RESUMO
Certain members of the cytochromes P450 superfamily metabolize polyunsaturated long-chain fatty acids to several classes of oxygenated metabolites. An approach based on in silico analysis predicted that Streptomyces peucetius CYP107N3 might be a fatty acid-metabolizing enzyme, showing high homology with epoxidase enzymes. Homology modeling and docking studies of CYP107N3 showed that oleic acid can fit directly into the active site pocket of the double bond of oleic acid within optimum distance of 4.6 Å from the Fe. In order to confirm the epoxidation activity proposed by in silico analysis, a gene coding CYP107N3 was expressed in Escherichia coli. The purified CYP107N3 was shown to catalyze C(9)-C(10) epoxidation of oleic acid in vitro to 9,10-epoxy stearic acid confirmed by ESI-MS, HPLC-MS and GC-MS spectral analysis.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Ácido Oleico/metabolismo , Streptomyces/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Compostos de Epóxi/metabolismo , Escherichia coli/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Oxirredutases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Ácidos Esteáricos/metabolismoRESUMO
Biocatalytic transfer of oxygen in isolated cytochrome P450 or whole microbial cells is an elegant and efficient way to achieve selective hydroxylation. Cytochrome P450 CYP105P2 was isolated from Streptomyces peucetius that showed a high degree of amino acid identity with hydroxylases. Previously performed homology modeling, and subsequent docking of the model with flavone, displayed a reasonable docked structure. Therefore, in this study, in a pursuit to hydroxylate the flavone ring, CYP105P2 was co-expressed in a two-vector system with putidaredoxin reductase (camA) and putidaredoxin (camB) from Pseudomonas putida for efficient electron transport. HPLC analysis of the isolated product, together with LCMS analysis, showed a monohydroxylated flavone, which was further established by subsequent ESI/MS-MS. A successful 10.35% yield was achieved with the whole-cell bioconversion reaction in Escherichia coli. We verified that CYP105P2 is a potential bacterial hydroxylase.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Flavonas/metabolismo , Streptomyces/enzimologia , Biotransformação , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Hidroxilação , Espectrometria de Massas , Engenharia Metabólica , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Pseudomonas putida/enzimologia , Streptomyces/genéticaRESUMO
Cytochrome P450 CYP107AJ1, which was isolated from Streptomyces peucetius and showed high homology with peroxygenases, catalyzed a dealkylation reaction with hydrogen peroxide to provide electrons, protons and oxygen, evading the requirement for a supporting redox protein. Preliminary investigation of its transcriptional level in S. peucetius showed significant expression. Homology modeling and subsequent docking with 7-ethoxycoumarin yielded a reasonable docked structure. cyp107AJ1 cloned into pET28a(+) was expressed in Escherichia coli, and soluble protein was subjected to column-chromatographic purification in order to carry out enzyme assays with 7-ethoxycoumarin. HPLC analysis of the extracted product, corresponding to its LC/MS analysis, showed the dealkylated 7-ethoxycoumarin, which was further established by subsequent GC/MS spectral analysis. We suggest that CYP107AJ1 bypassed the requirement for NAD(P)H and redox partners for generating novel analogues.
Assuntos
Biotecnologia/métodos , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Peróxido de Hidrogênio/farmacologia , Streptomyces/enzimologia , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/genética , Remoção de Radical Alquila/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Streptomyces/genética , Streptomyces/metabolismo , Homologia Estrutural de ProteínaRESUMO
Two superoxide dismutase (SOD) genes; sod1 and sod2, from Streptomyces peucetius ATCC 27952 show high similarity to other known SODs from Streptomyces coelicolor A3(2) and Streptomyces avermitilis MA-4680. These sod1 and sod2 were cloned into pIBR25 expression vector under a strong ermE* promoter to enhance secondary metabolites from Streptomyces strains. The recombinant expression plasmids; pIBR25SD1 and pIBR25SD2, were constructed to overexpress sod1 and sod2 respectively to enhance production of doxorubicin (DXR) in S. peucetius, clavulanic acid (CA) in Streptomyces clavuligerus NRRL 3585 and actinorhodin (ACT) and undecylprodigiosin (Red) in Streptomyces lividans TK24. Biomass variation, antibiotics production and transcriptional analysis of regulatory genes in recombinant strains have been studied to understand the effect of sod1 and sod2. The cell growth analysis shows that life span of all recombinant strains was found to be elevated as compared to wild type cells. In S. peucetius, overexpression of sod1 and sod2 was not effective in DXR production but in case of S. clavuligerus, CA production was increased by 2.5 and 1.5 times in sod1 and sod2 overexpression, respectively while in case of S. lividans, ACT production was increased by 1.4 and 1.6 times and Red production by 1.5 and 1.2 times upon sod1 and sod2 overexpressions, respectively as compared to the corresponding wild type strains.
