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

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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.

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Acknowledgements

This work was funded by the BIO.NRW Cluster Biotechnology North Rhine-Westphalia, Germany (support code: W0805wb001b).

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Authors and Affiliations

  1. Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, 52425, Jülich, Germany

    Solvej Siedler, Stephanie Bringer & Michael Bott

  2. Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany

    Stephanie Bringer & Michael Bott

Authors
  1. Solvej Siedler
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  2. Stephanie Bringer
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  3. Michael Bott
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Correspondence to Stephanie Bringer or Michael Bott.

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Table S1A

Intracellular 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)

Table S1B

Intracellular 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)

Fig. S1

Correlation of the [NADPH]/[NADP+] ratios before MAA addition to the biotransformation test mixtures (t 0) and the biotransformation yield (mole MHB per mole glucose) (PDF 19 kb)

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Siedler, S., Bringer, S. & Bott, M. Increased NADPH availability in Escherichia coli: improvement of the product per glucose ratio in reductive whole-cell biotransformation. Appl Microbiol Biotechnol 92, 929–937 (2011). https://doi.org/10.1007/s00253-011-3374-4

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