  claim 20 , further comprising the step of polymerizing the lactide to form a polylactide polymer or copolymer. 
     
     
         22 . A method for producing lactic acid comprising culturing the cell of  claim 1  under fermentation conditions in a medium containing a sugar that is fermentable by the cell. 
     
     
         23 . A method for producing lactic acid comprising culturing the cell of  claim 8  under fermentation conditions in a medium containing a sugar that is fermentable by the cell. 
     
     
         24 . A method for producing lactic acid comprising culturing the cell of  claim 9  under fermentation conditions in a medium containing a sugar that is fermentable by the cell. 
     
     
         25 . A method for producing lactic acid comprising culturing the cell of  claim 14  under fermentation conditions in a medium containing a sugar that is fermentable by the cell. 
     
     
         26 . A method according to  claim 22 ,  23 ,  24 , or  25 , further comprising the step of converting at least a portion of the lactic acid to lactide 
     
     
         27 . The method of  claim 26 , further comprising the step of polymerizing the lactide to form a polylactide polymer or copolymer.

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