Applied Microbiology and Biotechnology

, Volume 92, Issue 5, pp 929–937 | Cite as

Increased NADPH availability in Escherichia coli: improvement of the product per glucose ratio in reductive whole-cell biotransformation

Biotechnological Products and Process Engineering

Abstract

A basic requirement for the efficiency of reductive whole-cell biotransformations is the reducing capacity of the host. Here, the pentose phosphate pathway (PPP) was applied for NADPH regeneration with glucose as the electron-donating co-substrate using Escherichia coli as host. Reduction of the prochiral β-keto ester methyl acetoacetate to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) served as a model reaction, catalyzed by an R-specific alcohol dehydrogenase. The main focus was maximization of the reduced product per glucose yield of this pathway-coupled cofactor regeneration with resting cells. With a strain lacking the phosphoglucose isomerase, the yield of the reference strain was increased from 2.44 to 3.78 mol MHB/mol glucose. Even higher yields were obtained with strains lacking either phosphofructokinase I (4.79 mol MHB/mol glucose) or phosphofructokinase I and II (5.46 mol MHB/mol glucose). These results persuasively demonstrate the potential of NADPH generation by the PPP in whole-cell biotransformations.

Keywords

Escherichia coli Pathway engineering NADPH yield Resting cells Reductive whole-cell biotransformation pfkA pfkB pgi 

Supplementary material

253_2011_3374_MOESM1_ESM.pdf (16 kb)
Table S1AIntracellular concentrations of NADPH and NADP+ and ratios of [NADPH]/[NADP+] in E. coli reference strain and deletion mutants at three time points during biotransformation. All strains expressed the plasmid-encoded alcohol dehydrogenase gene from Lactobacillus brevis. Samples taken at the time point zero did not yet contain the biotransformation substrate MAA. Results were derived from at least two independent experiments (PDF 16 kb)
253_2011_3374_MOESM2_ESM.pdf (16 kb)
Table S1BIntracellular concentrations of NADH and NAD+ and ratios of [NADH]/[NAD+] in E. coli reference strain and deletion mutants at three time points during biotransformation. All strains expressed the plasmid-encoded alcohol dehydrogenase gene from Lactobacillus brevis. Samples taken at the time point zero did not yet contain the biotransformation substrate MAA. Results were derived from at least two independent experiments (PDF 16 kb)
253_2011_3374_MOESM3_ESM.pdf (19 kb)
Fig. S1Correlation of the [NADPH]/[NADP+] ratios before MAA addition to the biotransformation test mixtures (t0) and the biotransformation yield (mole MHB per mole glucose) (PDF 19 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Institut für Bio- und Geowissenschaften, IBG-1: BiotechnologieForschungszentrum JülichJülichGermany
  2. 2.Institut für Biotechnologie 1Forschungszentrum Jülich GmbHJülichGermany

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