Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains

Abstract

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.

Cofactor engineering for advancing chemical biotechnology

“…In another strategy, phosphofructokinase has been deleted from E. coli [49][50][51] and studied in various combinations with other alterations such as GAPDH overexpression, NAD kinase, glucose uptake systems and transhydrogenase alterations. The altered glycolysis in pfk mutants routed glucose through pentose phosphate pathway and led to higher NADPH dependent biosynthesis in systems sensitive to NADPH availability reported in [49][50][51] and found by our group [52]. Xylitol formation using NADPH-dependent xylose reductase from Candida boidinii was improved in an E. coli pfkA and sthA mutant [51].…”

NADPH-generating systems in bacteria and archaea

“…The apparent relationship between the rate of NADPH generation and actual carbon fluxes through the oxPPP suggests that the oxPPP can be a target for metabolic engineering for overproduction of NADPH. The oxPPP has been engineered to increase the NADPH/NADP + ratio, through overexpression of oxPPP enzymes (Lim et al, 2002 ; Choi et al, 2003 ; Becker et al, 2007 ; Lee et al, 2007a ) and through redirection of the carbon flux from glycolysis to the oxPPP, by disrupting phosphoglucose isomerase (Kabir and Shimizu, 2003 ; Marx et al, 2003 ; Chemler et al, 2010 ) or phosphofructokinase (Chin and Cirino, 2011 ; Wang et al, 2013d ), overexpressing fructose 1,6-bisphosphatase (Becker et al, 2005 ), or introducing glucose dehydrogenase (Zhang et al, 2011 ). Both strategies have been applied successfully in various prokaryotes, but reduction in growth is a common side effect (Lim et al, 2002 ; Lee et al, 2003 ; Marx et al, 2003 ).…”

Improvement of NADPH-dependent P450-mediated biotransformation of 7α,15α-diOH-DHEA from DHEA by a dual cosubstrate-coupled system

“…Both nicotinic acid and glucose showed beneficial effects on DHEA biotransformation, and the pathways for NADPH synthesis by these two cosubstrates were different [15,23]. Therefore, the dual cosubstrate combination of nicotinic acid and glucose was investigated to further improve the 7α,15α-diOH-DHEA molar conversion.…”