Skip to main content
SpringerLink
Log in

Growth ofZymomonas on lactose: Gene cloning in combination with mutagenesis

  • Original Papers
  • Published:
Journal of Industrial Microbiology

Summary

Wild-type strains ofZymomonas mobilis have a limited substrate range of glucose, fructose and sucrose. In order to expand this substrate range, transconjugants ofZ. mobilis containing Lac+ plasmids have been constructed. Although β-galactosidase is expressed in such strains, they lack the ability to grow on lactose. We now report the development ofZ. mobilis strains capable of growth on lactose. This was achieved in two stages. First, a broad host range plasmid was constructed (pRUT102) which contained the lactose operon under the control of aZ. mobilis promoter plus genes for galactose utilization.Z. mobilis CP4.45 containing pRUT102 was then subjected to mutagenesis combined with continued selection pressure for growth on lactose. One strain,Z. mobilis SB6, produced a turbid culture that yielded 0.25% ethanol from 5% lactose (plus 2% yeast extract) in 15 days.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Ahmed, A. 1984. Plasmid vectors for positive galactose-resistance selection of cloned DNA inEscherichia coli. Gene 28: 37–43.

    PubMed  Google Scholar 

  2. Bagdasarian, M. and K.N. Timmis. 1981. Host-vector systems for gene cloning inPseudomonas. Curr. Topics. Microbiol. Immunol. 96: 47–67.

    Google Scholar 

  3. Buchholz, S.E. and D.E. Eveleigh. 1986. Transfer of plasmids to an antibiotic-sensitive mutant ofZymomonas mobilis. Appl. Environ. Microbiol. 52: 366–370.

    Google Scholar 

  4. Buchholz, S.E., P. O'Mullan and D.E. Eveleigh. 1988. Growth ofZymomonas mobilis CP4 on mannitol. Appl. Microbiol. Biotechnol. (in press).

  5. Carey, V.C., S.K. Walia and L.O. Ingram. 1983. Expression of a lactose transposon (Tn951) inZymomonas mobilis. Appl. Environ. Microbiol. 45: 1163–1168.

    Google Scholar 

  6. Carr, J.G. 1981. The genusZymomonas. In: The Prokaryotes; a Handbook on Habitats, Isolation and Identification of Bacteria, Vol. 2 (Starr, M.P., H.G. Truper, A. Balows and H.G. Schlegel, eds.), pp. 1349–1354, Springer-Verlag, New York.

    Google Scholar 

  7. Chun, U.H. and P.L. Rogers. 1986. Kinetic studies on aLac-containing strain ofZymomonas mobilis. Biotechnol. Lett. 8: 807–810.

    Google Scholar 

  8. Cornelis, G., D. Ghosal and H. Saedler. 1978. Tn951: a new transposon carrying the lactose operon. Mol. Gen. Genet. 160: 215–224.

    PubMed  Google Scholar 

  9. Dawes, E.A., D.W. Ribbons and P.J. Large. 1966. The route of ethanol formation inZymomonas mobilis. Biochem. J. 98: 795–803.

    PubMed  Google Scholar 

  10. Dooley, M.M. and S.E. Buchholz. 1987. Fermentation of lactose byZymomonas mobilis: genetic approaches. Annu. Meet. Am. Soc. Microbiol. (Atlanta, Ga), Abstr. O-28.

  11. Eveleigh, D.E., H.W. Stokes and E.L. Dally. 1983. Recombinant DNA approaches for enhancing the ethanol productivity ofZymomonas mobilis. In: Organic Chemicals from Biomass (Wise, D.L., ed.), pp. 69–91, Benjamin/Cummings, Menlo Park, CA.

    Google Scholar 

  12. Freifelder, D. 1987. Molecular Biology: a Comprehensive Introduction to Prokaryotes and Eukaryotes, 2nd Ed. Jones and Bartlett Publishers, Boston, MA.

    Google Scholar 

  13. Goodman, A.E., P.L. Rogers and M.L. Skotnicki. 1982. Minimal medium for isolation of auxotrophicZymomonas mutants. Appl. Environ. Microbiol. 44: 496–498.

    PubMed  Google Scholar 

  14. Goodman, A.E., A.T. Strzelecki and P.L. Rogers. 1984. Formation of ethanol from lactose byZymomonas mobilis. J. Biotechnol. 1: 219–228.

    Google Scholar 

  15. Grote, W., L.J. Lee and P.L. Rogers. 1980. Continuous ethanol production by immobilized cells ofZymomonas mobilis. Biotechnol. Lett. 2: 481–486.

    Google Scholar 

  16. Kaback, H.R. 1985. Proton electrochemical gradients and active transport: the saga oflac permease. Ann. N.Y. Acad. Sci. 456: 291–304.

