RESUMO
Streptomyces venezuelae ATCC 10712 produces chloramphenicol in small amounts. To enhance chloramphenicol production, two genes, aroB and aroK, encoding rate-limiting enzymes of the shikimate pathway were overexpressed using the expression vector pIJ86 under the control of the strong constitutive ermE* promoter. The recombinant strains, S. venezuelae/pIJ86-aroB and S. venezuelae/pIJ86-aroK, produced 2.5- and 4.3-fold greater amounts respectively of chloramphenicol than wild type at early stationary phase of growth. High transcriptional levels of aroB and aroK genes were detected at the early exponential growth of both recombinant strains and consistent with the enhanced expression of pabB gene encoding an early enzyme in chloramphenicol biosynthesis. The results suggested that the increment of carbon flux was directed towards intermediates in the shikimate pathway required for the production of chorismic acid, and consequently resulted in the enhancement of chloramphenicol production. This work is the first report of a convenient genetic approach to manipulate primary metabolite genes in S. venezuelae in order to increase chloramphenicol production.
Assuntos
Cloranfenicol/biossíntese , Expressão Gênica , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ácido Chiquímico/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Catálise , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Redes e Vias Metabólicas , Transcrição GênicaRESUMO
A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His(6)-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline-serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V(max) and K(m) of 3.63 +/- 0.16 micromol min(-1) mg(-1) and 0.79 +/- 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50-55 degrees C and pH 7-8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm(-2) h(-1) for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.
Assuntos
Actinomycetales/enzimologia , Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Expressão Gênica , Pichia/genética , Actinomycetales/química , Actinomycetales/genética , Actinomycetales/isolamento & purificação , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Catálise , Clonagem Molecular , Dados de Sequência Molecular , Pichia/metabolismo , Alinhamento de Sequência , Ressonância de Plasmônio de SuperfícieRESUMO
Maklamicin is a spirotetronate-class antibiotic produced by Micromonospora sp. NBRC 110955, and a polyketide assembly line and a glycerate utilization system are involved in its biosynthesis. One tailoring step in the biosynthesis is predicted to be post-polyketide synthase (PKS) modification, which seems to be catalysed by putative cytochrome P450 monooxygenases, MakC2 and/or MakC3. In this study, we characterized makC2 and makC3 in the biosynthesis of maklamicin and identified a new maklamicin analogue from a makC2 disruptant. Gene deletion of makC2 resulted in the complete loss of maklamicin production with concomitant accumulation of a new compound (29-deoxymaklamicin), while gene deletion of makC3 did not affect the maklamicin production, indicating that 29-deoxymaklamicin is an intermediate in the biosynthetic pathway of maklamicin and should serve as the substrate of MakC2. 29-Deoxymaklamicin showed strong-to-modest anti-microbial activity against gram-positive bacteria. The fact that Streptomyces avermitilis heterologously expressing makC2 successfully converted 29-deoxymaklamicin into maklamicin confirmed that MakC2 is the final-step hydroxylase in the formation of mature maklamicin.
Assuntos
Antibacterianos/biossíntese , Macrolídeos/metabolismo , Micromonospora/genética , Micromonospora/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Vias Biossintéticas/genética , Sistema Enzimático do Citocromo P-450/deficiência , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Bactérias Gram-Positivas/efeitos dos fármacos , Hidroxilação , Macrolídeos/química , Macrolídeos/farmacologia , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Streptomyces/genética , Streptomyces/metabolismoRESUMO
Maklamicin, which is produced by the endophytic Micromonospora sp. NBRC 110955, is a spirotetronate-class antibiotic possessing anti-microbial activity against Gram-positive bacteria, and has several unique structural features different from other spirotetronates. Here we describe identification and characterization of the maklamicin biosynthetic (mak) gene cluster through draft genome sequencing, genomic library screening, and gene disruption. Sequence analysis revealed that a plausible maklamicin cluster resides in a 152 kb DNA region encoding 46 open reading frames, 24 of which can be assigned roles in the biosynthesis of polyketide backbone, spirotetronate or peripheral moieties, self-resistance and the regulation of maklamicin production. Disruption of the polyketide synthase (PKS) genes makA1 or makA4 resulted in a complete loss of maklamicin production, indicating that the type I modular PKS system is responsible for the biosynthesis of maklamicin. The mak gene cluster contained a set of biosynthetic genes for the formation of a tetronate moiety, which were found to be highly conserved in the gene clusters for spirotetronate antibiotics. Based on the estimated biosynthetic genes, we propose the biosynthetic pathway for maklamicin. Our findings provide not only insights on the biosynthetic mechanism of the unique structures in maklamicin, but also useful information to facilitate a comparative analysis of the spirotetronate biosynthetic pathways to expand the structural repertoire.
Assuntos
Antibacterianos/biossíntese , Macrolídeos/metabolismo , Micromonospora/genética , Micromonospora/metabolismo , Família Multigênica , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Vias Biossintéticas/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Macrolídeos/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
A new hexadienamide derivative named sarmentosamide (1) was identified from the culture of Streptomyces sp. SBI108 isolated from Thai soil under an herb. The structure was elucidated on the basis of spectroscopic data, and the absolute configuration was determined by chemical degradation.