RESUMEN
Enormous progress in the field of polyketide biosynthesis has led to the establishment of rules for general text book biosynthetic logic and consequently to the assumption that biosynthetic genes can be easily correlated with the corresponding natural products. However, non-textbook examples of polyketide assembly continue to be discovered suggesting the gene to product and product to gene predictions need improvement, especially as they are increasingly used in the post-genomic era. Here, we analyzed the genomic blueprint of a myxobacterial multi-producer of secondary metabolites, Stigmatella aurantiaca DW4/3-1, for its biosynthetic potential by genome-mining. In addition to the five polyketide synthase and/or nonribosomal peptide synthetase gene clusters of known function we identified a further 13 genomic regions exemplifying the enormous genetic potential for the production of additional chemical diversity by this strain. We show by gene inactivation and heterologous expression of the newly identified biosynthetic pathway for dawenol that the biosynthesis of this known polyene does not follow text book biosynthetic logic. Intriguingly, a genomic locus encoding an unusual polyketide synthase exhibiting similarity to gene loci involved in the formation of polyunsaturated fatty acids and secondary lipids was identified.
Asunto(s)
Polienos/metabolismo , Sintasas Poliquetidas/química , Stigmatella aurantiaca/enzimología , Secuencia de Aminoácidos/genética , Vías Biosintéticas , Complejos Multienzimáticos/metabolismo , Péptido Sintasas/genética , Polienos/química , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/aislamiento & purificaciónRESUMEN
Natural products of microbial origin continue to be an important source of pharmaceuticals and agrochemicals exhibiting potent activities and often novel modes of action. Due to their inherent structural complexity chemical synthesis is often hardly possible, leaving fermentation as the only viable production route. In addition, the pharmaceutical properties of natural products often need to be optimized for application by sophisticated medicinal chemistry and/or biosynthetic engineering. The latter requires a detailed understanding of the biosynthetic process and genetic tools to modify the producing organism that are often unavailable. Consequently, heterologous expression of complex natural product pathways has been in the focus of development over recent years. However, piecing together existing DNA cloned from natural sources and achieving efficient expression in heterologous circuits represent several limitations that can be addressed by synthetic biology. In this work we have redesigned and reassembled the 56 kb epothilone biosynthetic gene cluster from Sorangium cellulosum for expression in the high GC host Myxococcus xanthus. The codon composition was adapted to a modified codon table for M. xanthus, and unique restriction sites were simultaneously introduced and others eliminated from the sequence in order to permit pathway assembly and future interchangeability of modular building blocks from the epothilone megasynthetase. The functionality of the artificial pathway was demonstrated by successful heterologous epothilone production in M. xanthus at significant yields that have to be improved in upcoming work. Our study sets the stage for future engineering of epothilone biosynthesis and production optimization using a highly flexible assembly strategy.
Asunto(s)
Epotilonas/biosíntesis , Genes Sintéticos , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Algoritmos , Vías Biosintéticas/genética , Biotecnología , Codón/genética , Epotilonas/química , Epotilonas/genética , Ingeniería Genética , Familia de Multigenes , Myxococcales/genética , Myxococcales/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Policétidos/metabolismo , Biología SintéticaRESUMEN
Manipulation of secondary metabolite production in the rare actinomycete Actinoplanes friuliensis, the producer of the lipopeptide antibiotic friulimicin, is hampered by the lack of sophisticated genetic tools. Since no expression vectors have been developed from endogenous Actinoplanes plasmids and expression signals, engineering of antibiotic biosynthesis relies on the use of vector systems derived from Streptomyces. While PhiC31 derived vectors were shown to integrate efficiently into the chromosome of Actinoplanes, information on promoter activity is missing. The manuscript describes the investigation of several different promoter systems which are widely used in Streptomyces in A. friuliensis by promoter probe experiments using eGFP as a reporter. These experiments indicated that promoter strength in A. friuliensis did not correlate to activity in Streptomyces lividans. The ermE* promoter regarded as one of the strongest promoter in Streptomyces has only low activity in A. friuliensis. In contrast, the promoter of the apramycin resistance gene aac(3)IV, originating from the Gram-negative Escherichia coli had the highest activity. By real-time RT-PCR experiments the transcription activity of ermE* promoter in comparison to a native promoter of the friulimicin biosynthetic gene cluster was analysed. This confirmed the results of the promoter probe experiments that indicated quite weak promoter activity of P-ermE* in Actinoplanes.