RESUMO
Metabolic engineering of plant-specific phenylpropanoid biosynthesis has attracted an increasing amount of attention recently, owing to the vast potential of flavonoids as nutraceuticals and pharmaceuticals. Recently, we have developed a recombinant Streptomyces venezuelae as a heterologous host for the production of flavonoids. In this study, we successfully improved flavonoid production by expressing two sets of genes predicted to be involved in malonate assimilation. The introduction of matB and matC encoding for malonyl-CoA synthetase and the putative dicarboxylate carrier protein, respectively, from Streptomyces coelicolor into the recombinant S. venezuelae strains expressing flavanone and flavone biosynthetic genes resulted in enhanced production of both flavonoids.
Assuntos
Flavonoides/biossíntese , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Streptomyces/genética , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Expressão Gênica , Malonatos/metabolismoRESUMO
Streptomyces venezuelae YJ028, bearing a deletion of the entire biosynthetic gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes, was used as a bioconversion system for combinatorial biosynthesis of glycosylated derivatives of tylosin. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of TDP-3-O-demethyl-D-chalcose or TDP-Lrhamnose in conjunction with the glycosyltransferaseauxiliary protein pair DesVII/DesVIII were expressed in a S. venezuelae YJ028 mutant strain. Supplementation of each mutant strain capable of producing TDP-3-O-demethyl- D-chalcose or TDP-L-rhamnose with tylosin aglycone tylactone resulted in the production of the 3-O-demethyl- D-chalcose, D-quinovose, or L-rhamnose-glycosylated tylactone.
Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Streptomyces/genética , Streptomyces/metabolismo , Tilosina/biossíntese , Amino Açúcares/biossíntese , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Biotecnologia/métodos , Técnicas de Química Combinatória , Deleção de Genes , Engenharia Genética/métodos , Glicosilação , Macrolídeos/metabolismo , Família Multigênica , Policetídeo Sintases/biossíntese , Policetídeo Sintases/genética , Streptomyces/enzimologia , Tilosina/químicaRESUMO
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
Recently, recombinant Streptomyces venezuelae has been established as a heterologous host for microbial production of flavanones and stilbenes, a class of plant-specific polyketides. In the present work, we expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides including flavones and flavonols. A plasmid with the synthetic codon-optimized flavone synthase I gene from Petroselium crispum was introduced to S. venezuelae DHS2001 bearing a deletion of the native pikromycin polyketide synthase gene, and the resulting strain generated flavones from exogenously fed flavanones. In addition, a recombinant S. venezuelae mutant expressing a codon-optimized flavanone 3beta-hydroxylase gene from Citrus siensis and a flavonol synthase gene from Citrus unshius also successfully produced flavonols.
Assuntos
Flavonas/biossíntese , Flavonóis/biossíntese , Microbiologia Industrial/métodos , Streptomyces/metabolismo , Apiaceae/enzimologia , Apiaceae/genética , Citrus/enzimologia , Expressão Gênica , Genes de Plantas/genética , Engenharia Genética , Oxigenases de Função Mista/genética , Streptomyces/genéticaRESUMO
Using metabolic engineering, we developed Streptomyces venezuelae YJ028 as an efficient heterologous host to increase the malonyl-CoA pool to be directed towards enhanced production of various polyketides. To probe the applicability of newly developed hosts in the heterologous production of polyketides, we expressed type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase, in these hosts. Flaviolin production was doubled by expression of acetyl-CoA carboxylase (ACCase) and 4-fold by combined expression of ACCase, metK1-sp and afsR-sp. Thus, the newly developed Streptomyces venezuelae YJ028 hosts produce heterologous polyketides more efficiently than the parent strain.
Assuntos
Melhoramento Genético/métodos , Macrolídeos/metabolismo , Malonil Coenzima A/biossíntese , Metaboloma/fisiologia , Engenharia de Proteínas/métodos , Streptomyces/fisiologia , Malonil Coenzima A/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Arabidopsis/enzimologia , Flavanonas/metabolismo , Engenharia Genética/métodos , Estilbenos/metabolismo , Streptomyces/enzimologia , Aciltransferases/genética , Aciltransferases/metabolismo , Códon , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Escherichia coli/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
An unusual set of reduced macrolide antibiotics was discovered by combination of organic synthesis and a biosynthetic approach using the unique metabolic diversity of Streptomyces venezuelae; two unnatural 16-membered ring macrolides are also created by employing this bio-catalyst.
Assuntos
Antibacterianos/biossíntese , Macrolídeos/metabolismo , Streptomyces/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Macrolídeos/química , Macrolídeos/farmacologia , Família Multigênica , Streptomyces/genéticaRESUMO
Epothilones, produced from the myxobacterium Sorangium cellulosum, are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 microg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 microg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides.
