1887

Abstract

SUMMARY: Malate synthase is a key enzyme of the glyoxylate cycle, which is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, encoding malate synthase from Streptomyces clavuligems NRRL 3585, was cloned using PCR and fully sequenced. The ORF obtained encodes 541 amino acids with a deduced M, of 6OOOO, consistent with the observed M, (62000-64000) of most malate synthase enzymes reported so far. The aceB gene has a high G+C content (71.5 molO/O), especially in the third codon position. A 50 bp region upstream of the malate synthase ORF was predicted to be a prokaryotic promoter region. The relationship between carbon source, antibiotic (cephalosporin) biosynthesis and malate synthase activity was investigated. Growth of S. clavuligerus on acetate as the major carbon sorce was delayed, compared to that on glycerol. Furthermore, high levels of malate synthase activity were associated with the presence of acetate in the growth medium. Growth on acetate also resulted in lower levels of cephalosporin production, compared to that on glycerol. The cloned 5. clavuligerus aceB gene was expressed in Escherichia coli BL2l (DE3).Transformants exhibited an approximately 713old increase in malate synthase activity, compared to the control, thereby demonstrating high-level expression of soluble and enzymically active malate synthase in the heterologous host.

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1998-11-01
2024-04-20
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References

  1. Aharonowitz Y., Demain A.L. 1978; Carbon catabolite regulation of cephalosporin production in Streptomyces clavuligerus.. Antimicrob Agents Chemother 14:159–164
    [Google Scholar]
  2. Alves A.M.C.R., Euverink G.J.W., Hektor H.J., Hessels G.I., Vlag J.V.D., Vrijbloed J.W., Hondmann D., Visser J., Dijkhuizen L. 1994; Enzymes of glucose and methanol metab-olism in the actinomycete Amycolatopsis methanolica.. J Bacteriol 176:6827–6835
    [Google Scholar]
  3. Bibb M.J., Findlay P.R., Johnson M.W. 1984; The re-lationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences.. Gene 30:157–166
    [Google Scholar]
  4. Brana A.F., Wolfe S., Demain A.L. 1985; Ammonium repression of cephalosporin production by Streptomyces clavuligerus.. Can J Microbiol 31:736–743
    [Google Scholar]
  5. Brendel V., Brocchieri L., Sandler S.J., Clark A.J., Karlin S. 1997; Evolutionary comparisons of RecA-like proteins across all major kingdoms of living organisms.. J Mol Evol 44:528–541
    [Google Scholar]
  6. Byrne C., Stokes H.W., Ward K.A. 1988; Nucleotide sequence of the aceB gene encoding malate synthase A in Escherichia coli.. Nucleic Acids Res 16:9342
    [Google Scholar]
  7. Comai L., Baden C.S., Harada J.J. 1989; Deduced sequence of a malate synthase polypeptide encoded by a subclass of the gene family.. J Biol Chem 264:2778–2782
    [Google Scholar]
  8. Felsenstein J. 1993; PHYLIP (Phylogeny Inference Package) version 3.5c.. Seattle, WA: University of Washington;
    [Google Scholar]
  9. Fernandez E., Fernandez M., Rodicio R. 1993; Two structural genes are encoding malate synthase isoenzymes in Saccharomyces cerevisiae.. FEBS Lett 320:271–275
    [Google Scholar]
  10. Graham I.A., Smith L.M., Brown J.W.S., Leaver C.J., Smith S.M. 1989; The malate synthase gene of cucumber.. Plant Mol Biol 13:673–684
    [Google Scholar]
  11. Hartig A., Simon M.M., Schuster T., Daugherty J.R., Yoo H.S., Cooper T.G. 1992; Differentially regulated malate synthase genes participate in carbon and nitrogen metabolism of S. cerevisiae.. Nucleic Acids Res 20:5677–5686
    [Google Scholar]
