1887

Abstract

Modular polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for the biosynthesis of numerous bacterial natural products such as erythromycin and rifamycin. Advanced genetic analysis of these remarkable systems is often seriously hampered by the large size (>40 kb) of PKS gene clusters, and, notwithstanding their considerable fundamental and biotechnological significance, by the lack of suitable methods for engineering non-selectable modifications in chromosomally encoded PKS genes. The development of a facile host–vector strategy for genetic engineering of the rifamycin PKS in the producing organism, S699, is described here. The genes encoding all 10 modules of the rifamycin PKS were replaced with a hygromycin-resistance marker gene. In a similar construction, only the first six modules of the PKS were replaced. The deletion hosts retained the ability to synthesize the primer unit 3-amino-5-hydroxybenzoic acid (AHBA), as judged by co-synthesis experiments with a mutant strain lacking AHBA synthase activity. Suicide plasmids carrying a short fragment from the 5′ flanking end of the engineered deletion, an apramycin-resistance marker gene, and suitably engineered PKS genes could be introduced via electroporation into the deletion hosts, resulting in the integration of PKS genes and biosynthesis of a reporter polyketide in quantities comparable to those produced by the wild-type organism. Since this strategy for engineering recombinant PKSs in requires only a selectable single crossover and eliminates the need for tedious non-selectable double-crossover experiments, it makes rifamycin PKS an attractive target for extensive genetic manipulation.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-145-9-2335
1999-09-01
2019-10-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/145/9/1452335a.html?itemId=/content/journal/micro/10.1099/00221287-145-9-2335&mimeType=html&fmt=ahah

References

  1. Arora, S. K. ( 1985; ). Correlation of structure and activity in ansamycins: structure, conformation, and interactions of antibiotic rifamycin S. J Med Chem 28, 1099-1102.[CrossRef]
    [Google Scholar]
  2. Arora, S. K. & Main, P. ( 1984; ). Correlation of structure and activity in ansamycin: molecular structure of cyclized rifamycin SV. J Antibiot 37, 178-181.[CrossRef]
    [Google Scholar]
  3. August, P. R., Tang, L., Yoon, Y. J. & 9 other authors ( 1998; ). Biosynthesis of the ansamycin antibiotic rifamycin. Deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chem Biol 5, 69–79.[CrossRef]
    [Google Scholar]
  4. Bacchi, A., Pelizzi, G., Nebuloni, M. & Ferrari, P. ( 1998; ). Comprehensive study on structure-activity relationships of rifamycins: discussion of molecular and crystal structure and spectroscopic and thermochemical properties of rifamycin O. J Med Chem 41, 2319-2332.[CrossRef]
    [Google Scholar]
  5. Barna, J. C. J. & Williams, D. H. ( 1984; ). The structure and mode of action of glycopeptide antibiotics of the vancomycin group. Annu Rev Microbiol 38, 339-357.[CrossRef]
    [Google Scholar]
  6. Bartolucci, C., Cellai, L., Marzano, M. & 8 other authors ( 1995; ). Structure-activity relationships in open ansa-chain rifamycin S derivatives as inhibitors of HIV-1 reverse transcriptase. Farmaco 50, 587–593.
    [Google Scholar]
  7. Bedford, D., Jacobsen, J. R., Luo, G., Cane, D. E. & Khosla, C. ( 1996; ). A functional chimeric modular polyketide synthase generated via domain replacement. Chem Biol 3, 827-831.[CrossRef]
    [Google Scholar]
  8. Cane, D. E., Walsh, C. T. & Khosla, C. ( 1998; ). Harnessing the biosynthetic code. Combinations, permutations, mutations. Science 282, 63-68.[CrossRef]
    [Google Scholar]
  9. Cole, S. T. ( 1996; ). Rifamycin resistance in mycobacteria. Res Microbiol 147, 48-52.[CrossRef]
    [Google Scholar]
  10. Donadio, S., McAlpine, J. B., Sheldon, P. J., Jackson, M. & Katz, L. ( 1993; ). An erythromycin analog produced by reprogramming of polyketide synthesis. Proc Natl Acad Sci USA 90, 7119-7123.[CrossRef]
    [Google Scholar]
  11. Flett, F., Mersinias, V. & Smith, C. P. ( 1997; ). High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol Lett 155, 233-239.[CrossRef]
    [Google Scholar]
  12. Ghisalba, O. & Nuesch, J. ( 1981; ). A genetic approach to the biosynthesis of the rifamycin-chromophore in Nocardia mediterranei. IV. Identification of 3-amino-5-hydroxybenzoic acid as a direct precursor of the seven-carbon amino starter-unit. J Antibiot 34, 64-71.[CrossRef]
    [Google Scholar]
  13. Ghisalba, O., Fuhrer, H., Richter, W. J. & Moss, S. ( 1981; ). A genetic approach to the biosynthesis of the rifamycin-chromophore in Nocardia mediterranei. III. Isolation and identification of an early aromatic ansamycin-precursor containing the seven-carbon amino starter-unit and three initial acetate/propionate-units of the ansa chain. J Antibiot 34, 59-63.
