Monascus strains modified for organic acid production
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 genetically modified or recombinant micro-organism of the genus Monascus , wherein the micro-organism is tolerant to organic acid at a concentration of at least 50 g/L and a pH of less than 5.0, and wherein the micro-organism has been genetically modified for increased production of a lactic acid by the introduction of an exogenous lactate dehydrogenase (LDH) gene.
2. The micro-organism according to claim 1 , which comprises one or more of the following:
a) a recombinant gene encoding L-LDH; and
b) an engineered gene deletion and/or inactivation of: (i) an endogenous D-lactic acid production pathway; (ii) an endogenous L-lactic acid consumption pathway, or (iii) a gene encoding a product involved in an endogenous pathway which produces a metabolite other than lactic acid.
3. The micro-organism according to claim 1 , which comprises an engineered gene deletion and/or inactivation in a gene encoding pyruvate decarboxylase (pdc), fumarate reductase, alcohol dehydrogenase (adh), acetaldehydedehydrogenase, phosphoenolpyruvate carboxylase (ppc), D-lactate dehydrogenase (d-ldh), L-lactate dehydrogenase (L-ldh), and/or lactate 2-monooxygenase.
4. The micro-organism according to claim 1 , which comprises at least one engineered gene deletion and/or inactivation in an endogenous gene encoding pyruvate decarboxylase (pdc), D-lactate dehydrogenase (d-ldh) and/or L-lactate dehydrogenase (l-ldh).
5. The micro-organism according to claim 1 , which is capable of producing lactic acid at a yield of at least 0.5 g/L from hexose or pentose sugars or combinations of hexose and pentose sugars.
6. The micro-organism according to claim 4 , wherein the yield of lactic acid is at least 2 g/L.
7. The micro-organism according to claim 1 , wherein the micro-organism is of the species Monascus ruber.
8. The micro-organism according to claim 1 , wherein the micro-organism has been genetically modified for increased production of L-lactic acid.
9. The micro-organism according to claim 1 , the further comprising one or more of the following:
a) one or more engineered gene deletions and/or inactivation of genes encoding enzymes involved in an endogenous metabolic pathway which produces a metabolite other than lactic acid; and
b) one or more engineered gene deletions and/or inactivation of genes encoding enzymes involved in an endogenous metabolic pathway which consumes lactic acid.
10. The micro-organism according to claim 1 , wherein the micro-organism is derived from a strain selected from the strains deposited as CBS 127654, CBS 127565 and CBS 127566.
11. The micro-organism according to claim 1 , which has been selected to be tolerant to organic acid by incubation in a medium comprising a lactic acid at a concentration of at least 50 g/L and having a pH of less than 5.0.
12. The micro-organism according to claim 1 , which has been selected to be tolerant to organic acid by incubation in a medium comprising lactic acid at a concentration of at least 100 g/L and having a pH of less than 3.0.
13. The micro-organism according to claim 10 , which overexpresses L-lactate dehydrogenase and comprises an engineered gene deletion and/or inactivation in a gene encoding pyruvate decarboxylase (pdc) and/or cytochrome-dependent L-lactate dehydrogenase (L-ldh).