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Applied Microbiology and Biotechnology

, Volume 64, Issue 3, pp 333–339 | Cite as

Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for d-mannitol formation in a whole-cell biotransformation

  • B. Kaup
  • S. Bringer-MeyerEmail author
  • H. Sahm
Original Paper

Abstract

A whole-cell biotransformation system for the conversion of d-fructose to d-mannitol was developed in Escherichia coli by constructing a recombinant oxidation/reduction cycle. First, the mdh gene, encoding mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH), was expressed, effecting strong catalytic activity of an NADH-dependent reduction of d-fructose to d-mannitol in cell extracts of the recombinant E. coli strain. By contrast whole cells of the strain were unable to produce d-mannitol from d-fructose. To provide a source of reduction equivalents needed for d-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH), encoding formate dehydrogenase, was functionally co-expressed. FDH generates the NADH used for d-fructose reduction by dehydrogenation of formate to carbon dioxide. These recombinant E. coli cells were able to form d-mannitol from d-fructose in a low but significant quantity (15 mM). The introduction of a further gene, encoding the glucose facilitator protein of Zymomonas mobilis (GLF), allowed the cells to efficiently take up d-fructose, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM d-mannitol in 17 h. Due to equimolar formation of sodium hydroxide during NAD+-dependent oxidation of sodium formate to carbon dioxide, the pH value of the buffered biotransformation system increased by one pH unit within 2 h. Biotransformations conducted under pH control by formic-acid addition yielded d-mannitol at a concentration of 362 mM within 8 h. The yield Y D-mannitol/D-fructosewas 84 mol%. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for d-mannitol formation.

Keywords

Biotransformation Formate Dehydrogenase Sodium Formate Zymomonas Mobilis Mannitol Dehydrogenase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

We wish to thank N. Esaki for plasmid pMcFDH, G.A. Sprenger for plasmid pZY507glf. We also thank the Fonds der Chemischen Industrie. This work was supported by the Institut für Technologie der Kohlenhydrate–Zuckerinstitut e. V.–Braunschweig, Germany. The authors ensure that all experiments comply with the current German laws.

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Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Institut für Biotechnologie 1Forschungszentrum Jülich GmbHJülichGermany

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