Increasing bacterial succinate productivity
Improved bacteria for making succinate and other 4 carbon dicarboxylates from the Krebs cycle have modifications to reduce acetate, lactate, EtOH and formate, as well as turn on the glyoxylate shunt, produce more NADH and overexpress In one embodiment, the bacteria are ΔadhEΔldhAΔiclRΔack-pta plus PYC + and NAD + -dependant FDH + .
1. An engineered E. coli comprising reduced activity of alcohol dehydrogenase (ADH), reduced activity of lactate dehydrogenase (LDH), reduced activity of acetate kinase (ACK) or phosphotransacetylase (PTA) or both (ACK-PTA), reduced activity of the aceBAK operon repressor (ICLR) and overexpressed NAD + -dependent formate dehydrogenase (FDH + ) and overexpressed pyruvate carboxylase (PYC + ).
2. The E. coli of claim 1 , wherein said E. coli comprising ΔadhE, Δldh, Δack-pta, ΔiclR and FDH + and PYC + .
3. The E. coli of claim 1 , wherein the FDH has at least 65% identity to SEQ ID NO 1.
4. The E. coli of claim 1 , wherein the PYC has at least 63% identity to SEQ ID NO. 2.
5. The E. coli of claim 1 , wherein the FDH has SEQ ID NO 1.
6. The E. coli of claim 1 , wherein the PYC has SEQ ID NO. 2.
7. The E. coli of claim 2 , wherein the FDH has at least 65% identity to SEQ ID NO 1.
8. The E. coli of claim 2 , wherein the PYC has at least 63% identity to SEQ ID NO. 2.
9. The E. coli of claim 2 , wherein the FDH has SEQ ID NO 1.
10. The E. coli of claim 2 , wherein the PYC has SEQ ID NO. 2.
11. A method of making a four carbon dicarboxylic acid from the Krebs cycle, comprising culturing the E. coli of any one of claim 1 - 10 in a medium, and isolating a four carbon dicarboxylic acid from said E. coli or medium or both.
12. A method of making a succinate, comprising culturing the E. coli of any one of claim 1 - 10 in a medium, and isolating succinate from said E. coli or medium or both.
13. The method of claim 11 , further comprising supplementing said medium with 25-250 mM formate.
14. The method of claim 12 , further comprising supplementing said medium with 25-250 mM formate.
15. The method of claim 12 , wherein the yield of succinate is >1.6 moles/mole of glucose.
16. The method of claim 14 , wherein the yield of succinate is >1.7 moles/mole of glucose.
17. The method of claim 11 , wherein the E. coli comprising ΔadhE, Δldh, Δack-pta, ΔiclR and FDH + and PYC + .