Antonie van Leeuwenhoek

, Volume 79, Issue 3–4, pp 251–259

Genetic methods and strategies for secondary metabolite yield improvement in actinomycetes

  • Richard H. Baltz
Article

Abstract

The foundation for any strain improvement program is efficient random chemically-induced mutagenesis coupled with highly reproducible fermentation and product assays. The broad spectrum of spontaneous mutations can be leveraged in some cases by direct selection of mutants with desired traits. Transposons containing outward-reading promoter activity might be used to enhance yields by inducing promoter fusions, disrupting negative regulatory elements, or disrupting genes involved in competing pathways. Transposons might also be used to identify and clone positive regulatory genes. As knowledge of the key elements in the fermentation process and secondary metabolite biosynthesis grows, gene cloning and targeted gene duplication becomes an important tool. Duplication of genes involved in rate limiting steps can be achieved to improve product yields by inserting the desired gene(s) into neutral sites in the chromosome by homologous recombination or by site-specific integration. The probabilities and frequencies of success of the molecular genetic approaches should increase with an increasing knowledge of key factors influencing product yields. This knowledge can be broadened dramatically by a combination of structural and functional genomics, gene disruption analysis and metabolic modeling. Protoplast fusion can be used to recombine beneficial traits from any of the other approaches.

gene duplication microbial genomics mutagenesis protoplast fusion transposition 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Baltz RH (1978) Genetic recombination in Streptomyces fradiae by protoplast fusion and cell regeneration. J. Gen. Microbiol. 107: 93-102Google Scholar
  2. Baltz RH (1986a) Mutagenesis in Streptomyces. In: Demain AL & Soloman NA (Eds) Manual of Industrial Microbiology and Biotechnology (pp 184-190). American Society for Microbiology, Washington, DCGoogle Scholar
  3. Baltz RH (1986b) Mutation in Streptomyces. In: Queener SW & Day LE (Eds) The Bacteria, Vol. IX. Antibiotic-Producing Streptomyces (pp 61-93). Academic Press, New YorkGoogle Scholar
  4. Baltz RH (1995) Gene expression in recombinant Streptomyces. Bioprocess Technol. 22: 309-381Google Scholar
  5. Baltz RH (1997) Molecular genetic approaches to yield improvement in actinomycetes. Drug Pharmaceutical Sci. 82: 49-62Google Scholar
  6. Baltz RH (1998a) Genetic manipulation of antibiotic producing Streptomyces. Trends Microbiol. 6: 76-83Google Scholar
  7. Baltz RH (1998b) New genetic methods to improve secondary metabolite production in Streptomyces. J. Indust. Microbiol. Biotechnol. 20: 360-363Google Scholar
  8. Baltz RH (1999) Mutagenesis. In: Flickinger MC & Drew SW (Eds) Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Separation (pp 1819-1822). Wiley, New YorkGoogle Scholar
  9. Baltz RH (2000) Mutagenesis. In: Lederberg J (Ed) Encyclopedia of Microbiology (pp 307-311). Academic Press, San DiegoGoogle Scholar
  10. Baltz RH & Hosted TJ (1996) Molecular genetic methods for improving secondary-metabolite production in actinomycetes. Trends Biotechnol. 14: 245-249Google Scholar
  11. Baltz RH & Matsushima P (1981) Protoplast fusion in Streptomyces: conditions for efficient genetic recombination and cell regeneration. J. Gen. Microbiol. 127: 137-146Google Scholar