Assuntos
Epotilonas/metabolismo , Engenharia Genética , Microbiologia Industrial , Streptomyces/genética , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Epotilonas/química , Dosagem de Genes , Vetores Genéticos/genética , Fases de Leitura Aberta , Oxirredutases/genética , Oxirredutases/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Deleção de Sequência , Streptomyces/enzimologiaRESUMO
To elevate the production level of heterologous polyketide in Streptomyces venezuelae, an additional copy of the positive regulatory gene pikD was introduced into the pikromycin (Pik) polyketide synthase (PKS) deletion mutant of S. venezuelae ATCC 15439 expressing tylosin PKS genes. The resulting mutant strain showed enhanced production of both tylactone (TL) and desosaminyl tylactone (DesTL) of 2.7- and 17.1-fold, respectively. The notable increase in DesTL production strongly suggested that PikD upregulates the expression of the desosamine (des) biosynthetic gene cluster. In addition, two hydroxylated forms of DesTL were newly detected from the extract of this mutant. These hydroxylated forms presumably resulted from a PikD-dependent increase in expression of the pikC gene that encodes P450 hydroxylase. Gene expression analysis by reverse transcriptase PCR and bioconversion experiments of 10-deoxymethynolide, narbonolide, and TL into the corresponding desosaminyl macrolides indicated that PikD is a positive regulator of the des and pikC genes, as well as the Pik PKS genes. These results demonstrate the role of PikD as a pathway-specific positive regulator of the entire Pik biosynthetic pathway and its usefulness in the development of a host-vector system for efficient heterologous production of desosaminyl macrolides and novel hydroxylated compounds.
Assuntos
Amino Açúcares/biossíntese , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Macrolídeos , Streptomyces/metabolismo , Fatores de Transcrição/metabolismo , Amino Açúcares/metabolismo , Antibacterianos/biossíntese , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Streptomyces/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
To develop a system for combinatorial biosynthesis of glycosylated macrolides, Streptomyces venezuelae was genetically manipulated to be deficient in the production of its macrolide antibiotics by deletion of the entire biosynthetic gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of thymidine diphosphate (TDP)-D-quinovose or TDP-D-olivose in conjunction with the glycosyltransferase-auxiliary protein pair DesVII/DesVIII derived from S. venezuelae were expressed in the mutant strain. Feeding the representative 12-, 14-, and 16-membered ring macrolactones including 10-deoxymethynolide, narbonolide, and tylactone, respectively, to each mutant strain capable of producing TDP-D-quinovose or TDP-D-olivose resulted in the successful production of the corresponding quinovose- and olivose-glycosylated macrolides. In mutant strains where the DesVII/DesVIII glycosyltransferase-auxiliary protein pair was replaced by TylMII/TylMIII derived from Streptomyces fradiae, quinovosyl and olivosyl tylactone were produced; however, neither glycosylated 10-deoxymethynolide nor narbonolide were generated, suggesting that the glycosyltransferase TylMII has more stringent substrate specificity toward its aglycones than DesVII. These results demonstrate successful generation of structurally diverse hybrid macrolides using a S. venezuelae in vivo system and provide further insight into the substrate flexibility of glycosyltransferases.
Assuntos
Macrolídeos/química , Macrolídeos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Deleção de Genes , Engenharia Genética , Glicosilação , Estrutura Molecular , Especificidade por SubstratoRESUMO
The pSKC2 cosmid, which has 32 kb and 28 open-reading frames, was isolated from Streptomyces kanamyceticus ATCC12853 as the gene cluster of kanamycin. This gene cluster includes the minimal biosynthetic genes of kanamycin with the resistance and regulatory genes. It was heterologously expressed in Streptomyces venezuelae YJ003, which has the advantage of fast growth, good efficiency of the transformation host, and rapid production of the aminoglycosides antibiotic. The isolated compound was analyzed by electrospray ionization-mass spectrometry, liquid chromatography-mass spectrometry, high-performance liquid chromatography, and tandem mass spectrometry and shows a molecular weight of 485 as kanamycin A.
Assuntos
Proteínas de Bactérias/genética , Canamicina/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Carboidratos , Regulação Bacteriana da Expressão Gênica , Canamicina/química , Família MultigênicaRESUMO
Two new macrolides from the pikromycin biosynthetic pathway of Streptomyces venezuelae, neopikromycin (9) and novapikromycin (10), were identified and structurally characterized through mass spectrometry and NMR spectroscopy. The established structures showed that 9 and 10 have hydroxyl groups at C-14 (9) and at both C-12 and C-14 (10), on the basis of a comparison with narbomycin (7). The purified PikC cytochrome P450 monooxygenase catalyzes the in vitro hydroxylation of 7 and pikromycin (8) to yield 9 and 10, respectively, thus expanding the substrate- and regio-flexibility of this enzyme.