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. 1993 May;175(9):2552–2563. doi: 10.1128/jb.175.9.2552-2563.1993

Deletion analysis of the avermectin biosynthetic genes of Streptomyces avermitilis by gene cluster displacement.

T MacNeil 1, K M Gewain 1, D J MacNeil 1
PMCID: PMC204556  PMID: 8478321

Abstract

Streptomyces avermitilis produces a group of glycosylated, methylated macrocyclic lactones, the avermectins, which have potent anthelmintic activity. A homologous recombination strategy termed gene cluster displacement was used to construct Neor deletion strains with defined endpoints and to clone the corresponding complementary DNA encoding functions for avermectin biosynthesis (avr). Thirty-five unique deletions of 0.5 to > 100 kb over a continuous 150-kb region were introduced into S. avermitilis. Analysis of the avermectin phenotypes of the deletion-containing strains defined the extent and ends of the 95-kb avr gene cluster, identified a regulatory region, and mapped several avr functions. A 60-kb region in the central portion determines the synthesis of the macrolide ring. A 13-kb region at one end of the cluster is responsible for synthesis and attachment of oleandrose disaccharide. A 10-kb region at the other end has functions for positive regulation and C-5 O methylation. Physical analysis of the deletions and of in vivo-cloned fragments refined a 130-kb physical map of the avr gene cluster region.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anzai H., Kumada Y., Hara O., Murakami T., Itoh R., Takano E., Imai S., Satoh A., Nagaoka K. Replacement of Streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences. J Antibiot (Tokyo) 1988 Feb;41(2):226–233. doi: 10.7164/antibiotics.41.226. [DOI] [PubMed] [Google Scholar]
  2. Baltz R. H., Seno E. T. Genetics of Streptomyces fradiae and tylosin biosynthesis. Annu Rev Microbiol. 1988;42:547–574. doi: 10.1146/annurev.mi.42.100188.002555. [DOI] [PubMed] [Google Scholar]
  3. Bevitt D. J., Cortes J., Haydock S. F., Leadlay P. F. 6-Deoxyerythronolide-B synthase 2 from Saccharopolyspora erythraea. Cloning of the structural gene, sequence analysis and inferred domain structure of the multifunctional enzyme. Eur J Biochem. 1992 Feb 15;204(1):39–49. doi: 10.1111/j.1432-1033.1992.tb16603.x. [DOI] [PubMed] [Google Scholar]
  4. Blanco G., Pereda A., Méndez C., Salas J. A. Cloning and disruption of a fragment of Streptomyces halstedii DNA involved in the biosynthesis of a spore pigment. Gene. 1992 Mar 1;112(1):59–65. doi: 10.1016/0378-1119(92)90303-7. [DOI] [PubMed] [Google Scholar]
  5. Burg R. W., Miller B. M., Baker E. E., Birnbaum J., Currie S. A., Hartman R., Kong Y. L., Monaghan R. L., Olson G., Putter I. Avermectins, new family of potent anthelmintic agents: producing organism and fermentation. Antimicrob Agents Chemother. 1979 Mar;15(3):361–367. doi: 10.1128/aac.15.3.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buttner M. J., Chater K. F., Bibb M. J. Cloning, disruption, and transcriptional analysis of three RNA polymerase sigma factor genes of Streptomyces coelicolor A3(2). J Bacteriol. 1990 Jun;172(6):3367–3378. doi: 10.1128/jb.172.6.3367-3378.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chater K. F., Bruton C. J. Resistance, regulatory and production genes for the antibiotic methylenomycin are clustered. EMBO J. 1985 Jul;4(7):1893–1897. doi: 10.1002/j.1460-2075.1985.tb03866.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chater K. F. The improving prospects for yield increase by genetic engineering in antibiotic-producing Streptomycetes. Biotechnology (N Y) 1990 Feb;8(2):115–121. doi: 10.1038/nbt0290-115. [DOI] [PubMed] [Google Scholar]
  9. Chen T. S., Inamine E. S. Studies on the biosynthesis of avermectins. Arch Biochem Biophys. 1989 May 1;270(2):521–525. doi: 10.1016/0003-9861(89)90534-1. [DOI] [PubMed] [Google Scholar]
  10. Donadio S., Katz L. Organization of the enzymatic domains in the multifunctional polyketide synthase involved in erythromycin formation in Saccharopolyspora erythraea. Gene. 1992 Feb 1;111(1):51–60. doi: 10.1016/0378-1119(92)90602-l. [DOI] [PubMed] [Google Scholar]
  11. Donadio S., Staver M. J., McAlpine J. B., Swanson S. J., Katz L. Modular organization of genes required for complex polyketide biosynthesis. Science. 1991 May 3;252(5006):675–679. doi: 10.1126/science.2024119. [DOI] [PubMed] [Google Scholar]
