Yeast cells having disrupted pathway from dihydroxyacetone phosphate to glycerol

Abstract

Yeast cells are genetically modified to disrupt a native metabolic pathway from dihydroxyacetone to glycerol. In certain aspects, the yeast cell is of the genera Kluyueromyces, Candida or Issatchenkia . In other aspects, the yeast cell is capable of producing at least one organic acid, such as lactate. The yeast cells produce significantly less glycerol than the wild-type strains, and usually produce greater yields of desired fermentation products. Yeast cells of the invention often grow well when cultivated, despite their curtailed glycerol production.

1 . A cell of a pre-whole genome duplication hemiascomycetous yeast, which is genetically modified to delete or disrupt a native metabolic pathway from dihydroxyacetone phosphate to glycerol, which cell, when cultivated in the presence of a carbon source that can be metabolized by the cell, metabolizes less than 2% of the weight of carbon source that is consumed by the cell to glycerol. 
     
     
         2 - 4 . (canceled) 
     
     
         5 . The cell of  claim 1  which contains a functional LDH gene cassette, and which produces lactate. 
     
     
         6 . The cell of  claim 5  wherein the deletion or disruption of the native metabolic pathway includes a deletion or disruption of at least one native glycerol-3-phosphate dehydrogenase gene. 
     
     
         7 . The cell of  claim 5  wherein the deletion or disruption of the native metabolic pathway includes a deletion or disruption of at least one native glycerol-3-phosphatase gene. 
     
     
         8 . The cell of  claim 5  wherein the deletion or disruption of the native metabolic pathway includes a deletion or disruption of at least one native glycerol-3-phosphate dehydrogenase gene and of at least one native glycerol-3-phosphatase gene. 
     
     
         9 . The cell of  claim 5  wherein the deletion of disruption of the native metabolic pathway may include a deletion or disruption of at least one native dihydroxyacetone phosphatase gene, a deletion or disruption of a native glycerol dehydrogenase gene, or a deletion or disruption of both a native dihydroxyacetone phosphatase gene and a native glycerol dehydrogenase gene. 
     
     
         10 . The cell of any of claims  4 - 9  which contains a deletion or disruption of a native metabolic pathway from pyruvate to ethanol. 
     
     
         11 . The cell of  claim 1 , which cell is within the  Zygosaccharomyces, Zygotorulaspora, Torulaspora, Lachancea, Kluyveromyces, Eremothecium  or  Hanseniaspora  clades of the  Saccharomyces  complex (Kurtzman 2003) and which, when cultivated in the presence of a carbon source that can be metabolized by the cell, metabolizes less than 2% of the weight of carbon source that is consumed by the cell to glycerol. 
     
     
         12 - 19 . (canceled) 
     
     
         20 . The cell of  claim 1 , which cell is of the genera  Kluyveromyces, Candida  or  Issatchenkia , which is genetically modified to delete or disrupt a native metabolic pathway from dihydroxyacetone phosphate to glycerol and which when cultivated in the presence of a carbon source that can be metabolized by the cell, metabolizes less than 2% of the weight of carbon source that is consumed by the cell to glycerol. 
     
     
         21 - 27 . (canceled) 
     
     
         28 . The cell of  claim 1 , which cell is within the  I. orientalis/P. fermentans  clade (Kurtzman and Robnett 1998) or within the  Kluyveromyces  clade of the  Saccharomyces  complex (Kurtzman 2003), which is genetically modified to delete or disrupt a native metabolic pathway from dihydroxyacetone phosphate to glycerol, and which, when cultivated in the presence of a carbon source that can be metabolized by the cell, metabolizes less than 2% of the weight of carbon source that is consumed by the cell to glycerol. 
     
     
         29 - 30 . (canceled) 
     
     
         31 . The cell of  claim 28  which contains a functional LDH gene cassette, and which produces lactate. 
     
     
         32 - 34 . (canceled) 
     
     
         35 . The cell of  claim 31 , which is a  K. marxianus  cell. 
     
     
         36 . The cell of  claim 35 , which contains a deletion or disruption of a native metabolic pathway from pyruvate to ethanol. 
     
     
         37 . The cell of  claim 31 , which is an  I. orientalis  cell. 
     
     
         38 . The cell of  claim 37 , which contains a deletion or disruption of a native metabolic pathway from pyruvate to ethanol. 
     
     
         39 . A genetically modified cell of a pre-whole genome duplication yeast species, which produces less than 2.0 g/L of glycerol when cultivated under the following standard microaerobic conditions: 
       A. defined aqueous medium containing, at the start of cultivation, 5 g/L ammonium sulfate, 3 g/L potassium dihydrogen phosphate, 0.5 g/L magnesium sulfate, trace elements, vitamins, 150 g/L glucose; 
       B. pH of 3.5 at the start of cultivation, with the fermentation medium being buffered if necessary to prevent the pH from falling below 3.0 or rising above 7.0 during the cultivation; 
       C. Cultivation inoculated with the yeast cell to an OD 600  of 1.0; 
       D. Cultivation temperature 30° C.; 
       E. Cultivation is continued until the glucose concentration is reduced to 10 g/L, but is not continued for longer than 120 hours; 
       F. Aeration and agitation sufficient to produce an oxygen uptake rate of 5.0±1.0 mmol/L/hr. 
     
     
         40 - 44 . (canceled) 
     
     
         45 . A genetically modified cell of a pre-whole genome duplication yeast species, which genetically modified cell lacks the ability to produce an active glycerol-3-phosphate dehydrogenase enzyme that is natively produced by cells of the wild-type yeast species. 
     
     
         46 - 49 . (canceled) 
     
     
         50 . A cell of a pre-whole genome duplication yeast species, which lacks the ability to produce an active glycerol-3-phosphatase enzyme that is natively produced by wild-type cells of the yeast species. 
     
     
         51 - 54 . (canceled) 
     
     
         55 . A yeast cell that is genetically modified to produce a product organic acid, said yeast cell further having a deletion or disruption of a native metabolic pathway from dihydroxyacetone phosphate to glycerol and a deletion or disruption of a native metabolic pathway from pyruvate to ethanol. 
     
     
         56 - 59 . (canceled) 
     
     
         60 . The yeast cell of  claim 55  wherein the organic acid is lactate. 
     
     
         61 . A fermentation process wherein a cell of  claim 1  is cultivated under fermentation conditions and in the presence of a carbon source to produce a desired fermentation product, wherein the glycerol yield is less than 2% based on the weight of the carbon source that is consumed by the cell. 
     
     
         62 - 66 . (canceled) 
     
     
         67 . A fermentation process wherein a cell of  claim 5  is cultivated under fermentation conditions and in the presence of a carbon source to produce lactate, wherein the glycerol yield is less than 2% based on the weight of the carbon source that is consumed by the cell. 
     
     
         68 - 69 . (canceled)