    [Google Scholar]
  14. Hatano, K., Akiyama, S., Asai, M. & Rickards, R. W. ( 1982; ). Biosynthetic origin of aminobenzenoid nucleus (C7N-unit) of ansamitocin, a group of novel maytansinoid antibiotics. J Antibiot 35, 1415-1417.[CrossRef]
    [Google Scholar]
  15. Hunziker, D., Yu, T. W., Hutchinson, C. R., Floss, H. G. & Khosla, C. ( 1998; ). Primer unit specificity in rifamycin biosynthesis principally resides in the later stages of the biosynthetic pathway. J Am Chem Soc 120, 1092-1093.[CrossRef]
    [Google Scholar]
  16. Kao, C. M., Katz, L. & Khosla, C. ( 1994; ). Engineered biosynthesis of a complete macrolactone in a heterologous host. Science 265, 509-512.[CrossRef]
    [Google Scholar]
  17. Kao, C. M., Pieper, R., Cane, D. E. & Khosla, C. ( 1996; ). Evidence for two catalytically independent clusters of active sites in a functional modular polyketide synthase. Biochemistry 135, 12363-12368.
    [Google Scholar]
  18. Kim, C. G., Yu, T. W., Fryhle, C. B., Handa, S. & Floss, H. G. ( 1998; ). 3-Amino-5-hydroxybenzoic acid synthase, the terminal enzyme in the formation of the precursor of mC7N units in rifamycin and related antibiotics. J Biol Chem 273, 6030-6040.[CrossRef]
    [Google Scholar]
  19. Lal, R., Lal, S. & Grund, E. ( 1991; ). Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei. Appl Environ Microbiol 57, 665-671.
    [Google Scholar]
  20. Lal, R., Khanna, R., Dhingra, N., Khanna, M. & Lal, S. ( 1998; ). Development of an improved cloning vector and transformation system in Amycolatopsis mediterranei (Nocardia mediterranei). J Antibiot 51, 161-169.[CrossRef]
    [Google Scholar]
  21. Lowder, J. F. & Johnson, R. S. ( 1987; ). The generation of the rifamycin binding site in the beta subunit of E. coli RNA polymerase through subunit interactions. Biochem Biophys Res Commun 147, 1129-1136.[CrossRef]
    [Google Scholar]
  22. McDaniel, R., Ebert-Khosla, S., Hopwood, D. A. & Khosla, C. ( 1993; ). Engineered biosynthesis of novel polyketides. Science 262, 1546-1550.[CrossRef]
    [Google Scholar]
  23. Madon, J. & Hutter, R. ( 1991; ). Transformation system for Amycolatopsis (Nocardia) mediterranei: direct transformation of mycelium with plasmid DNA. J Bacteriol 173, 6325-6331.
    [Google Scholar]
  24. Malpartida, F., Zalacain, M., Jimenen, A. & Davies, J. ( 1983; ). Molecular cloning and expression in Streptomyces lividans of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus. Biochem Biophys Res Commun 117, 6-12.[CrossRef]
    [Google Scholar]
  25. Marsden, A. F., Wilkinson, B., Cortes, J., Dunster, N. J., Staunton, J. & Leadlay, P. F. ( 1998; ). Engineering broader specificity into an antibiotic-producing polyketide synthase. Science 279, 199-202.[CrossRef]
    [Google Scholar]
  26. Matsushima, P., McHenney, M. A. & Baltz, R. H. ( 1987; ). Efficient transformation of Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmids. J Bacteriol 169, 2298-2300.
