Methods and materials for the production of organic products in cells of
View Patent ↗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 genetically modified cell from genera Candida, comprising a recombinant expression construct comprising a nucleic acid having a nucleotide sequence encoding a lactate dehydrogenase protein, wherein the nucleic acid encodes a lactate dehydrogenase protein from Bacillus megaterium, Lactobacillus helveticus, or Rhizopus orvzae, and wherein the lactate dehydrogenase protein-encoding nucleic acid is operatively linked to a promoter functional in a yeast cell from a Candida species that is a Candida sonorensis, Candida parapsilosis, Candida naeodendra, Candida methanosorbosa, Candida entomophila, Candida krusei, Candida blankii, or Candida diddensiae cell wherein the cell expresses a metabolic phenotype that is the Crabtree-negative phenotype.
2. The cell of claim 1 , wherein the cell further expresses reduced pyruvate decarboxylase (PDC) activity.
3. The cell of claim 2 , wherein the reduced PDC activity results from deletion in at least one pyruvate decarboxylase gene.
4. The cell of claim 2 , wherein the reduced PDC activity results from genetic disruption of at least one pyruvate decarboxylase gene.
5. A Candida cell according to claim 1 , comprising a deletion at a first locus that is the pdc1 gene locus, a disruption at a second locus that is the pdc2 gene locus and insertion of two or more copies of a nucleic acid that encodes a lactate dehydrogenase protein in the cellular genome at each of the pdc1 and pdc2 loci.
6. The cell of claim 5 , wherein each of the two or more copies of a nucleic acid that encodes a lactate dehydrogenase protein are operably linked to a promoter that is transcriptionally active in the Candida cell.
7. A Candida cell according to claim 1 that is genetically modified to contain non-functional or deleted pdc1 or pdc2 gene, characterized by at least a 10-fold reduction of ethanol production when cultured in the presence of a defined glucose or rich glucose medium.
8. The Candida cell of claim 1 , wherein the cell has increased lactic acid dehydrogenase activity relative to an untransformed Candida cell.