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 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.
Kaup
B
B
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Bringer-Meyer
S
S
Sahm
H
H
D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D001709:Biotechnology / Q000379:methods*; D001711:Biotransformation; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005285:Fermentation; D005560:Formate Dehydrogenases / Q000235:genetics / Q000502:physiology; D005632:Fructose / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007934:Leuconostoc / Q000201:enzymology / Q000235:genetics; D008353:Mannitol / Q000378:metabolism*; D008354:Mannitol Dehydrogenases / Q000235:genetics / Q000502:physiology; D009161:Mycobacterium / Q000201:enzymology / Q000235:genetics; D009876:Operon; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D012972:Sodium Hydroxide / Q000378:metabolism; D014169:Transformation, Bacterial; D017355:Zymomonas / Q000235:genetics / Q000502:physiology
D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; C086512:glucose facilitator protein, Zymomonas; D005632:Fructose; D008353:Mannitol; D012972:Sodium Hydroxide; D008354:Mannitol Dehydrogenases; D005560:Formate Dehydrogenases