RESUMO
This paper focuses on study of second and third ring cyclization in anthracycline biosynthesis by a heterologous gene expression. Firstly, anthracycline non-producing Streptomyces peucetius mutant, D2 was heterologously complemented to produce daunomycins with plasmids pSgs44 and pSYE66, which contain putative cyclase genes of S. galilaeus and S. nogalater, respectively. A point mutation in the cyclase gene dpsY of D2 has changed glycine to serine resulting inactivation of the enzyme. Secondly, the putative cyclase gene snoaM from S. nogalater, was expressed in a gene cassette in S. lividans TK24 and S. coelicolor CH999 to study the influence of the cyclase gene on auramycinone production and the impact of endogenous genes on production profiles. The results obtained confirms that a cyclase closing the second and third ring of a polyketide is essential in anthracycline biosynthesis.
Assuntos
Antraciclinas/metabolismo , Antibióticos Antineoplásicos/metabolismo , Proteínas de Bactérias/metabolismo , Streptomyces/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Ciclização , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , Teste de Complementação Genética , Mutagênese Insercional , Ressonância Magnética Nuclear Biomolecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Streptomyces/enzimologia , Streptomyces/genéticaRESUMO
The biosynthesis pathways of two anthracyclines, nogalamycin and aclacinomycin, were directed toward angucyclines by using an angucycline-specific cyclase, pgaF, isolated from a silent antibiotic biosynthesis gene cluster. Addition of pgaF to a gene cassette that harbored the early biosynthesis genes of nogalamycin resulted in the production of two known angucyclinone metabolites, rabelomycin and its precursor, UWM6. Substrate flexibility of pgaF was demonstrated by replacement of the nogalamycin minimal polyketide synthase genes in the gene cassette with the equivalent aclacinomycin genes together with aknE2 and aknF, which specify the unusual propionate starter unit in aclacinomycin biosynthesis. This modification led to the production of a novel angucyclinone, MM2002, in which the expected ethyl side chain was incorporated into the fourth ring.
Assuntos
Aclarubicina/análogos & derivados , Aclarubicina/biossíntese , Antibacterianos/biossíntese , Antibióticos Antineoplásicos/biossíntese , Nogalamicina/biossíntese , Streptomyces/metabolismo , Engenharia Genética , Família MultigênicaRESUMO
The anthracycline skeleton is biosynthesized by aromatic (type II) polyketide synthases. Furthermore, three post-polyketide steps are needed to form the basic aglycone of anthracyclines. Auramycinone was produced in Streptomyces lividans by introducing nine structural genes from three different anthracycline-producing Streptomyces species. The genes used to construct the auramycinone biosynthesis cluster were derived from nogalamycin-, daunomycin- and aclacinomycin-producing Streptomyces strains. The biosynthetic stages were divided into polyketide and post-polyketide steps on the assumption that the first stable intermediate would be nogalonic acid, named analogously to aklanonic acid, the precursor of several anthracyclines. Single genes were cloned in the expression construct in the order determined by the proposed biosynthetic pathway. This facilitated investigation of the products formed in the heterologous host after addition of each separate gene to the construct. The results thus elucidate the biosynthesis steps, products and the genes responsible for the reactions needed to build up an anthracyclinone.
