The Effect Of Reducing Power Generating Enzymes On Fatty Acid Biosynthesis In Escherichia Coli

Reducing power is an essential factor for fatty acid biosynthesis. In the process of fattyacid chain extending, the ongoing of reduction reactions must be supported by reducingpower supply. In the bacterial cells, reducing power generating enzymes includingNADP+-dependent malic enzyme (NADP-ME), NADP+-dependent isocitrate dehydrogenase(NADP-IDH), glucose6-phosphate dehydrogenase (G6PD),6-phosphogluconatedehydrogenase (PGD) and NAD+-dependent malic enzyme (NAD-ME) are all potentialsources of reducing power for fatty acid biosynthesis. But the actual role these five reducingpower generating enzymes play in bacterial fatty acid biosynthesis was not clear. In order toinvestigate their effect on fatty acid biosynthesis in Escherichia coli, endogenous NAD-ME,NADP-ME, NADP-IDH, G6PD and PGD were overexpressed respectively and the total fattyacid production of the reconstructed strains were analyzed in this study.NAD-ME, NADP-ME, NADP-IDH, G6PD and PGD were overexpressed in E. coliBL21(DE3) respectively, resulting recombinant strains ZY2to ZY6. Overexpression ofNAD-ME, NADP-ME, NADP-IDH, G6PD and PGD in the recombinant strains led to19-,37-,3-,315-and52-fold increase of the corresponding enzyme activity, respectively. Incomparison to the control strain ZY1, the total fatty acid production of strains ZY2to ZY6didn’t increase. Even by adding different concentrations of corresponding substrate to theculture, total fatty acid production of strains ZY3, ZY4, and ZY5didn’t increase. Thereforeoverexpressing reducing power generating enzymes in E. coli BL21(DE3) couldn’t lead tofatty acid yield increase and the wild type E. coli BL21(DE3) is not suitable as the host strainin this study. Strain BL21and ZY1to ZY6synthesized eight fatty acids: C12:0, C14:0, C16:0,C16:1, C17:0△, C18:0, C18:1and C19:0△.Then strain BL21△fadE/pTE which possesses the ability to accumulate fatty acids wasconstructed to serve as the new host strain, and the five reducing power generating enzymeswere overexpressed in it, resulting recombinant strains YS2to YS6. Overexpression ofNAD-ME, NADP-ME, NADP-IDH, G6PD and PGD in the recombinant strains led to2-,826-,11-,96-and14-fold increase of the corresponding enzyme activity, respectively. Thetotal fatty acid production of strains YS2to YS6presented significant differences:overexpression of NAD-ME (YS2), G6PD (YS5) and NADP-IDH (YS4) resulted in2.15-,1.6-and1.16-fold increase in total fatty acid production respectively, while overexpression ofPGD (YS6) didn’t lead to a significant increase of fatty acid production and overexpression ofNADP-ME (YS3) led to a15%decline in fatty acid yield. In strains YS1to YS2, C12:1andC14:1were detected besides the mentioned eight fatty acids existed in ZY strains. Comparedto the control strain YS1, the percentages of C12and C14fatty acids increased significantly.Total fatty acid yield increase of strains YS2and YS5was mainly caused by the increase ofthe C12and C14fatty acids production.This study demonstrated that NADH can work as the reducing power for fatty acidbiosynthesis in E.coli, and in the regard of reducing power supply, NAD-ME and G6PDprobably play a more important role than other reducing power generating enzymes (NADP-ME, NADP-IDH and PGD) in E.coli fatty acid biosynthesis. This work also providesa feasible strategy for enhancing fatty acid production in engineered E. coli strain byoverexpressing the reducing power generating enzymes NAD-ME and G6PD.