    PubMed  Google Scholar 

  17. Krug, T.A. and A.J. Daugulis. 1983. Ethanol production usingZymomonas mobilis immobilized on an ion exchange resin. Biotechnol. Lett. 5: 159–184.

    Google Scholar 

  18. Lawford, G.R., B.H. Lavers, D. Good, R. Charley, J. Fein and H.G. Lawford. 1983.Zymomonas ethanol fermentations: biochemistry and bioengineering. In: Ethanol from Biomass International Symposium (Duckworth, H.E. and E.A. Thompson, eds.), pp. 482–507, Royal Society, Canada, Ottawa.

    Google Scholar 

  19. Maniatis, T., E.F. Fritsch and J. Sambrook. 1982. Molecular Cloning. Cold Spring Harbor Laboratories. Cold Spring Harbor, NY.

    Google Scholar 

  20. McGhee, J.E., G.St. Julian, R.W. Detroy and R.J. Bothast, 1982. Ethanol production by immobilizedSaccharomyces cerevisiae, Saccharomyces uvarum andZymomonas mobilis. Biotechnol. Bioeng. 29: 370–382.

    Google Scholar 

  21. Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Springer Harbor Laboratories, Cold Spring Harbor, NY.

    Google Scholar 

  22. Millis, N.F. 1956. A study of cider-sickness bacillus—a new variety ofZymomonas anaerobia. J. Gen. Microbiol. 15: 521–528.

    PubMed  Google Scholar 

  23. Montenecourt, B.S. 1985.Zymomonas, a unique genus of bacteria. In: Biology of Industrial Microorganisms (Demain, A.L. and N.A. Solomon, eds.), pp. 261–289, Benjamin/Cummings, Menlo Park, CA.

    Google Scholar 

  24. Palva, E.T. and T.J. Silhavy. 1984.lacZ fusions to genes that specify exported proteins: a general technique. Mol. Gen. Genet. 194: 388–394.

    PubMed  Google Scholar 

  25. Perbal, B. 1984. A Practical Guide to Molecular Cloning. John Wiley and Sons, New York.

    Google Scholar 

  26. Pugsley, A. P. and M. Schwartz. 1985. Export and secretion of proteins by bacteria. FEMS Microbiol. Rev. 32: 3–38.

    Google Scholar 

  27. Rogers, P.L., K.J. Lee, J.H. Lee and M.L. Skotnicki. 1984. Recent developments in theZymomonas process for ethanol production. CRC Crit. Rev. Biotechnol. 1: 273–288.

    Google Scholar 

  28. Rogers, P.L., K.J. Lee and D.E. Tribe. 1979. Kinetics of alcohol production byZymomonas mobilis at high sugar concentrations. Biotechnol. Lett. 1: 165–170.

    Google Scholar 

  29. Romano, A.H. 1986. Microbial sugar transport systems and their importance in biotechnology. Trends Biotechnol. 4: 207–213.

    Google Scholar 

  30. Stokes, H.W., E.L. Dally, M.D. Yablonsky and D.E. Eveleigh. 1983. Comparison of plasmids of strains ofZymomonas mobilis. Plasmid 9: 138–146.

    PubMed  Google Scholar 

  31. Strzelecki, A.T., A.E. Goodman and P.L. Rogers. 1986. Stability of thelac operon inZymomonas mobilis in batch and continuous culture. J. Biotechnol. 3: 197–205.

    Google Scholar 

  32. Swings, J. and J. De Ley. 1977. The biology ofZymomonas. Bacteriol. Rev. 41: 1–46.

    PubMed  Google Scholar 

  33. Vieira, J. and J. Messing. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: 259–268.

    PubMed  Google Scholar 

  34. Yanase, H., M. Yasui, T. Miyazaki and K. Tonomua. 1985. Fusion of spheroplasts and genetic recombination ofZymomonas mobilis. Agric. Biol. Chem. 49: 133–140.

    Google Scholar 

Download references

Author information

Author notes

  1. Steven E. Buchholz

    Present address: Experimental Station, E228/339, E.I. du Pont de Nemours & Co., 19898, Wilmington, DE, U.S.A.

  2. Margaret M. Dooley

    Present address: Biology Department, The College of Staten Island, 10301, Staten Island, NY, U.S.A.

Authors and Affiliations

  1. Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, NJ, U.S.A.

    Steven E. Buchholz, Margaret M. Dooley & Douglas E. Eveleigh

Authors
  1. Steven E. Buchholz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Margaret M. Dooley
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Douglas E. Eveleigh
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Buchholz, S.E., Dooley, M.M. & Eveleigh, D.E. Growth ofZymomonas on lactose: Gene cloning in combination with mutagenesis. Journal of Industrial Microbiology 4, 19–27 (1989). https://doi.org/10.1007/BF01569689

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01569689

Key words

Search

Navigation