Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains
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NADPH-dependent reactions play important roles in production of industrially valuable compounds. In this study, we used phosphofructokinase (PFK)-deficient strains to direct fructose-6-phosphate to be oxidized through the pentose phosphate pathway (PPP) to increase NADPH generation. pfkA or pfkB single deletion and double-deletion strains were tested for their ability to produce lycopene. Since lycopene biosynthesis requires many NADPH, levels of lycopene were compared in a set of isogenic strains, with the pfkA single deletion strain showing the highest lycopene yield. Using another NADPH-requiring process, a one-step reduction reaction of 2-chloroacrylate to 2-chloropropionic acid by 2-haloacrylate reductase, the pfkA pfkB double-deletion strain showed the highest yield of 2-chloropropionic acid product. The combined effect of glucose-6-phosphate dehydrogenase overexpression or lactate dehydrogenase deletion with PFK deficiency on NADPH bioavailability was also studied. The results indicated that the flux distribution of fructose-6-phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis.
KeywordsPFK NADPH bioavailability pfkA pfkB G6PDH E. coli
This work was supported in part by National Science Foundation CBET0828516. Y. Wang was partially supported by a postdoctoral fellowship from the HHMI Beyond Traditional Borders program and by a John S. Dunn Foundation Collaborative Research Award. The authors want to thank Prof. Kurata for providing plasmid pET101-D-topo-CAA43 and Prof. Benning for the gift of pASK-IBA3-G6PD1.
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