Assuntos
Metaboloma , Streptomyces/enzimologia , Streptomyces/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Antibacterianos/biossíntese , Biomassa , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Expressão Gênica , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Análise de Sequência de DNA , Streptomyces/genéticaRESUMO
Doxorubicin (DXR) is an anthracycline-type polyketide, typically produced by Streptomyces peucetius ATCC 27952. Like the biosynthesis of other secondary metabolites in Streptomyces species, DXR biosynthesis is tightly regulated, and a very low level of DXR production is maintained in the wild-type strain. Despite that DXR is one of the most broadly used and clinically important anticancer drugs, a traditional strain improvement strategy has long been practiced via recursive random mutagenesis, with little understanding of the molecular genetic basis underlying such enhanced DXR production. Since DXR titer enhancement is imperative in the fermentation industry, attaining a comprehensive understanding and its application of the specific regulatory systems that govern secondary metabolite production is an important aspect of metabolic engineering that can efficiently improve fermentation titers. In this mini-review, various efforts to improve the titers of DXR have been summarized based on biosynthetic and regulatory studies including transcriptional and product analyses.
Assuntos
Antibióticos Antineoplásicos/biossíntese , Doxorrubicina/biossíntese , Engenharia Genética , Microbiologia Industrial , Streptomyces/genética , Streptomyces/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Annotation of genome of Streptomyces peucetius revealed a putative phenylacetic acid degradation NADH oxidoreductase. RT-PCR analysis of the gene readily showed notable transcription in its native state. The transcription level of paaE when the host is grown on phenylacetic acid showed increased transcription. paaE was cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in Escherichia coli BL21(DE3) and purified by immobilized metal affinity chromatography using His-tag. The flavin released from heat-denatured PaaE was identical to that of authentic FAD in HPLC analysis. The purified protein efficiently reduced p-nitroblue tetrazolium (an electron acceptor) in presence of NADH. Cell growth analysis of S. peucetius in phenylacetic acid evidently revealed its involvement in degradation of phenylacetic acid - a key environmental pollutant.
Assuntos
NADH NADPH Oxirredutases/metabolismo , Fenilacetatos/metabolismo , Streptomyces/enzimologia , Streptomyces/metabolismo , Cromatografia de Afinidade , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/isolamento & purificação , Nitroazul de Tetrazólio/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Streptomyces/genética , Transcrição GênicaRESUMO
Doxorubicin (DXR), produced by Streptomyces peucetius ATCC 27952, exhibits potent antitumor activity against various cancer cell lines. Considerable time has lapsed since the biosynthesis of DXR and its overproduction was first summarized. Based on biosynthetic studies and product analysis, various factors affecting its production by the parental strain, S. peucetius ATCC 27952, are reviewed to better circumvent any bottlenecks in DXR production, thereby providing ideas to genetically engineered industrial strains of S. peucetius.
Assuntos
Doxorrubicina/biossíntese , Streptomyces/metabolismo , Antraciclinas/metabolismo , Vias Biossintéticas , Inibidores das Enzimas do Citocromo P-450 , Doxorrubicina/antagonistas & inibidores , Retroalimentação Fisiológica , Glicosilação , Hexosaminas/biossínteseRESUMO
Biosynthesis of doxorubicin (DXR) is tightly regulated, limiting its production in Streptomyces peucetius cultures. The regulatory genes dnrN, dnrI, afsR, and metK1-sp from S. peucetius ATCC 27952 were cloned into the pIBR25 expression vector under the control of the strong ermE* promoter to enhance DXR production. The constructed expression plasmids, pNI25 (with dnrN-dnrI), pNIS25 (with dnrN-dnrI-metK1-sp), pNIR25 (with dnrN-dnrI-afsR), pRS25 (with afsR-metK1-sp) and pNIRS25 (with dnrN-dnrI-afsR-metK1-sp), were transformed into S. peucetius. The recombinant strains NI, NIS and NIR produced greater amounts of DXR than the parental strain with an increment of 1.2-fold by pNI25, 1.4-fold by pNIS25 and 4.3-fold by pNIR25, whereas pRS25 and pNIRS25 had no significant effect on DXR production. We also studied the transcriptional level of overexpressed regulatory genes and relative production of DXR, daunorubicin (DNR) and epsilon-rhodomycinone (RHO) in each recombinant strain.
