Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 64, Issue 3, Pages 333-339Publisher
SPRINGER
DOI: 10.1007/s00253-003-1470-9
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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.
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