  12. Baltz RH & Matsushima P (1983) Advances in protoplast fusion and transformation in Streptomyces. Experientia Suppl. 46: 143-148Google Scholar
  13. Baltz RH & Stonesifer J (1985a) Mutagenic and error-free DNA repair in Streptomyces. Mol. Gen. Genet. 200: 351-355Google Scholar
  14. Baltz RH & Stonesifer J (1985b) Adaptive response and enhancement of N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis by chloramphenicol in Streptomyces fradiae. J. Bacteriol. 164: 944-946Google Scholar
  15. Baltz RH, Hahn DR, McHenney MA & Solenberg PJ (1992) Transposition of Tn5096 and related transposons in Streptomyces species. Gene 115: 61-65Google Scholar
  16. Baltz RH, McHenney MA, Cantwell CA, Queener SW & Solenberg PJ (1997) Applications of transposition mutagenesis in antibiotic producing streptomycetes. Antonie Leeuwenhoek 71: 179-187Google Scholar
  17. Baltz RH, McHenney MA & Solenberg PJ (1993) Properties of transposons derived from IS493 and applications in streptomycetes. In: Baltz RH, Hegeman G & Skatrud PL (Eds) Industrial Microorganisms: Basic and Applied Molecular Genetics (pp 51-56). American Society for Microbiology, Washington, DCGoogle Scholar
  18. Bibb, M (1996) 1995 Colworth Prize Lecture. The regulation of antibiotic production in Streptomyces coelicolor A3(2). Microbiology 142: 1335-1344Google Scholar
  19. Coulondre C & Miller, JH (1977) Genetic studies of the lac repressor IV. Mutagenic specificity in the lacl gene of Escherichia coli. J. Mol. Biol. 117: 577-606Google Scholar
  20. De Saizieu A, Certa U, Warrington J, Gray C, Keck W & Mous J (1998) Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays. Nature Biotechnol. 16: 45-48Google Scholar
  21. Gravius B, Glocker D, Pigac J, Pandza K, Hranueli D & Cullum J (1994) The 387 kb linear plasmid of Streptomyces rimosus and its interactions with the chromosome. Microbiology 140: 2271-2277Google Scholar
  22. Hahn DR, Solenberg PJ & Baltz RH (1991) Tn5099, a xylE promoter probe transposon for Streptomyces spp. J Bacteriol 173: 5573-5577Google Scholar
  23. Hesketh A & Ochi K (1997) A novel method for improving Streptomyces coelicolor A3(2) for production of actinorhodin by introduction of rpsL (encoding ribosomal protein S12) mutations conferring resistance to streptomycin. J. Antibiot. 50: 532-535Google Scholar
  24. Hosoya Y, Okamoto S, Muramatsu H & Ochi K (1998) Acquisition of certain streptomycin-resistant (str) mutations enhances antibiotic production in bacteria. Antimicrob. Agent Chemother. 42: 2041-2047Google Scholar
  25. Hosted TJ & Baltz RH (1996) Mutants of Streptomyces roseosporus that express enhanced recombination within partially homologous genes. Microbiology 142: 2803-2813Google Scholar
  26. Hosted TJ & Baltz RH (1997) Use of rpsL for dominance selection and gene replacement in Streptomyces roseosporus. J. Bacteriol. 179: 180-186Google Scholar
  27. Matsushima P & Baltz RH (1986) Protoplast fusion. In: Demain AL and Solomon NA (Eds) Manual of Industrial Microbiology and Biotechnology (pp 170-183). American Society for Microbiology, Washington, DCGoogle Scholar
  28. Matsushima P, Broughton CM, Turner JR & Baltz RH (1994) Conjugal transfer of cosmid DNA from Escherichia coli to Saccharopolyspora spinosa: effects of chromosomal insertions on macrolide A83543 production. Gene 146: 39-45Google Scholar
  29. McHenney MA & Baltz RH (1996) Gene transfer and transposition mutagenesis in Streptomyces roseosporus: mapping of insertions that influence daptomycin or pigment production. Microbiology 142: 2363-2373Google Scholar
  30. Miller J (1983) Mutational specificity in bacteria. Annu. Rev. Genet. 17: 215-238Google Scholar
