Metabolic engineering for microbial production of terpenoid products

1. A method for production of a terpenoid product, comprising:

providing a bacterial strain expressing a terpenoid biosynthesis pathway, the terpenoid biosynthesis pathway comprising an upstream methylerythritol phosphate pathway (MEP pathway) and a recombinant downstream synthesis pathway, wherein the bacterial strain overexpresses IspG and IspH and one or more recombinant oxidoreductase enzymes enhancing supply and/or transfer of electrons to the IspG and IspH; and

culturing the bacterial strain to produce the terpene or terpenoid product,

wherein said one or more recombinant oxidoreductase is a pyruvate:flavodoxin oxidoreductase (PFOR).

2. The method according to claim 1 , wherein the PFOR comprises an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from Ec. YdbK (SEQ ID NO: 9), Scy.pfor ( Synechocystis sp.) (SEQ ID NO: 29), Ki.pfor ( Kluyvera intermedia ) (SEQ ID NO: 30), Da.pfor ( Desulfovibrio africanus ) (SEQ ID NO: 31), Ns.pfor ( Nostoc sp.) (SEQ ID NO: 32), Ec.ydhV ( E. coli ) (SEQ ID NO: 33), Ga.pfor ( Gilliamella apicola ) (SEQ ID NO: 35), and Sco.pfor ( Synechococcus sp.).

3. The method according to claim 1 , wherein the PFOR comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 9.

4. The method according to claim 1 , wherein a polynucleotide encoding the PFOR is integrated into a chromosome or expressed from a plasmid.

5. The method according to claim 1 , wherein the bacterial strain expresses two or more recombinant electron carriers.

6. The method according to claim 5 , wherein at least one recombinant electron carrier has a redox potential in the range of about 400 to 550 mV.

7. The method according to claim 5 , wherein at least one recombinant electron carrier is a recombinant ferrodoxin (fdx) or recombinant flavodoxin (Fld).

8. The method according to claim 7 , wherein the recombinant fdx is selected from Hm.fdx1 ( Heliobacterium modesticaldum ), Pa.fdx ( Pseudomonas aeruginosa ), Cv.fdx ( Allochromatium vinosum ), Ca.fdx ( Clostridium acetobutylicum ), Cp.fdx ( Clostridium pasteurianum ), Ev2.fdx ( Ectothiorhodospira shaposhnikovii ), Pp1.fdx ( Pseudomonas putida ) and Pp2.fdx ( Pseudomonas putida ), or a derivative thereof.

9. The method according to claim 5 , wherein the recombinant fld is selected from Ac.fldA2 ( Azotobacter chroococcum ), Av.fldA2 ( Azotobacter vinelandii ), and Bs.fldA ( B. subtilis ), or a derivative thereof.

10. The method according to claim 1 , wherein the downstream pathway comprises one or more P450 enzymes for the production of a terpenoid compound.

11. The method of claim 1 , wherein the bacterial strain is a bacteria selected from Escherichia spp., Bacillus spp., Corynebacterium spp., Rhodobacter spp., Zymomonas spp., Vibrio spp., and Pseudomonas spp.

12. The method of claim 11 , wherein the bacterial strain is a species selected from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis, Vibrio natriegens , or Pseudomonas putida.

13. The method of claim 12 , wherein the bacterial strain is E. coli.

14. The method of claim 1 , wherein the bacterial strain has reduced or eliminated pyruvate dehydrogenase (PDH) mediated conversion of pyruvate to acetyl-CoA.

15. The method of claim 14 , wherein the bacterial strain expresses a mutant aceE gene.

16. The method of claim 15 , wherein the bacterial strain has a deletion or inactivation of aceE.

17. The method of claim 1 , wherein the IspG and IspH enzymes are overexpressed in the bacterial strain by complementation with recombinant ispG and ispH genes, such that IspH activity is higher than IspG activity.

18. The method of claim 17 , wherein the bacterial strain expresses dxs, ispD, ispF, and idi as recombinant genes.

19. The method of claim 18 , wherein the bacterial strain expresses dxs, dxr, ispD, ispE, ispF, and idi as recombinant genes.

20. The method of claim 19 , wherein the expression or activity of IspG and IspH is balanced with respect to the expression or activity of Dxr, Dxs, IspD, IspE and IspF to reduce MEcPP metabolite in the culture.

21. The method of claim 1 , wherein culturing step is a fed-batch process comprising a biomass production phase where bacterial biomass is created, followed by a terpene or terpenoid production phase.

22. The method of claim 21 , wherein the culture is at least about 100 L.

23. The method of claim 21 , wherein the culture is maintained under aerobic conditions.

24. The method of claim 21 , wherein the culture is maintained under micro-aerobic conditions.

25. The method of claim 23 , wherein the biomass production phase occurs under aerobic conditions, followed by reducing oxygen levels after from about 10 to about 20 hours.

26. The method of claim 21 , wherein the production phase includes feeding a nitrogen source.

27. The method of claim 21 , wherein the production phase includes feeding a carbon source.

28. The method of claim 1 , further comprising, recovering the terpene or terpenoid product.

29. The method according to claim 2 , wherein the PFOR comprises an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from Ec. YdbK (SEQ ID NO: 9), Scy.pfor ( Synechocystis sp.) (SEQ ID NO: 29), Ki.pfor ( Kluyvera intermedia ) (SEQ ID NO: 30), Da.pfor ( Desulfovibrio africanus ) (SEQ ID NO: 31), Ns.pfor ( Nostoc sp.) (SEQ ID NO: 32), Ec.ydhV ( E. coli ) (SEQ ID NO: 33), Ga.pfor ( Gilliamella apicola ) (SEQ ID NO: 35), and Sco.pfor ( Synechococcus sp.).

30. The method according to claim 6 , wherein at least one recombinant electron carrier has a redox potential in the range of about 400 to 500 mV.

31. The method according to claim 6 , wherein at least one recombinant electron carrier has a redox potential in the range of about 400 to 450 mV.

32. The method according to claim 21 , wherein the culture is at least about 10,000 L.

33. The method according to claim 21 , wherein the culture is at least about 100,000 L.