Expression of the Escherichia coli pmi gene, encoding phosphomannose-isomerase in Zymomonas mobilis, leads to utilization of mannose as a novel growth substrate, which can be used as a selective marker.
Wild-type Zymomonas mobilis can utilize only three substrates (sucrose, glucose, and fructose) as sole carbon sources, which are largely converted into ethanol and carbon dioxide. Here, we show that although D-mannose is not used as a growth substrate, it is taken up via the glucose uniport system (glucose facilitator protein) with a Vmax similar to that of glucose. Moreover, D-mannose was phosphorylated by a side activity of the resident fructokinase to mannose-6-phosphate. Fructokinase was purified to homogeneity from an frk-recombinant Z. mobilis strain showing a specific activity of 205 +/- 25 U of protein mg-1 with fructose (K(m), 0.75 +/- 0.06 mM) and 17 +/- 2 U mg-1 (relative activity, 8.5%) with mannose (K(m), 0.65 +/- 0.08 mM). However, no phosphomannoseisomerase activity could be detected for Z. mobilis, and this appeared to be the reason for the lack of growth on mannose. Therefore, we introduced the Escherichia coli gene pmi (manA) in Z. mobilis under the control of a lacIq-Ptac system on a broad-host-range plasmid (pZY507; Cmr). Subsequently, in pmi-recombinant cells of Z. mobilis, phosphomannoseisomerase was expressed in a range of from 3 U (without isopropyl-beta-D-thiogalactopyranoside [IPTG]) to 20 U mg-1 of protein in crude extracts (after IPTG induction). Recombinant cells of different Z. mobilis strains utilized mannose (4%) as the sole carbon source with a growth rate of 0.07 h-1, provided that they contained fructokinase activity. When the frk gene was additionally expressed from the same vector, fructokinase activities of as much as 9.7 U mg-1 and growth rates of as much as 0.25 h-1 were detected, compared with 0.34 h-1 on fructose for wild-type Z. mobilis. Selection for growth on mannose was used to monitor plasmid transfer of pZY507pmi from E. coli to Z. mobilis strains and could replace the previous selection for antibiotic resistance.
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, Germany.
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Weisser
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Krämer
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Sprenger
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D001693:Biological Transport, Active; D015415:Biomarkers; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005631:Fructokinases / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D007700:Kinetics; D008358:Mannose / Q000378:metabolism* / Q000493:pharmacokinetics; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D013379:Substrate Specificity; D014994:Xylose / Q000378:metabolism; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development
D001426:Bacterial Proteins; D015415:Biomarkers; D017931:DNA Primers; C086512:glucose facilitator protein, Zymomonas; D014994:Xylose; D005631:Fructokinases; C046173:fructokinase; D008359:Mannose-6-Phosphate Isomerase; D008358:Mannose