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

• Published in: Applied microbiology and biotechnology 2004-04, Vol.64 (3), p.333-339
• Main Authors: Kaup, B, Bringer-Meyer, S, Sahm, H
• Format: Article
• Language: English
• Subjects: Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - physiology; Biocatalysts; Biological and medical sciences; Biotechnology; Biotechnology - methods; Biotransformation; Carbon dioxide; Cloning, Molecular; Culture Media; Dehydrogenase; Dehydrogenases; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Formate Dehydrogenases - genetics; Formate Dehydrogenases - physiology; Fructose - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Bacterial; genetic recombination; Hydrogen-Ion Concentration; Leuconostoc - enzymology; Leuconostoc - genetics; mannitol; Mannitol - metabolism; Mannitol Dehydrogenases - genetics; Mannitol Dehydrogenases - physiology; Microbiology; Mycobacterium - enzymology; Mycobacterium - genetics; Operon; Oxidation; Oxidation-Reduction; Recombinant Proteins - metabolism; Sodium hydroxide; Sodium Hydroxide - metabolism; Transformation, Bacterial; Zymomonas - genetics; Zymomonas - physiology
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-003-1470-9

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-fructose) was 84 mol%. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for D-mannitol formation.