The present invention provides tools and methods for producing organic acids using strains of Monascus which are tolerant to high organic acid concentrations at low pH.
1. A method of producing a composition comprising an organic acid, the method comprising the steps of:
(i) providing a Monascus micro-organism strain deposited as CBS 127564, CBS 127565 or CBS 127566, that has been genetically modified to comprise a recombinant gene involved in the production of an organic acid that is lactic acid, succinic acid, or fumaric acid so as to produce, under anaerobic conditions, an increased yield of such the organic acid as compared to the corresponding wild-type strain;
(ii) culturing the micro-organism in a medium having at a pH less than 1.5 units above the pKa value of the organic acid in the presence of hexose, pentose, or a combination thereof, as the sole carbon source; and
(iii) recovering the organic acid.
2. The method of claim 1 , wherein the said micro-organism has been further genetically modified to delete or inactivate a gene involved in an endogenous metabolic pathway that consumes the organic acid or produces a metabolite other than the organic acid.
3. The method of claim 1 , wherein the micro-organism has been genetically modified to delete or inactivate a gene involved in the endogenous production of ethanol.
4. The method of claim 3 , wherein the micro-organism has been genetically modified to delete or inactivate a PDC1, PDC2 or PDC4 gene.
5. The method of claim 1 , wherein:
the organic acid is lactic acid; and
the micro-organism has been genetically modified to: (a) comprise a recombinant gene encoding L-lactate dehydrogenase; or (b) delete or inactivate a gene involved in an endogenous D-lactic acid production or L-lactic acid consumption pathway or an endogenous pathway that produces a metabolite other than lactic acid.
6. The method of claim 5 , wherein the recombinant gene encoding L-LDH is an exogenous LDH gene.
7. The method of claim 5 , wherein the micro-organism has been genetically modified to delete or inactivate a gene encoding pyruvate decarboxylase, fumarate reductase, alcohol dehydrogenase, acetaldehydedehydrogenase, phosphoenolpyruvate carboxylase, D-lactate dehydrogenase, or L-lactate dehydrogenase.
8. The method of claim 5 , wherein the micro-organism has been genetically modified to delete or inactivate a gene encoding pyruvate decarboxylase, D-lactate dehydrogenase, or L-lactate dehydrogenase.
9. The method of claim 1 , wherein the organic acid is succinic acid or fumaric acid; and the micro-organism has been genetically modified to: (a) comprise an exogenous gene encoding phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarase, fumarate reductase, isocitrate lyase or malate synthase; or (b) delete or inactivate a gene involved in an endogenous succinic acid or fumaric acid consumption pathway or an endogenous pathway that produces a metabolite other than succinic acid or fumaric acid.
10. The method of claim 9 , wherein the organic acid is fumaric acid and the micro-organism comprises an exogenous gene encoding pyruvate decarboxylase, malate dehydrogenase or fumarase.
11. The method of claim 9 , wherein the micro-organism has been genetically modified to delete or inactivate a gene encoding pyruvate decarboxylase, alcohol dehydrogenase, acetaldehydedehydrogenase, phosphoenolpyruvate carboxylase, D-lactate dehydrogenase, L-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase, or isocitrate dehydrogenase.
12. The method of claim 9 , wherein the organic acid is succinic acid and the micro-organism has been genetically modified to delete or inactivate a gene encoding succinate dehydrogenase or fumarate reductase.
13. The method of claim 1 , wherein the organic acid is produced at a yield of at least 0.5 g/L.
14. The method of claim 13 , wherein the yield is at least 2 g/L.
15. The method of claim 1 , wherein the recovery of the organic acid is from the cultivation medium.