Increasing intracellular NADPH availability in
A method of increasing cellular NADPH levels by expressing one or more genes that encode a enzyme that causes the production of NADPH. The system can be combined with other enzymes that require NADPH, thus improving the overall production of the desired protein.
1. A method of increasing the NADPH levels in a bacterial cell, comprising
a) providing a bacterial host cell,
b) transforming the host cell with an expression vector encoding a pyridine nucleotide transhydrogenase that catalyzes the following reaction:
and,
c) transforming the host cell with one or more genes that encode at least one enzyme that uses NADPH as a cofactor,
wherein the bacterial host cell produces at least 50% more NADPH after transformation than it did prior to transformation.
2. The method of claim 1 , wherein at least one enzyme is overexpressed.
3. The method of claim 2 , wherein at least one enzyme is selected from the group consisting of formate dehydrogenase, glucose-6 phosphate dehydrogenase, mannitol dehydrogenase, erythritol-4-phosphate dehydrogenase, glucose dehydrogenase (GDH) and NADPH forming glyceraldehyde phosphate dehydrogenase.
4. The method of claim 3 , wherein the host cell is E. coli.
5. The method of claim 1 , wherein the genes are the PHB operon.
6. A genetically engineered cell that produces more NADPH than the same cell prior to genetic engineering, wherein said cell comprises one or two expression vectors encoding:
a) a pyridine nucleotide transhydrogenase; and
b) phb operon including β-ketothiolase, acetoacetyl-CoA reductase and PHB synthase, wherein said cell increases NADPH production as measured by increased PHB synthesis.
7. The genetically engineered cell of claim 6 , that produces at least 50% more NADPH than the same cell prior to genetic engineering.
8. The genetically engineered cell of claim 6 , that produces at least 75% more NADPH than the same cell prior to genetic engineering.
9. The genetically engineered cell of claim 8 , that is a bacterial cell.
10. The genetically engineered cell of claim 9 , additionally comprising an enzyme selected from the group consisting of formate dehydrogenase, glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, erythritol-4-phosphate dehydrogenase, and glucose dehydrogenase, wherein the enzyme is overexpressed.
11. The genetically engineered cell of claim 6 , comprising an E. coli cell that overexpresses pyridine nucleotide transhydrogenase.
12. A genetically engineered bacterial cell, comprising overexpression of pyridine nucleotide transhydrogenase so that at least 50% more NADPH is expressed in said cell as compared with the same cell prior to genetic engineering; and overexpression of one or more genes that encode at least one enzyme that uses NADPH as a cofactor.