title: 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 (13)C 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.
authors: Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, Jülich, Germany.
authors: Solvej
authors: S
authors: Siedler
authors: Stephanie
authors: S
authors: Bringer
authors: Lars M
authors: LM
authors: Blank
authors: Michael
authors: M
authors: Bott
mesh_terms: D000090:Acetoacetates / Q000378:metabolism*; D001711:Biotransformation; D002247:Carbon Isotopes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005947:Glucose / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism*; D060847:Metabolic Engineering; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010427:Pentose Phosphate Pathway / Q000235:genetics; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism
chemical_list: C539306:3-hydroxybutyrate methyl ester; D000090:Acetoacetates; D002247:Carbon Isotopes; D006885:Hydroxybutyrates; D009249:NADP; C005217:methyl acetoacetate; D010770:Phosphotransferases; D005947:Glucose