RESUMO
The biosynthetic gene cluster for the angiogenesis inhibitor borrelidin has been cloned from Streptomyces parvulus Tü4055. Sequence analysis indicates that the macrolide ring of borrelidin is formed by a modular polyketide synthase (PKS) (borA1-A6), a result that was confirmed by disruption of borA3. The borrelidin PKS is striking because only seven rather than the nine modules expected for a nonaketide product are encoded by borA1-A6. The starter unit of the PKS has been verified as trans-cyclopentane-1,2-dicarboxylic acid (trans-1,2-CPDA), and the genes involved in its biosynthesis identified. Other genes responsible for biosynthesis of the nitrile moiety, regulation, and self-resistance were also identified.
Assuntos
Inibidores da Angiogênese/biossíntese , Álcoois Graxos/metabolismo , Genes Bacterianos , Família Multigênica , Streptomyces/genética , Inibidores da Angiogênese/química , Clonagem Molecular , Ciclopentanos/síntese química , Ácidos Dicarboxílicos/síntese química , Álcoois Graxos/química , Modelos Químicos , Dados de Sequência Molecular , Estrutura Molecular , Complexos Multienzimáticos/genética , Análise de Sequência de DNA , Streptomyces/enzimologia , Streptomyces/metabolismoRESUMO
Novel spinosyns have been prepared by biotransformation, using a genetically engineered strain of Saccharopolyspora erythraea, in which the beta-D-forosamine moiety in glycosidic linkage to the hydroxy group at C17 is replaced by alpha-L-mycarose.
Assuntos
Antibacterianos/biossíntese , Desoxiaçúcares/metabolismo , Engenharia Genética , Biotransformação , Fermentação , Glicosiltransferases/genética , Hexoses/metabolismo , Macrolídeos , Saccharopolyspora/genéticaAssuntos
Sirolimo/química , Sirolimo/metabolismo , Streptomyces/química , Clonagem Molecular , Cromatografia Gasosa-Espectrometria de Massas , Genes Bacterianos/genética , Imunossupressores/química , Imunossupressores/metabolismo , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Sirolimo/análogos & derivados , Sirolimo/isolamento & purificação , Estereoisomerismo , Streptomyces/genética , Streptomyces/metabolismoRESUMO
The function of gene products involved in the biosynthesis of the clinically important polyketide rapamycin were elucidated by biotransformation and gene complementation.
Assuntos
Genes , Sirolimo/metabolismo , Teste de Complementação Genética , Espectrometria de Massas , Sirolimo/análogos & derivados , Sirolimo/químicaRESUMO
The 18-membered polyketide macrolide borrelidin exhibits a number of important biological activities, including potent angiogenesis inhibition. This has prompted two recent total syntheses as well as the cloning of the biosynthetic gene cluster from Streptomyces parvulus Tü4055. Borrelidin possesses some unusual structural characteristics, including a cyclopentane carboxylic acid moiety at C17 and a nitrile moiety at C12 of the macrocyclic ring. Nitrile groups are relatively rare in nature, and little is known of their biosynthesis during secondary metabolism. The nitrile group of borrelidin is shown here to arise from the methyl group of a methylmalonyl-CoA extender unit incorporated during polyketide chain extension. Insertional inactivation of two genes in the borrelidin gene cluster, borI (coding for a cytochrome P450 monooxygenase) and borJ (coding for an aminotransferase), generated borrelidin non-producing mutants. These mutants accumulated different compounds lacking the C12 nitrile moiety, with the product of the borI-minus mutant (12-desnitrile-12-methyl-borrelidin) possessing a methyl group and that of the borJ-minus mutant (12-desnitrile-12-carboxyl-borrelidin) a carboxyl group at C12. The former but not the latter was converted into borrelidin when biotransformed by an S. parvulus mutant that is deficient in the biosynthesis of the borrelidin starter unit. This suggests that 12-desnitrile-12-methyl-borrelidin is a competent biosynthetic intermediate, whereas the carboxylated derivative is a shunt metabolite. Bioconversion of 12-desnitrile-12-methyl-borrelidin into borrelidin was also achieved in a heterologous system co-expressing borI and borJ in Streptomyces albus J1074. This bioconversion was more efficient when borK, which is believed to encode a dehydrogenase, was simultaneously expressed with borI and borJ. On the basis of these findings, a pathway is proposed for the formation of the nitrile moiety during borrelidin biosynthesis.
Assuntos
Álcoois Graxos/química , Álcoois Graxos/metabolismo , Nitrilas/química , Nitrilas/metabolismo , Streptomyces/metabolismo , Acil Coenzima A/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotransformação , Clonagem Molecular , Genes Bacterianos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mutagênese Insercional , Mutação , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/genética , Transaminases/genética , Transaminases/metabolismoRESUMO
Ivermectin, a mixture of 22,23-dihydroavermectin B1a9 with minor amounts of 22,23-dihydroavermectin B1b 10, is one of the most successful veterinary antiparasitic drugs ever produced. In humans, ivermectin has been used for the treatment of African river blindness (onchocerciasis) resulting in an encouraging decrease in the prevalence of skin and eye diseases linked to this infection. The components of ivermectin are currently synthesized by chemical hydrogenation of a specific double bond at C22-C23 in the polyketide macrolides avermectins B1a 5 and B1b 6, broad-spectrum antiparasitic agents isolated from the soil bacterium Streptomyces avermitilis. We describe here the production of such compounds (22,23-dihydroavermectins B1a 9 and A1a 11) by direct fermentation of a recombinant strain of S. avermitilis containing an appropriately-engineered polyketide synthase (PKS). This suggests the feasibility of a direct biological route to this valuable drug.