High molar succinate yield bacteria by increasing the intracellular NADH availability
The invention relates to increasing the yield of succinate in bacteria by increasing the intracellular availability of cofactors such as NADH.
1. An engineered Escherichia coli ( E. coli ), said E. coli comprising a DNA that expresses pyruvate carboxylase (pyc), further comprising inactivated E. coli genes encoding alcohol dehydrogenase (adh) and lactate dehydrogenase (ldh), wherein said E. coli produces greater than 1 mole of succinate per mole of glucose under anaerobic culture conditions.
2. The engineered E. coli of claim 1 , wherein said E. coli produces at least 1.3 mole of succinate per mole of glucose.
3. The engineered E. coli of claim 1 , wherein said E. coli produces at least 1.4 mole of succinate per mole of glucose.
4. The engineered E. coli of claim 1 , wherein said E. coli produces at least 1.5 mole of succinate per mole of glucose.
5. The engineered E. coli of claim 1 , further comprising an inactivated E. coli gene encoding phosphotransferase G (ptsG).
6. The engineered E. coli of claim 5 , wherein said Escherichia coli produces greater than 1.3 mole of succinate per mole of glucose.
7. The engineered E. coli of claim 1 , further transformed with a DNA encoding phosphoenolpyruvate carboxylase (pepc).
8. The engineered E. coli of claim 1 , further transformed with a DNA encoding formate dehydrogenase (fdh).
9. An engineered E. coli , comprising an inactivating deletion of all or part of the E. coli genes encoding alcohol dehydrogenase (adh) and lactate dehydrogenase (ldh) and a plasmid encoding pyruvate carboxylase (pyc), wherein said E. coli overexpresses pyc and produces greater than 1 mole of succinate per mole of glucose under anaerobic culture conditions.
10. The engineered E. coli of claim 9 , further comprising an inactivating deletion of all or part of the E. coli gene encoding phosphotransferase G (ptsG).
11. The engineered E. coli of claim 9 , which produces greater than 1.3 mole of succinate per mole of glucose.
12. The engineered E. coli of claim 10 , which produces greater than 1.3 mole of succinate per mole of glucose.
13. The engineered E. coli of claim 10 , which produces greater than 1.5 mole of succinate per mole of glucose.
14. The engineered Escherichia coli of claim 9 , further transformed with a DNA encoding phosphoenolpyruvate carboxylase (pepc).
15. The engineered Escherichia coli of claim 6 , further transformed with a DNA encoding formate dehydrogenase (fdh).
16. The engineered E. coli of claim 1 , consisting essentially of:
a) a DNA that expresses pyruvate carboxylase (pyc);
b) inactivated E. coli genes encoding alcohol dehydrogenase (adh) and lactate dehydrogenase (ldh); and
c) optionally one or more of an inactivated E. coli gene encoding phosphotransferase G (ptsG) or a DNA encoding phosphoenolpyruvate carboxylase (pepc) or a DNA encoding formate dehydrogenase (fdh),
wherein said E. coli produces greater than 1 mole of succinate per mole of glucose under anaerobic culture conditions.
17. A method of producing succinate comprising culturing the Escherichia coli of any one of claim 1 - 15 or 16 under anaerobic conditions in the presence of glucose, and isolating succinate from said culture.
18. A method of producing succinate comprising:
a) culturing the Escherichia coli of any one of claim 1 - 15 or 16 under aerobic conditions,
b) transferring said Escherichia coli to anaerobic cultures with glucose, and
c) culturing said Escherichia coli anaerobically to produce greater than 1 mole of succinate per mole of glucose.