    [Google Scholar]
  27. Moretti, P., Hintermann, G. & Huttler, R. ( 1985; ). Isolation and characterization of an extrachromosomal element from Nocardia mediterranei. Plasmid 14, 126-133.[CrossRef]
    [Google Scholar]
  28. Nadkarni, S. R., Patel, M. V., Chatterjee, S., Vijayakumar, E. K. S., Desikan, K. R., Blumbach, J. & Ganguli, B. N. ( 1994; ). Balhimycin, a new glycopeptide antibiotics produced by Amycolatopsis sp. Y-86,21022. Taxonomy, production, isolation and biological activity. J Antibiot 47, 334-341.[CrossRef]
    [Google Scholar]
  29. Pelzer, S., Reichert, W., Huppert, M., Heckmann, D. & Wohlleben, W. ( 1997; ). Cloning and analysis of a peptide synthetase gene of the balhimycin producer Amycolatopsis mediterranei DSM5908 and development of a gene disruption/replacement system. J Biotechnol 56, 115-128.[CrossRef]
    [Google Scholar]
  30. Riva, S., Fietta, A. & Silvestri, L. G. ( 1972; ). Mechanism of action of a rifamycin derivative (AF-013) which is active on the nucleic acid polymerases insensitive to rifampicin. Biochem Biophys Res Commun 49, 1263-1271.[CrossRef]
    [Google Scholar]
  31. Rowe, C. J., Cortes, J., Gaisser, S., Staunton, J. & Leadlay, P. F. ( 1998; ). Construction of new vectors for high-level expression in actinomycetes. Gene 216, 215-223.[CrossRef]
    [Google Scholar]
  32. Ruan, X., Pereda, A., Stassi, D. L. & 8 other authors ( 1997; ). Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives. J Bacteriol 179, 6416–6425.
    [Google Scholar]
  33. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  34. Schupp, T. & Diver, M. ( 1986; ). Protoplast preparation and regeneration in Nocardia mediterranei. FEMS Microbiol Lett 36, 159-165.[CrossRef]
    [Google Scholar]
  35. Schupp, T., Toupet, C., Engel, N. & Goff, S. ( 1998; ). Cloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster from Amycolatopsis mediterranei. FEMS Microbiol Lett 157, 201-207.
    [Google Scholar]
  36. Sensi, P., Margalith, P. & Timbal, M. T. ( 1959; ). Rifamycin, a new antibiotic. Preliminary report. Farm Ed Sci 14, 146-147.
    [Google Scholar]
  37. Tang, L., Yoon, Y. J., Choi, C. Y. & Hutchinson, C. R. ( 1998; ). Characterization of the enzymatic domains in the modular polyketide synthase involved in rifamycin B biosynthesis by Amycolatopsis mediterranei. Gene 216, 255-265.[CrossRef]
    [Google Scholar]
  38. Ueno, M., Iijima, M., Masuda, T., Kinoshita, N., Iinuma, H., Naganawa, H., Hamada, M., Ishizuka, M. & Takeuchi, T. ( 1992; ). Dethymicin, a novel immunosuppressant isolated from an Amycolatopsis. Fermentation, isolation, physico-chemical properties and biological activities. J Antibiot 12, 1819-1826.
    [Google Scholar]
  39. Van Wageningen, A. M., Kirkpatrick, P. N., Williams, D. H., Harris, B. R., Kershaw, J. K., Lennard, N. J., Jones, M., Jones, S. J. & Solenberg, P. J. ( 1998; ). Sequencing and analysis of genes involved in the biosynthesis of a vancomycin group antibiotic. Chem Biol 5, 133-162.
    [Google Scholar]
  40. Vrijbloed, J. W., Madon, J. & Dijkhuizen, L. ( 1994; ). A plasmid from the methylotrophic actinomycete Amycolatopsis methanolica capable of site-specific integration. J Bacteriol 176, 7087-7090.
    [Google Scholar]
  41. Yu, T.-W., Shen, Y., Doi-Katayama, Y., Tang, L., Park, C., Moore, B. S., Hutchinson, C. R. & Floss, H. G. (1999). Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively. Proc Natl Acad Sci USA (in press).
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-145-9-2335
Loading
/content/journal/micro/10.1099/00221287-145-9-2335
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error