  12. Fishman S. E., Cox K., Larson J. L., Reynolds P. A., Seno E. T., Yeh W. K., Van Frank R., Hershberger C. L. Cloning genes for the biosynthesis of a macrolide antibiotic. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8248–8252. doi: 10.1073/pnas.84.23.8248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Geistlich M., Losick R., Turner J. R., Rao R. N. Characterization of a novel regulatory gene governing the expression of a polyketide synthase gene in Streptomyces ambofaciens. Mol Microbiol. 1992 Jul;6(14):2019–2029. doi: 10.1111/j.1365-2958.1992.tb01374.x. [DOI] [PubMed] [Google Scholar]
  14. Gewain K. M., Occi J. L., Foor F., MacNeil D. J. Vectors for generating nested deletions and facilitating subcloning G+C-rich DNA between Escherichia coli and Streptomyces sp. Gene. 1992 Sep 21;119(1):149–150. doi: 10.1016/0378-1119(92)90083-2. [DOI] [PubMed] [Google Scholar]
  15. Hillemann D., Pühler A., Wohlleben W. Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation of integration vectors. Nucleic Acids Res. 1991 Feb 25;19(4):727–731. doi: 10.1093/nar/19.4.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ikeda H., Kotaki H., Omura S. Genetic studies of avermectin biosynthesis in Streptomyces avermitilis. J Bacteriol. 1987 Dec;169(12):5615–5621. doi: 10.1128/jb.169.12.5615-5621.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  18. MacNeil D. J. Characterization of a unique methyl-specific restriction system in Streptomyces avermitilis. J Bacteriol. 1988 Dec;170(12):5607–5612. doi: 10.1128/jb.170.12.5607-5612.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. MacNeil D. J., Gewain K. M., Ruby C. L., Dezeny G., Gibbons P. H., MacNeil T. Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene. 1992 Feb 1;111(1):61–68. doi: 10.1016/0378-1119(92)90603-m. [DOI] [PubMed] [Google Scholar]
  20. MacNeil D. J., Occi J. L., Gewain K. M., MacNeil T., Gibbons P. H., Ruby C. L., Danis S. J. Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase. Gene. 1992 Jun 15;115(1-2):119–125. doi: 10.1016/0378-1119(92)90549-5. [DOI] [PubMed] [Google Scholar]
  21. Malpartida F., Niemi J., Navarrete R., Hopwood D. A. Cloning and expression in a heterologous host of the complete set of genes for biosynthesis of the Streptomyces coelicolor antibiotic undecylprodigiosin. Gene. 1990 Sep 1;93(1):91–99. doi: 10.1016/0378-1119(90)90141-d. [DOI] [PubMed] [Google Scholar]
  22. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  23. Matsushima P., Cox K. L., Baltz R. H. Highly transformable mutants of Streptomyces fradiae defective in several restriction systems. Mol Gen Genet. 1987 Mar;206(3):393–400. doi: 10.1007/BF00428877. [DOI] [PubMed] [Google Scholar]
  24. Meselson M., Yuan R. DNA restriction enzyme from E. coli. Nature. 1968 Mar 23;217(5134):1110–1114. doi: 10.1038/2171110a0. [DOI] [PubMed] [Google Scholar]
  25. Piret J. M., Chater K. F. Phage-mediated cloning of bldA, a region involved in Streptomyces coelicolor morphological development, and its analysis by genetic complementation. J Bacteriol. 1985 Sep;163(3):965–972. doi: 10.1128/jb.163.3.965-972.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Richardson M. A., Kuhstoss S., Huber M. L., Ford L., Godfrey O., Turner J. R., Rao R. N. Cloning of spiramycin biosynthetic genes and their use in constructing Streptomyces ambofaciens mutants defective in spiramycin biosynthesis. J Bacteriol. 1990 Jul;172(7):3790–3798. doi: 10.1128/jb.172.7.3790-3798.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schulman M. D., Acton S. L., Valentino D. L., Arison B. H. Purification and identification of dTDP-oleandrose, the precursor of the oleandrose units of the avermectins. J Biol Chem. 1990 Oct 5;265(28):16965–16970. [PubMed] [Google Scholar]
  28. Schulman M. D., Ruby C. Methylation of demethylavermectins. Antimicrob Agents Chemother. 1987 Jun;31(6):964–965. doi: 10.1128/aac.31.6.964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Weber J. M., Leung J. O., Maine G. T., Potenz R. H., Paulus T. J., DeWitt J. P. Organization of a cluster of erythromycin genes in Saccharopolyspora erythraea. J Bacteriol. 1990 May;172(5):2372–2383. doi: 10.1128/jb.172.5.2372-2383.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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