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. 1996 Nov;62(11):4155–4161. doi: 10.1128/aem.62.11.4155-4161.1996

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.

P Weisser 1, R Krämer 1, G A Sprenger 1
PMCID: PMC168237  PMID: 8900006

Abstract

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.

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Selected References

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  1. Aulkemeyer P., Ebner R., Heilenmann G., Jahreis K., Schmid K., Wrieden S., Lengeler J. W. Molecular analysis of two fructokinases involved in sucrose metabolism of enteric bacteria. Mol Microbiol. 1991 Dec;5(12):2913–2922. doi: 10.1111/j.1365-2958.1991.tb01851.x. [DOI] [PubMed] [Google Scholar]
  2. Bork P., Sander C., Valencia A. Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases. Protein Sci. 1993 Jan;2(1):31–40. doi: 10.1002/pro.5560020104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Brickman E., Soll L., Beckwith J. Genetic characterization of mutations which affect catabolite-sensitive operons in Escherichia coli, including deletions of the gene for adenyl cyclase. J Bacteriol. 1973 Nov;116(2):582–587. doi: 10.1128/jb.116.2.582-587.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Curtis S. J., Epstein W. Phosphorylation of D-glucose in Escherichia coli mutants defective in glucosephosphotransferase, mannosephosphotransferase, and glucokinase. J Bacteriol. 1975 Jun;122(3):1189–1199. doi: 10.1128/jb.122.3.1189-1199.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dimarco A. A., Romano A. H. d-Glucose Transport System of Zymomonas mobilis. Appl Environ Microbiol. 1985 Jan;49(1):151–157. doi: 10.1128/aem.49.1.151-157.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eddy C. K., Smith O. H., Noel K. D. Cosmid cloning of five Zymomonas trp genes by complementation of Escherichia coli and Pseudomonas putida trp mutants. J Bacteriol. 1988 Jul;170(7):3158–3163. doi: 10.1128/jb.170.7.3158-3163.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Markovitz A., Sydiskis R. J., Lieberman M. M. Genetic and biochemical studies on mannose-negative mutants that are deficient in phosphomannose isomerase in Escherichia coli K-12. J Bacteriol. 1967 Nov;94(5):1492–1496. doi: 10.1128/jb.94.5.1492-1496.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Miles J. S., Guest J. R. Nucleotide sequence and transcriptional start point of the phosphomannose isomerase gene (manA) of Escherichia coli. Gene. 1984 Dec;32(1-2):41–48. doi: 10.1016/0378-1119(84)90030-1. [DOI] [PubMed] [Google Scholar]
  12. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  13. Parker C., Barnell W. O., Snoep J. L., Ingram L. O., Conway T. Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport. Mol Microbiol. 1995 Mar;15(5):795–802. doi: 10.1111/j.1365-2958.1995.tb02350.x. [DOI] [PubMed] [Google Scholar]
  14. Porter E. V., Chassy B. M., Holmlund C. E. Partial purification and properties of a mannofructokinase from Streptococcus mutans SL-1. Infect Immun. 1980 Oct;30(1):43–50. doi: 10.1128/iai.30.1.43-50.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Scopes R. K., Testolin V., Stoter A., Griffiths-Smith K., Algar E. M. Simultaneous purification and characterization of glucokinase, fructokinase and glucose-6-phosphate dehydrogenase from Zymomonas mobilis. Biochem J. 1985 Jun 15;228(3):627–634. doi: 10.1042/bj2280627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sebastian J., Asensio C. Purification and properties of the mannokinase from Escherichia coli. Arch Biochem Biophys. 1972 Jul;151(1):227–233. doi: 10.1016/0003-9861(72)90492-4. [DOI] [PubMed] [Google Scholar]
  17. Snoep J. L., Arfman N., Yomano L. P., Fliege R. K., Conway T., Ingram L. O. Reconstruction of glucose uptake and phosphorylation in a glucose-negative mutant of Escherichia coli by using Zymomonas mobilis genes encoding the glucose facilitator protein and glucokinase. J Bacteriol. 1994 Apr;176(7):2133–2135. doi: 10.1128/jb.176.7.2133-2135.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Swings J., De Ley J. The biology of Zymomonas. Bacteriol Rev. 1977 Mar;41(1):1–46. doi: 10.1128/br.41.1.1-46.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tanaka S., Lerner S. A., Lin E. C. Replacement of a phosphoenolpyruvate-dependent phosphotransferase by a nicotinamide adenine dinucleotide-linked dehydrogenase for the utilization of mannitol. J Bacteriol. 1967 Feb;93(2):642–648. doi: 10.1128/jb.93.2.642-648.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Uhlenbusch I., Sahm H., Sprenger G. A. Expression of an L-alanine dehydrogenase gene in Zymomonas mobilis and excretion of L-alanine. Appl Environ Microbiol. 1991 May;57(5):1360–1366. doi: 10.1128/aem.57.5.1360-1366.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Walsh M. C., Smits H. P., Scholte M., van Dam K. Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose. J Bacteriol. 1994 Feb;176(4):953–958. doi: 10.1128/jb.176.4.953-958.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Weisser P., Krämer R., Sahm H., Sprenger G. A. Functional expression of the glucose transporter of Zymomonas mobilis leads to restoration of glucose and fructose uptake in Escherichia coli mutants and provides evidence for its facilitator action. J Bacteriol. 1995 Jun;177(11):3351–3354. doi: 10.1128/jb.177.11.3351-3354.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Zembrzuski B., Chilco P., Liu X. L., Liu J., Conway T., Scopes R. Cloning, sequencing, and expression of the Zymomonas mobilis fructokinase gene and structural comparison of the enzyme with other hexose kinases. J Bacteriol. 1992 Jun;174(11):3455–3460. doi: 10.1128/jb.174.11.3455-3460.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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