RESUMO
Streptomyces venezuelae ATCC 15439, which produces 12- and 14-membered ring macrolide antibiotics, is a platform strain for heterologous expression of secondary metabolites. Its 9.05-Mb genome sequence revealed an abundance of genes involved in the biosynthesis of secondary metabolites and their precursors, which should be useful for the production of bioactive compounds.
Assuntos
Genoma Bacteriano , Análise de Sequência de DNA/métodos , Streptomyces/genética , Composição de Bases , Tamanho do Genoma , Metabolismo SecundárioRESUMO
Streptomyces venezuelae has an inherent advantage as a heterologous host for polyketide production due to its fast rate of growth that cannot be endowed easily through metabolic engineering. However, the utility of S. venezuelae as a host has been limited thus far due to its inadequate intracellular reserves of the (2S)-ethylmalonyl-CoA building block needed to support the biosynthesis of polyketides preventing the efficient production of the desired metabolite, such as tylactone. Here, via precursor supply engineering, we demonstrated that S. venezuelae can be developed into a more efficient general heterologous host for the quick production of polyketides. We first identified and functionally characterized the ethylmalonyl-CoA pathway which plays a major role in supplying the (2S)-ethylmalonyl-CoA extender unit in S. venezuelae. Next, S. venezuelae was successfully engineered to increase the intracellular ethylmalonyl-CoA concentration by the deletion of the meaA gene encoding coenzyme B12-dependent ethylmalonyl-CoA mutase in combination with ethylmalonate supplementation and was engineered to upregulate the expression of the heterologous tylosin PKS by overexpression of the pathway specific regulatory gene pikD. Thus, a dramatic increase (â¼10-fold) in tylactone production was achieved. In addition, the detailed insights into the role of the ethylmalonyl-CoA pathway, which is present in most streptomycetes, provides a general strategy to increase the ethylmalonyl-CoA supply for polyketide biosynthesis in the most prolific family of polyketide-producing bacteria.
Assuntos
Acil Coenzima A/metabolismo , Antibacterianos/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Policetídeos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Deleção de Genes , Expressão Gênica , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Doxorubicin, one of the most widely used anticancer drugs, is composed of a tetracyclic polyketide aglycone and l-daunosamine as a deoxysugar moiety, which acts as an important determinant of its biological activity. This is exemplified by the fewer side effects of semisynthetic epirubicin (4'-epi-doxorubicin). An efficient combinatorial biosynthetic system that can convert the exogenous aglycone ε-rhodomycinone into diverse glycosylated derivatives of doxorubicin or its biosynthetic intermediates, rhodomycin D and daunorubicin, was developed through the use of Streptomyces venezuelae mutants carrying plasmids that direct the biosynthesis of different nucleotide deoxysugars and their transfer onto aglycone, as well as the postglycosylation modifications. This system improved epirubicin production from ε-rhodomycinone by selecting a substrate flexible glycosyltransferase, AknS, which was able to transfer the unnatural sugar donors and a TDP-4-ketohexose reductase, AvrE, which efficiently supported the biosynthesis of TDP-4-epi-l-daunosamine. Furthermore, a range of doxorubicin analogs containing diverse deoxysugar moieties, seven of which are novel rhodomycin D derivatives, were generated. This provides new insights into the functions of deoxysugar biosynthetic enzymes and demonstrates the potential of the S. venezuelae-based combinatorial biosynthetic system as a simple biological tool for modifying structurally complex sugar moieties attached to anthracyclines as an alternative to chemical syntheses for improving anticancer agents.
Assuntos
Doxorrubicina/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Antraciclinas/metabolismo , Daunorrubicina/metabolismo , Doxorrubicina/química , Epirubicina , Engenharia Genética , Glicosilação , Glicosiltransferases/metabolismo , Família Multigênica , Plasmídeos/genéticaRESUMO
Phenylpropanoids, including flavonoids and stilbenes, are plant secondary metabolites with potential pharmacological and nutraceutical properties. To expand the applicability of Streptomyces venezuelae as a heterologous host to plant polyketide production, flavonoid and stilbene biosynthetic genes were expressed in an engineered strain of S. venezuelae DHS2001 bearing a deletion of native pikromycin polyketide synthase gene. A plasmid expressing the 4-coumarate/cinnamate:coenzyme A ligase from Streptomyces coelicolor (ScCCL) and the chalcone synthase from Arabidopsis thaliana (atCHS) under the control of a single ermE* promoter was constructed and introduced into S. venezuelae DHS2001. The resulting strain produced racemic naringenin and pinocembrin from 4-coumaric acid and cinnamic acid, respectively. Placement of an additional ermE* promoter upstream of the codon-optimized atCHS (atCHS(op)) gene significantly increased the yield of both flavanones. Expression of codon-optimized chalcone isomerase gene from Medicago sativa, together with ScCCL and atCHS(op) genes led to production of (2S)-flavanones, but the yield was reduced. On the other hand, a recombinant strain harboring the ScCCL and codon-optimized stilbene synthase gene from Arachis hypogaea generated stilbenes such as resveratrol and pinosylvin. This is the first report on the heterologous expression of plant phenylpropanoid biosynthetic pathways in Streptomyces genus.