  12. Hashimoto T., Nakamura Y., Kamaishi T., Nakamura F., Adachi J., Okamoto K., Hasegawa M. 1995; Phylogenetic place of mitochondrion-lacking protozoan, Gardia lamblia, inferred from amino acid sequences of elongation factor 2.. Mol Biol Evol 12:782–793
    [Google Scholar]
  13. Hikida M., Atomi H., Fukuda Y., Aoki A., Hishida T., Teranishi Y., Ueda M., Tanaka A. 1991; Presence of two transcribed malate synthase genes in an N-alkane-utilizing yeast, Candida tropicalis.. J Biochem 110:909–914
    [Google Scholar]
  14. Hüttner S., Mecke D., Fröhlich K.U. 1997; Gene cloning and sequencing, and enzyme purification of the malate synthase of Streptomyces arenae.. Gene 188:239–246
    [Google Scholar]
  15. Jennings D.H., Lysek G. 1996 Fungal Biology. Oxford, UK: BIOS;
    [Google Scholar]
  16. Jensen S.E., Leskiw B.K., Vining L.C., Aharanowitz Y., Westlake D.W.S., Wolfe S. 1986; Purification of isopenicillin N synthetase from Streptomyces clavuligerus.. J Bacteriol 172:7269–7271
    [Google Scholar]
  17. Kornberg H.L. 1966; The role and control of the glyoxylate cycle in Escherichia coli.. Biochem J 99:1–11
    [Google Scholar]
  18. Maloy S.R., Bohlander M., Nunn W.D. 1980; Elevated levels of glyoxylate shunt enzymes in Escherichia coli strains constitutive for fatty acid degradation.. J Bacteriol 143:720–725
    [Google Scholar]
  19. Moat A.G., Foster J.W. 1995; Carbohydrate metabolism and energy production.. In: Microbial Physiology, 3rd edn.. pp. 319–321 Moat A.G., Foster J.W. Edited by New York: Wiley-Liss;
    [Google Scholar]
  20. Molina I., Pellicer M.T., Aguilar J., Baldoma L. 1994; Molecular characterization of Escherichia coli malate synthase G: differentiation with the malate synthase A isoenzyme.. Eur J Biochem 224:541–548
    [Google Scholar]
  21. Obradors N., Aguilar J. 1991; Efficient biodegradation of high-molecular-weight polyethylene glycols by pure cultures of Pseudomonas stutzeri.. Appl Environ Microbiol 57:2383–2388
    [Google Scholar]
  22. Ornston L.N., Ornston M.K. 1969; Regulation of glyoxylate metabolism in Escherichia coli K-12.. J Bacteriol 98:1098–1108
    [Google Scholar]
  23. Reinscheid D.J., Eikmanns B.J., Sahm H. 1994; Malate synthase from Cory neb acterium glutamicum: sequence analysis of the gene and biochemical characterization of the enzyme.. Microbiology 140:3099–3108
    [Google Scholar]
  24. Rijik P.D., Peer Y.V.D., Broeck I.V.D., Wachter R.D. 1995; Evolution according to large ribosomal subunit RNA.. J Mol Evol 41:366–375
    [Google Scholar]
  25. Saitou N., Nei M. 1987; The neighbour-joining method: a new method for reconstructing phylogenetic trees.. Mol Biol Evol 4:406–425
    [Google Scholar]
  26. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  27. Sandemann R.A., Hynes M.J., Fincham J.R.S., Connerton I.F. 1991; Molecular organization of the malate synthase genes of Aspergillus nidulans and Neurospora crassa.. Mol Gen Genet 228:445–452
    [Google Scholar]
  28. Strohl W.R. 1992; Compilation and analysis of DNA sequences associated with apparent streptomycete promoters.. Nucleic Acids Res 20:961–974
    [Google Scholar]
  29. Thomas G.H., Connerton I.F., Fincham J.R.S. 1988; Molecular cloning, identification and transcriptional analysis of genes involved in acetate utilization in Neurospora crassa.. Mol Microbiol 2:599–606
    [Google Scholar]
  30. Thompson J.D., Higgins D.G., Gibson T.J. 1994; CLUSTAL W : improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.. Nucleic Acids Res 22:4673–4680
    [Google Scholar]
  31. Woese C.R. 1987; Bacterial evolution.. Microbiol Rev 51:221–271
    [Google Scholar]
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