Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation.
A whole-cell biotransformation system for the conversion of D-fructose to D-mannitol was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. First, the mdh gene encoding for the mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH) was expressed, effecting a strong catalytic activity of a NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant E. coli strain but not enabling whole cells of the strain to produce D-mannitol from D-fructose. To provide a source for reduction equivalents needed for D-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH) encoding formate dehydrogenase was functionally co-expressed. FDH generates 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 for the glucose facilitator protein of Zymomonas mobilis (GLF) enabled the cells to efficiently take up D-fructose into the cells, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM D-mannitol in 17 hours. Biotransformations conducted under pH-control by formic acid addition yielded D-mannitol at a concentration of 362 mM within 8 hours. 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.
Institute of Biotechnologie 1, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany.
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B
Kaup
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Bringer-Meyer
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H
Sahm
D001711:Biotransformation; D004738:Engineering / Q000379:methods; D004926:Escherichia coli / Q000254:growth & development* / Q000302:isolation & purification / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000378:metabolism; D005561:Formates / Q000378:metabolism*; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D005561:Formates; D011994:Recombinant Proteins; D008353:Mannitol; D005560:Formate Dehydrogenases