  31. Miller J (1996) Spontaneous mutators in bacteria. Annu. Rev. Microbiol. 50: 625-643Google Scholar
  32. Overbeek R, Fonstein M, D'sousza M, Pusch GD & Malstev N (1999) The use of gene clusters to infer functional coupling. Proc. Nat. Acad. Sci. USA 96: 2896-2901Google Scholar
  33. Overbeek R, Larsen N, Smith W, Maltzev N & Selkov E (1997) Representation of function: the next step. Gene 191: GC1-GC9Google Scholar
  34. Peschke U, Schmidt H, Zhang H-Z & Piepersberg W (1995) Molecular characterization of the lincomycin-production gene cluster of Streptomyces lincolnensis 78-11. Mol. Microbiol. 16: 1137-1158Google Scholar
  35. Queener SW & Lively DH (1986) Screening and selection for strain improvement. In: Demain AL and Solomon NA (Eds) Manual of Industrial Microbiology and Biotechnology (pp 155-169). American Society for Microbiology, Washington, DCGoogle Scholar
  36. Schaaper RM, Bond BI & Fowler RG (1989) AT-CG transversions and their prevention by the Escherichia coli mutT and mutHLS pathways. Mol. Gen. Genet. 219: 256-262Google Scholar
  37. Schilling CH, Edwards JS & Palsson BO (1999) Toward metabolic phenomics: analysis of genomic data using flux balances. Biotechnol. Prog. 15: 288-295Google Scholar
  38. Selkov E Jr, Grechkin Y, Mikhailova N & Selkov E (1998) MPWW: the metabolic pathways database. Nucl. Acid Res. 26: 43-45Google Scholar
  39. Selkov E, Maltzev N, Olsen GJ, Overbeek R & Whitman WB (1997) A reconstruction of the metabolism of Methanococcus jannaschii. Gene 197: GC11-GC26Google Scholar
  40. Sezonov G, Blanc V, Bamas-Jacques N, Friedman A, Pernodet JL & Guerineau M (1997) Complete conversion of antibiotic precursor to pristinamycin IIA by overexpression of Streptomyces pristinaespiralis biosynthetic genes. Nature Biotechnol. 15: 349-353Google Scholar
  41. Shima J, Hesketh A, Okamoto S, Kawamoto S & Ochi K (1996) Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces coelicolor A3(2). J. Bacteriol. 178: 7276-7284Google Scholar
  42. Solenberg PJ & Baltz RH (1991) Transposition of Tn5096 and other IS493 derivatives in Streptomyces griseofuscus. J. Bacteriol. 173: 1096-1104Google Scholar
  43. Solenberg PJ & Baltz RH (1994) Hyper-transposing derivatives of the streptomycete insertion sequence IS493. Gene 147: 47-54Google Scholar
  44. Solenberg PJ, Cantwell CA, Tietz AJ, McGilvray D, Queener SW & Baltz RH (1996) Transposition mutagenesis in Streptomyces fradiae: identification of a neutral site for the stable insertion of DNA by transposon exchange. Gene 168: 67-72Google Scholar
  45. Stonesifer J & Baltz RH (1985) Mutagenic DNA repair in Streptomyces. Proc. Nat. Acad. Sci. USA 82: 1180-1182Google Scholar
  46. Tao H, Bausch C, Richmond C, Blattner FR & Conway T (1999) Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media. J. Bacteriol. 181: 6425-6440Google Scholar
  47. VanBogelen RA, Schiller EE, Thomas JD & Neidhardt FC (1999) Diagnosis of cellular states of microbial organisms using proteomics. Electrophoresis 20: 2149-2159Google Scholar
  48. Vinci VA & Byng G (1999) Strain improvement by nonrecombinant methods. In: Demain AL & Davies JE (Eds) Manual of Industrial Microbiology and Biotechnology, Second Edition (pp 103-113). ASM Press, Washington, DCGoogle Scholar
  49. Wright F & Bibb MJ (1992) Codon usage in the G+C-rich Streptomyces genome. Gene 113: 55-65Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Richard H. Baltz
    • 1
  1. 1.CognoGen Biotechnology ConsultingIndianapolisUSA

Personalised recommendations