Molecular analysis and heterologous expression of the gene encoding methylmalonyl—coenzyme a mutase from rifamycin SV-producing strain Amycolatopsis mediterranei U32
The conversion of succinyl-coenzyme A (CoA) into methylmalonyl-CoA, catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM), represents an important source of building blocks for rifamycin SV biosynthesis. The structural gene for MCM from rifamycin SV—producing strain Amycolatopsis mediterranei U32 was isolated by using a heterologous gene probe encoding the MCM of Streptomyces cinnamonensis. A 7.8-kbp fragment was sequenced and four complete open reading frames (ORFs) and two incomplete ORFs were found. Two central ORFs, ORF3 and ORF4, overlap by four nucleotides and were found to encode MCM small (602 residues) and large (721 residues) subunits, respectively. Comparison showed that the MCM gene of A. mediterranei U32 was quite similar to those from other sources. The functionally unknown ORF5, immediately downstream of the mut AB gene, was quite similar to the ORFs downstream of mut AB from S. cinnamonensis and Mycobacterium tuberculosis. Such a striking cross-species conservation of gene order suggested that ORF5 could also be involved in the metabolism of methylmalonyl-CoA. MCM gene was overexpressed in Escherichia coli under T7 promoter, and MCM activity could be detected in the recombinant E. coli clone harboring MCM gene after the addition of coenzyme B12. A purification procedure based on the B12 affinity column was established to purify the MCM from E. coli. The molecular weight of purified MCM from E. coli was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, which corresponds to that calculated from the MCM protein sequence and is also the same size as that of the enzyme purified directly from A. mediterranei U32. MCM gene was overexpressed in polyketide monensin producing S. cinnamonensis, and the total monensin production was increased by 32%.
Index EntriesMethylmalonyl—Coenzyme A mutase cloning expression rifamycin SV monensin biosynthesis
Unable to display preview. Download preview PDF.
- 5.March, E. N., McKie, N., Davis, N. K., and Leadlay, P. F. (1990), Biochem. J. 260, 345–352.Google Scholar
- 7.Wilkemeyer, M. F., Crane, A. M., and Leadley, F. D. (1990), Biochem. J. 271, 449–455.Google Scholar
- 10.Birch, A., Leiser, A., and Robinson, J. A. (1993), J. Bacteriol. 175, 3511–3519.Google Scholar
- 11.Roy, I. and Leadley, P. F. (1992), J. Bacteriol. 174, 5763, 5764.Google Scholar
- 13.Han, L. and Reynolds, K. A. (1997), J. Bacteriol. 179, 5157–5164.Google Scholar
- 14.Zhang, W. and Chiao, J. S. (1996), Acta Microbiologica Sinica 36, 199–206.Google Scholar
- 15.Zhang, W. and Chiao, J. S. (1996), Acta Microbiologica Sinica 36, 276–282.Google Scholar
- 16.Zhang, W. and Chiao, J. S. (1996), Chin. J. Biochem. 22, 167–172.Google Scholar
- 17.Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring, NY.Google Scholar
- 20.Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M., and Schrempf, H. (1985), Genetic Manipulation of Streptomyces: A Laboratory Manual, John Inns Foundation, Norwich, England.Google Scholar
- 21.Reynolds, K. A., O’Hagan, D., Gani, D., and Robinson, J. A., (1988), Chem. Soc. Perkin. Trans. 1, 3194–3207.Google Scholar
- 27.Roy, I. (1996), FEMS Lett. 394, 126–128.Google Scholar