Assuntos
Doxorrubicina/biossíntese , Dosagem de Genes , Regulação Bacteriana da Expressão Gênica , Plasmídeos , Streptomyces/genética , Streptomyces/metabolismo , Vias Biossintéticas/genética , Perfilação da Expressão Gênica , Engenharia Genética , Humanos , Estrutura Molecular , Recombinação GenéticaRESUMO
Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Here, we report the identification, cloning, and characterization of panK-sp from Streptomyces peucetius ATCC 27952. The gene encoded a protein of 332 amino acids with a calculated molecular mass of 36.8 kDa and high homology with PanK from S. avermitilis and S. coelicolor A3(2). To elucidate the putative function of PanK-sp, it was cloned into pET32a(+) to construct pPKSP32, and the PanK-sp was then expressed in E. coli BL21(DE3) as a His-tag fusion protein and purified by immobilized metal affinity chromatography. The enzyme assay of PanK-sp was carried out as a coupling assay. The gradual decrease in NADH concentration with time clearly indicated the phosphorylating activity of PanK-sp. Furthermore, the ca. 1.4-fold increase of DXR and the ca. 1.5-fold increase of actinorhodin by in vivo overexpression of panK-sp, constructed in pIBR25 under the control of a strong ermE* promoter, established its positive role in secondary metabolite production from S. peucetius and S. coelicolor, respectively.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Streptomyces/enzimologia , Antraquinonas/metabolismo , Cromatografia de Afinidade , Clonagem Molecular , Coenzimas/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Expressão Gênica , Dados de Sequência Molecular , Peso Molecular , NAD/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The resistance genes drrABC from Streptomyces peucetius ATCC 27952 were cloned into the pIBR25 expression vector under a strong ermE* promoter to enhance doxorubicin (DXR) production. The recombinant expression plasmids, pDrrAB25, pDrrC25 and pDrrABC25, were constructed to overexpress drrAB, drrC and drrABC, respectively, in S. peucetius ATCC 27952. The recombinant strains produced more DXR than the parental strain: a 2.2-fold increase with pDrrAB25, a 5.1-fold increase with pDrrC25, and a 2.4-fold increase with pDrrABC25. We also studied the relative ratios of doxorubicin, daunorubicin and epsilon-rhodomycinone produced in these recombinant strains.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibióticos Antineoplásicos/biossíntese , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Doxorrubicina/biossíntese , Streptomyces/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Antibióticos Antineoplásicos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Doxorrubicina/metabolismo , Farmacorresistência Bacteriana/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Streptomyces/genéticaRESUMO
To enhance doxorubicin (DXR) production, the structural sugar biosynthesis genes desIII and desIV from Streptomyces venezuelae ATCC 15439 and the glycosyltransferase pair dnrS/dnrQ from Streptomyces peucetius ATCC 27952 were cloned into the expression vector pIBR25, which contains a strong ermE promoter. The recombinant plasmids pDnrS25 and pDnrQS25 were constructed for overexpression of dnrS and the dnrS/dnrQ pair, whereas pDesSD25 and pDesQS25 were constructed to express desIII/desIV and dnrS/dnrQ-desIII/desIV, respectively. All of these recombinant plasmids were introduced into S. peucetius ATCC 27952. The recombinant strains produced more DXR than the S. peucetius parental strain: a 1.2-fold increase with pDnrS25, a 2.8-fold increase with pDnrQS25, a 2.6-fold increase with pDesSD25, and a 5.6-fold increase with pDesQS25. This study showed that DXR production was significantly enhanced by overexpression of potential biosynthetic sugar genes and glycosyltransferase.
