The Streptomyces glaucescens tcmKL Polyketide Synthase and tcmN Polyketide Cyclase Genes Govern the Size and Shape of Aromatic Polyketides

نویسنده

  • B. Shen
چکیده

The mechanism of a type I1 polyketide synthase was analyzed by combinatorially expressing components of the tetracenomycin (tcm) and the actinorhodin (act) polyketide synthase genes in various mutants or heterologous hosts. Structural analysis of metabolites produced by the recombinant organisms provided evidence to dissect the function of individual components of a type II PKS. Complementation studies with the S. glaucescens TcmKand TcmLmutants and constructs harboring the tcmK and actl-ORF2 or actl-ORF1 and tcmL genes demonstrated that a heterologous pair of genes encoding a j3-ketoacy1:acyl carrier protein synthase and chain length factor is often (but not always) nonfunctional. Isolations of the octaketides 6 and 7 from strains bearing actl-ORFl, actl-ORF2, and tcmM (pWHM766) or t c d , actl-ORF1, actl-ORF2, and tcmM (pWHM768), and the decaketides 8 and 9 from strains bearing tcmK, tcmL, and tcmM (pELE37) or t c d , tcmK, tcmL, and tcmM (pWHM731) established that the j3-ketoacy1:acyl carrier protein synthase and chain length factor proteins determine the chain length of the polyketide. While the addition of the t c d gene to the polyketide synthase core proteins consisting of the j3-ketoacy1:acyl carrier protein synthase, chain length factor and acyl carrier protein had no effect on the structures of the resulting metabolites, adding the tcmN gene to either pWHM766 and pWHM768 or pELE37 and pWHM731 resulted in the synthesis of octaketide 5 or decaketide 2, respectively. TcmN thus can alter the regiospecificity for the f i s t aldol cyclization from C-7/C-12 to C-9/C-14, suggesting that cyclases like TcmN determine the folding pattem of the linear polyketide intermediate. These activities, along with the choice of the starter unit, the loading of the extender unit to the PKS complex, and the function of T c d are discussed in an attempt to provide a rationale for the engineered biosynthesis of novel polyketides. Polyketide metabolites are a large and diverse family of secondary metabolites found in bacteria, fungi, and plants.’ Many of them are clinically valuable antibiotics or chemotherapeutic agents or have other useful pharmacological activities (immunosuppressive, antiparasitic, insecticidal, etc).2 Ever since Birch3 outlined his “acetate hypothesis” in the early 1950s that polyketides could be biosynthetically derived from short fatty acids such as acetate, propionate, or butyrate, activated as acylthioesters, chemists and biochemists have been searching for a unified biosynthetic mechanism to account for the huge structural diversity found in polyketides. From the results of isotope labeling experiments, it has been evident for some time that the mechanism of polyketide biosynthesis is analogous to that of long-chain fatty acid biosynthesis catalyzed by the fatty acid synthases and that both polyketide and fatty acid biosynthesis utilize largely the same precursors. However, direct evidence supporting the analogy * Corresponding author: C. R. Hutchinson, School of Pharmacy, University of Wisconsin, 425 North Charter St., Madison, WI 53706. Phone: (608)262-7582. Fax: (608)262-3134. email: crhutchi@facstaff. wisc.edu. School of Pharmacy. * Department of Bacteriology. 5 Current address: Deuartment 47N. Abbott Laboratories AP9A. Abbott Park, IL 60064. @ Abstract published in Advance ACS Abstracts, June 15, 1995. (1) O’Hagen, D. The Polyketide Metabolites; Ellis Horwood: Chichester, U.K., 1991. (2) Monaghan, R. L.; Tkacz, J. S . Annu. Rev. Microbiol. 1990,44,271301. (3) Birch, A. J.; Donovan, F. W. Aus. J. Chem. 1953,6,360-368,373378; Birch, A. J.; Elliot, P. Aus. J. Chem. 1953, 6, 369-372; Birch, A. J. Science 1967, 156, 202-206. between polyketide and fatty acid biosynthesis has come only recently from the molecular genetic and biochemical studies of antibiotic biosynthesis in Streptomyces species, principally. Sequence analysis of several sets of bacterial genes (plus a few from fungi) encoding polyketide synthases has revealed a highly conserved gene organization and a high degree of amino acid sequence similarity among the enzymes. Comparison of the polyketide synthases and fatty acid synthases has led to the conclusion that these systems must share a common mechanism of carbon chain assembly from similar if not identical precursor^.^-^ Yet, the details of how a polyketide synthase assembles the polyketide carbon skeleton remain obscure. Most of the mechanistic insights have been deduced from the effects of introducing mutations into the chromosomal copy of the polyketide synthase genes or by overexpressing the native or mutant polyketide synthase genes in various background^.^ Consequently, our understanding of the enzymology and biochemistry of polyketide synthases is rudimentary, and the purification or reconstitution of a complete polyketide synthase complex from a bacterium has so far not been reported, although significant progress has recently been made in this directi~n.’~-’~ (4) Hopwood, D. A.; Sherman, D. H. Annu. Rev. Genet. 1990,24, 37( 5 ) Katz, L.; Donadio, S. Annu. Rev. Microbiol. 1993, 47, 875-912. (6) Hopwood, D. A.; Khosla, C. Ciba Found. Symp. 1992, 171, 88(7) Hopwood, D. A. Curr. Opin. Biotechnoi. 1994, 4, 531-537. (8J Vining, L. C.; Stuttard, C. Genetics and Biochemistry ofAntibioric Production; Butterworth-Heinemann: Boston, 1995; pp 323-498. (9) Hutchinson, C. R.; Fujii, I. Annu. Rev. Microbiol. 1995, 47, 201238. 66.

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تاریخ انتشار 2001