RESUMO
The actVI genetic region of Streptomyces coelicolor A3(2) is part of the biosynthetic gene cluster of actinorhodin (ACT), the act cluster, consisting of six ORFs: ORFB, ORFA, ORF1, ORF2, ORF3, ORF4. A newly devised method of ACT detection with a combination of HPLC and LC/MS was applied to the analysis of the disruptants of each ORF. ACT was produced by those of ORFB, ORFA, ORF3, and ORF4. Instead of ACT, the ORF1 disruptant produced 3,8-dihydroxy-1-methylanthraquinone-2-carboxylic acid (DMAC) and aloesaponarin II as shunt products. The ORF2 disruptant gave 4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho-[2,3-c]-pyran-3-(S)-acet ic acid, (S)-DNPA. These results support our previous proposal for stereospecific pyran ring formation in the biosynthesis of ACT, most importantly suggesting that the actVI-ORF2 product would recognize (S)-DNPA as a substrate for stereospecific reduction at C-15. The disruptant of ORFA produced (S)-DNPA together with ACT, suggesting that actVI-ORFA might play a role such as stabilising the multicomponent, type II PKS complex.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Antraquinonas/análise , Antraquinonas/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Meios de Cultura , Regulação Bacteriana da Expressão Gênica , Espectrometria de Massas/métodos , Família Multigênica , Mutação , Naftalenos/análise , Naftalenos/metabolismo , Piranos/análise , Piranos/metabolismoRESUMO
Pyran ring formation in the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2) was studied using the act cluster deficient strain, CH999, carrying pRM5-based plasmids harbouring combinations of the actVI genes. The strain, CH999/pIJ5660 (pRM5 + actVI-ORF1), produced a chiral intermediate, (S)-DNPA, suggesting that the actVI-ORF1 product is a reductase determining the C-3 stereochemical centre.
