Methods and materials for the production of organic products in cells of Candida species

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 Candida cells, methods for making the Candida cells, and their use in production of organic products, particularly lactic acid.

1 . A method for producing lactic acid comprising the steps of 
 a) culturing a genetically modified cell from genera  Candida , comprising at least one exogenous nucleic acid encoding a lactate dehydrogenase protein under conditions that allow the cell to proliferate; and    b) fermenting the cell culture of (a) in a nutrient medium comprising a sugar, under conditions whereby the amount of the sugar converted by the cell to lactic acid is increased, relative to the amount of the sugar converted to lactic acid by an untransformed  Candida  cell.    
     
     
         2 . The method according to  claim 1 , wherein the lactic acid is L-lactic acid.  
     
     
         3 . The method of  claim 1 , wherein the cell is a  Candida  sonorensis cell.  
     
     
         4 . The method of  claim 3 , wherein the cell comprises at least one exogenous nucleic acid encoding a lactate dehydrogenase protein that is a  L. helveticus, B. megaterium , or  R. oryzae  lactate dehydrogenase protein, or combinations thereof.  
     
     
         5 . The method of  claim 1 , wherein the cell is a  Candida methanosorbosa  cell.  
     
     
         6 . The method of  claim 5 , wherein the cell comprises at least one exogenous nucleic acid encoding a lactate dehydrogenase protein that is a  L. helveticus, B. megaterium , or  R. oryzae  lactate dehydrogenase protein, or combinations thereof.  
     
     
         7 . The method of  claim 1 , wherein the cells are cultivated in a medium that is a buffered medium, wherein the medium is buffered to maintain a pH in the nutrient medium from about pH 5 to about pH 9.  
     
     
         8 . The method of  claim 1 , wherein the final pH of the culture medium after lactic acid production is from about pH 2.6 to about pH 5.  
     
     
         9 . The method of  claim 1 , wherein fermenting step is performed under an atmosphere that contains no more than 2% oxygen.  
     
     
         10 . The method of  claim 1 , wherein the fermenting step is performed under anaerobic conditions.  
     
     
         11 . The method of  claim 1 , wherein the sugar in the nutrient medium is one or a plurality of hexoses, one or a plurality of pentoses, or combinations thereof.  
     
     
         12 . The method of  claim 1 , wherein the sugar in the nutrient medium is glucose, xylose, or L-arabinose, or combinations thereof.  
     
     
         13 . The method of  claim 12 , wherein the sugar in the nutrient medium is glucose, and wherein the yield of lactic acid relative to the amount of glucose consumed by the cell is at least 60% by weight.  
     
     
         14 . The method of  claim 12 , wherein the sugar in the nutrient medium is xylose, and wherein the yield of lactic acid relative to the amount of xylose consumed by the cell is at least 15% by weight.  
     
     
         15 . The method of  claim 12 , wherein the sugar in the nutrient medium is L-arabinose, and wherein the yield of lactic acid relative to the amount of L-arabinose consumed by the cell is at least 20% by weight.  
     
     
         16 . The method of  claim 1 , wherein the  Candida  cell is a  Candida diddensiae, Candida parapsilosis, Candida naeodendra, Candida krusei, Candida blankii, Candida methanosorbosa  or  Candida entomophila  cell.  
     
     
         17 . A method of reducing pyruvate decarboxylase activity in a cell from genera  Candida  comprising transforming the cell with a recombinant nucleic acid construct, wherein the nucleic acid construct comprises a selectable gene flanked by 5′ and 3′ flanking sequences from at least one pyruvate decarboxylase gene native to the genera  Candida.    
     
     
         18 . The method of  claim 17 , wherein the at least one pyruvate decarboxylase gene is selected from the group consisting of pyruvate decarboxylase 1 (pdc1), pyruvate decarboxylase 2 (pdc2), or both pdc1 and pdc2.  
     
     
         19 . The method of  claim 17 , wherein the flanking sequences are a promoter and a terminator for at least one pyruvate decarboxylase gene native to the genera  Candida.    
     
     
         20 . A genetically modified  Candida  cell made by the method of  claim 17 .  
     
     
         21 . A genetically modified  Candida  cell made by the method of  claim 18 .  
     
     
         22 . A genetically modified  Candida  cell made by the method of  claim 19.