Cloning and Characterization of Actinorhodin Biosynthetic Gene Clusters from Streptomyces lividans TK24

  • Park, Kie-In (Faculty of Biological Sciences, College of Natural Sciences, Chonbuk national University)
  • Published : 2002.12.01

Abstract

Actinorhodin antibiotics produced by Streptomyces lividans TK24 are blue pigments with a weak antibiotic activity, derived from one acetyl-CoA and 15 malonyl-CoA units via a typical ployketide pathway. In an attempt to clone polyketide biosynthetic genes of S. lividans TK24, hybridizing fragments in the genomic DNA of S. lividans TK24 were detected by use of acn and act III polyketide synthase gene probes. Since typical aromatic polyketide bio-synthetic gene clusters are roughly 22-34 Kb long, we constructed in E. coli XL-Blue MR using the Streptomyces-E. coli bifunctional shuttle cosmid vector (pojn46). Then, about 5,000 individual E. coii colonies were thor-oughly screened with acrl-ORFI and actIII probes. From these cosmid libra-ries, 12 positive clones were identified. Restriction analysis and southern hybridization showed two polyketide biosynthetic gene clusters in this organism. These cosmid clones can be transformed into Streptomyces parvulus 12434 for expression test that identify product of actinorhodin biosynthetic genes by heterologous expression. Thus, heterologous expres-sion of a derivative compound of a actinorhodin biosynthetic intermediate was obtained in pKE2430. Expression of these compounds by the trans-formants was detected by photodiode array HPLC analysis of crude extracts.

Keywords

References

  1. Berdy J (1974) Recent developments of antibiotic research and classification of antibiotics according to chemical structure. Adv Appl Microbiol 18: 309-406 https://doi.org/10.1016/S0065-2164(08)70573-2
  2. Davis NK and Chater KF (1990) Spore colour in Streptomyces coelicolor A3(2) involves the developmentally regulated synthesis of a compound biosynthetically related to polyketide antibiotics. Mol Microbial 4: 1679-1691 https://doi.org/10.1111/j.1365-2958.1990.tb00545.x
  3. Fernandez-Moreno MA, Martinez E, Boto L, Hopwood DA, and Malpartida F (1992) Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin. J Biol Chem 267: 19278-19290
  4. Hong ST, Carney JR, and Gould SJ (1997) Cloning and heterologous expression of the entire gene clusters for PD 116740 from Streptomyces strain WP 4669 and tetragulol and tetrangomycin from Streptomyces rimosus NRRL 3016. J Bacteriol 179: 470-476 https://doi.org/10.1128/jb.179.2.470-476.1997
  5. Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ, Kieser HM, Lydiate OJ, Smith CP, Ward JM, and Schrempf H (1985) Genetic Manipulation of Streptomyces: a Laboratory Manual. John Innes Foundation, Norwich
  6. Hopwood DA and Sherman DH (1990) Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Genet 24: 37-66 https://doi.org/10.1146/annurev.ge.24.120190.000345
  7. Hutchinson CR and McDaniel R (2001) Combinatorial biosynnthesis in microorganisms as a route to new antimicrobial, antitumor and neuroregenerative drugs. Curr Opin Investig Drugs 2: 1681-1690.
  8. Kao CM, Katz L, and Khosla C (1994) Engineered biosynthesis of a complete macrolactone in a heterologous host. Science 265: 509-512 https://doi.org/10.1126/science.8036492
  9. Malpartida F and Hopwood DA (1986) Physical and genetic characterisation of the gene cluster for the antibiotic actinoorhodin in Streptomyces coelicolor A3(2). Mol Gen Genet 205: 66-73 https://doi.org/10.1007/BF02428033
  10. McDaniel R, Ebert-Khosla S, Hopwood DA, and Khosla C (1993) Engineered biosynthesis of novel polyketides. Science 262: 1546-1550 https://doi.org/10.1126/science.8248802
  11. O'Hagan D (1993) Biosynthesis of fatty acid and polyketide metabolites. Nat Prod Rep 10: 593-624 https://doi.org/10.1039/NP9931000593
  12. Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning: a Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, New York
  13. Tsoi CJ and Khosla C (1995) Combinatorial biosynthesis of 'unnatural' natural products: the polyketide example. Chem Biol 2: 355-362 https://doi.org/10.1016/1074-5521(95)90214-7
  14. Walsh CT (2002) Combinatorial biosynthesis of antibiotics: challenges and opportunities. Chembiochem 3: 125-134 https://doi.org/10.1002/1439-7633(20020301)3:2/3<124::AID-CBIC124>3.0.CO;2-J
  15. Chembiochem v.3 Combinatorial biosynthesis of antibiotics: challenges and opportunities Walsh CT