Cloning, nucleotide sequence, and heterologous expression of the biosynthetic gene cluster for R1128, a non-steroidal estrogen receptor antagonist. Insights into an unusual priming mechanism.
R1128 substances are anthraquinone natural products that were previously reported as non-steroidal estrogen receptor antagonists with in vitro and in vivo potency approaching that of tamoxifen. From a biosynthetic viewpoint, these polyketides possess structurally interesting features such as an unusual primer unit that are absent in the well studied anthracyclic and tetracyclic natural products. The entire R1128 gene cluster was cloned and expressed in Streptomyces lividans, a genetically well developed heterologous host. In addition to R1128C, a novel optically active natural product, designated HU235, was isolated. Nucleotide sequence analysis of the biosynthetic gene cluster revealed genes encoding two ketosynthases, a chain length factor, an acyl transferase, three acetyl-CoA carboxylase subunits, two cyclases, two oxygenases, an amidase, and remarkably, two acyl carrier proteins. Feeding studies indicate that the unusual 4-methylvaleryl side chain of R1128C is derived from valine. Together with the absence of a dedicated ketoreductase, dehydratase, or enoylreductase within the R1128 gene cluster, this suggests a functional link between fatty acid biosynthesis and R1128 biosynthesis in the engineered host. Specifically, we propose that the R1128 synthase recruits four subunits from the endogenous fatty acid synthase during the biosynthesis of this family of pharmacologically significant natural products.
Marti
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Departments of Chemical Engineering and Chemistry and Biochemistry, Stanford University, Stanford, California 94305-5025, USA.
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Pohl
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Khosla
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D000880:Anthraquinones / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004965:Estrogen Antagonists / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D005810:Multigene Family; D011960:Receptors, Estrogen / Q000037:antagonists & inhibitors*; D013302:Streptomyces / Q000235:genetics*
D000880:Anthraquinones; D004965:Estrogen Antagonists; D005227:Fatty Acids; D011960:Receptors, Estrogen; C082781:1,3,6-trihydroxy-8-n-propylanthraquinone; C082782:1,3,6-trihydroxy-8-n-butylanthraquinone; C082783:1,3,6-trihydroxy-8-(3-methylbutyl)anthraquinone; C082784:1,3,6-trihydroxy-8-n-pentylanthraquinone