Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport

Abstract

Optimization of yields and productivities in reductive whole-cell biotransformations is an important issue for the industrial application of such processes. In a recent study with Escherichia coli, we analyzed the reduction of the prochiral β-ketoester methyl acetoacetate by an R-specific alcohol dehydrogenase (ADH) to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) using glucose as substrate for the generation of NADPH. Deletion of the phosphofructokinase gene pfkA almost doubled the yield to 4.8 mol MHB per mole of glucose, and it was assumed that this effect was due to a partial cyclization of the pentose phosphate pathway (PPP). Here, this partial cyclization was confirmed by 13C metabolic flux analysis, which revealed a negative net flux from glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. For further process optimization, the genes encoding the glucose facilitator (glf) and glucokinase (glk) of Zymomonas mobilis were overexpressed in recombinant E. coli strains carrying ADH and deletions of either pgi (phosphoglucose isomerase), or pfkA, or pfkA plus pfkB. In all cases, the glucose uptake rate was increased (30–47%), and for strains Δpgi and ΔpfkA also, the specific MHB production rate was increased by 15% and 20%, respectively. The yield of the latter two strains slightly dropped by 11% and 6%, but was still 73% and 132% higher compared to the reference strain with intact pgi and pfkA genes and expressing glf and glk. Thus, metabolic engineering strategies are presented for improving yield and rate of reductive redox biocatalysis by partial cyclization of the PPP and by increasing glucose uptake, respectively.

Mutational Analysis of the Pentose Phosphate and Entner-Doudoroff Pathways in Gluconobacter oxydans Reveals Improved Growth of a Δ edd Δ eda Mutant on Mannitol

“…In the absence of phosphofructokinase, the PPP operates cyclically, as fructose-6-phosphate formed by transaldolase or transketolase is converted to glucose-6-phosphate, which enters the oxidative PPP again. This has recently been shown for an Escherichia coli ⌬pfkA mutant (23). Therefore, conversion of 1 mol glucose-6-phosphate via the PPP in the absence of phosphofructokinase yields 1 mol acetate or acetyl-CoA ϩ 4 mol CO 2 ϩ 8 mol NAD(P)H ϩ 2 mol ATP (13) (Fig.…”

Cofactor engineering for advancing chemical biotechnology

“…We have shown that replacement of the E. coli native NAD + dependent GAPDH gapA by NADP + dependent GAPDH gapB from Bacillus subtilis increased product formation and the strain showed further increased NADPH dependent biosynthesis when NAD kinase was co-expressed (unpublished results). 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].…”

“…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].…”

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

“…Normally, the fructose 6-phosphate resulting from the non-oxidative stage of PPP will enter glycolysis, but in the absence of both pfkA and pfkB, theoretically, it will be converted to glucose 6-phophate and can proceed back to PPP again, which will lead to more NADPH generation. This has been recently described as a partial cyclization of the pentose phosphate pathway (Siedler et al 2012). Glyceraldehyde 3-phosphate resulting from PPP will reenter the glycolytic pathway and TCA cycle in the end to generate NADH and ATP.…”