Methods and materials for the synthesis of organic products
View Patent ↗This invention provides biocatalysts that are recombinant yeast cells comprising recombinant expression vectors encoding heterologous lactate dehydrogenase genes for producing lactate.
1. An isolated nucleic acid encoding a yeast lactate dehydrogenase protein having an amino acid sequence Seq. ID No. 22.
2. An isolated nucleic acid according to claim 1 encoding a yeast lactate dehydrogenate protein that hybridizes to the nucleic acid probe of Seq. ID No. 21 under conditions of a temperature of 42° C. in a solution containing 50% formamide, 5× Denhardt's, 5×SSPE, 0.1% SDS, 100 μg/mL herring sperm DNA, 1 μg/mL polyA DNA, wherein hybridization is detected after washing under conditions of room temperature in a solution of 2×SSC for 5 min and repeated, followed by two 30 minute washes in a solution of 1×SSC −0.1% SDS at 68° C.
3. A recombinant expression construct comprising the nucleic acid having the nucleotide sequence encoding a yeast lactate dehydronase protein according to claim 1 , wherein the nucleic acid is expressed in a yeast cell.
4. A recombinant expression construct according to claim 3 , further comprising a yeast promoter operably linked to the nucleic acid encoding a yeast lactate dehydronase protein.
5. A recombinant expression construct according to claim 3 , further comprising a yeast transcriptional terminator element operably linked to the nucleic acid encoding a yeast lactate dehydronase protein.
6. A recombinant expression construct according to claim 3 , further comprising a yeast replication element of a yeast 2-micron circle plasmid.
7. A yeast cell transformed with the recombinant expression construct of claim 3 wherein the transformed cell expresses the yeast lactate dehydrogenate protein.
8. A yeast cell according to claim 7 , wherein the yeast cell is a yeast from genera Saccharomyces, Kluyveromyces, Hansenula, Candida, Trichosporon, Yamadazyma, Torulaspora or Pichia.
9. A yeast cell according to claim 7 , wherein the yeast cell expresses a crabtree-negative phenotype.
10. A yeast cell according to claim 7 , wherein the yeast cell is a yeast species selected from the group consisting of C. soronensis and K. marxianus.
11. A yeast cell according to claim 7 , wherein the yeast cell produces a glycolytic enzyme selected from the group consisting of pyruvate decarboxylase, alcohol dehydrogenate, and acetyl-CoA synthase in an amount lower than the amount produced in a C. soronensis or K. marxianus yeast cell.
12. A method for producing lactic acid comprising the step of fermenting the yeast cell culture according to claim 7 in a nutrient medium containing a sugar under conditions whereby at least 50% of the sugar is converted by the yeast cell to lactic acid.
13. The method of claim 12 , wherein the yeast cell is grown at a temperature from about 35° C. to about 55° C.
14. The method of claim 12 , wherein the nutrient culture has a pH less than about pH 5.0.
15. The method of claim 12 , wherein the yeast is grown under substantially anaerobic conditions.
16. The method of claim 12 , wherein the yeast cell is a crabtree-negative yeast cell.
17. The method of claim 16 , wherein the yeast cell is K. marxianus or C. sonorensis.
18. The method of claim 12 , wherein the yeast cell produces a glycolytic enzyme selected from the group consisting of pyruvate decarboxylase, alcohol dehydrogenate, and acetyl-CoA synthase in an amount lower than the amount produced in a C. soronensis or K. marxianus yeast cell.
19. The method of claim 12 , wherein the sugar is glucose, xylose, ribose, arabinose, mannose, galactose, fructose, maltose or lyxose.