RESUMO
Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4''-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4''-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Evolução Molecular Direcionada , Inseticidas/metabolismo , Ivermectina/análogos & derivados , Biotransformação , Dissacarídeos/metabolismo , Biblioteca Gênica , Genes Bacterianos , Ivermectina/metabolismo , Dados de Sequência Molecular , Mutação , Oxirredução , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por SubstratoRESUMO
4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Ivermectina/análogos & derivados , Streptomyces/classificação , Streptomyces/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Ivermectina/química , Ivermectina/metabolismo , Cinética , Dados de Sequência Molecular , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Estereoisomerismo , Streptomyces/genéticaRESUMO
The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Ivermectina/análogos & derivados , Pseudomonas putida/enzimologia , Streptomyces/enzimologia , Streptomyces/genética , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/genética , Dissacarídeos/química , Dissacarídeos/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica , Engenharia Genética/métodos , Microbiologia Industrial/métodos , Ivermectina/química , Ivermectina/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Regiões Promotoras Genéticas , Pseudomonas putida/genética , Pseudomonas putida/crescimento & desenvolvimento , Alinhamento de Sequência , Streptomyces/classificação , Streptomyces/crescimento & desenvolvimento , Transformação BacterianaRESUMO
A 108-kb genomic DNA region of Saccharopolyspora spinosa NRRL 18395, producer of the agriculturally important insecticidal antibiotics spinosyns, has been cloned, sequenced and analyzed to reveal clustered genes encoding a type I polyketide synthase (PKS) complex. The genes for the PKS are flanked by genes encoding homologs of enzymes that are involved in the urea cycle, valine, leucine and isoleucine biosynthesis and energy metabolism. While the disruption of the PKS genes by insertional inactivation was not expected to abolish the production of spinosyns, no differences were found in the antibacterial, antifungal, or insecticidal activities either of the parental and the knockout mutant strains under the growth conditions tested. Deduction of the most likely structure of the polyketide core of the cryptic metabolite, termed obscurin, from the predicted modules and domains of the PKS suggests the formation of a highly unsaturated substituted C22 carboxylic acid that might undergo further processing after its release from the PKS.
Assuntos
Genoma Bacteriano , Complexos Multienzimáticos/genética , Saccharopolyspora/genética , Clonagem Molecular , Sequência Conservada/genética , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Análise de Sequência de DNARESUMO
The antifungal polyketide soraphen A is produced by the myxobacterium Sorangium cellulosum So ce26. The slow growth, swarming motility and general intransigence of the strain for genetic manipulations make industrial strain development, large-scale fermentation and combinatorial biosynthetic manipulation of the soraphen producer very challenging. To provide a better host for soraphen A production and molecular engineering, the biosynthetic gene cluster for this secondary metabolite was integrated into the chromosome of Streptomyces lividans ZX7. The upstream border of the gene cluster in Sor. cellulosum was defined by disrupting sorC, which is proposed to take part in the biosynthesis of methoxymalonyl-coenzyme A, to yield a Sor. cellulosum strain with abolished soraphen A production. Insertional inactivation of orf2 further upstream of sorC had no effect on soraphen A production. The genes sorR, C, D, F and E thus implicated in soraphen biosynthesis were then introduced into an engineered Str. lividans strain that carried the polyketide synthase genes sorA and sorB, and the methyltransferase gene sorM integrated into its chromosome. A benzoate-coenzyme A ligase from Rhodopseudomonas palustris was also included in some constructs. Fermentations with the engineered Str. lividans strains in the presence of benzoate and/or cinnamate yielded soraphen A. Further feeding experiments were used to delineate the biosynthesis of the benzoyl-coenzyme A starter unit of soraphen A in the heterologous host.
Assuntos
Antifúngicos/metabolismo , Macrolídeos/metabolismo , Myxococcales/genética , Myxococcales/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Acil Coenzima A/metabolismo , Sequência de Bases , Benzoatos/metabolismo , DNA Bacteriano/genética , Fermentação , Genes Bacterianos , Engenharia Genética , Família Multigênica , Mutagênese InsercionalRESUMO
The myxobacterium Sorangium cellulosum So ce26, the producer of the agriculturally important fungicide antibiotic soraphen A, displays coordinated gliding motility (swarming) on agar surfaces. The consequent failure to form detached colonies represents a major obstacle for microbiological and genetic studies, since single cells representing discrete genetic events cannot be reliably separated and propagated as clones. The MglA protein, the product of the mglA gene, has been shown to be a central regulator of gliding motility and swarming in the related myxobacterium Myxococcus xanthus. We have cloned and sequenced a chromosomal locus from S. cellulosum So ce26 that shows similarity to the M. xanthus mglA locus. Insertional inactivation of the chromosomal copy of the S. cellulosum So ce26 mglA homolog resulted in a strain with a non-swarming colony phenotype. This strain is able to form distinct colonies presumably derived from single cells. This is the first report on the characterization of a genetic element of the gliding motility system in the myxobacterial suborder Sorangineae.
RESUMO
2-Hexyl-5-propylresorcinol is the predominant analog of several dialkylresorcinols produced by Pseudomonas aurantiaca (Pseudomonas fluorescens BL915). We isolated and characterized three biosynthetic genes that encode an acyl carrier protein, a beta-ketoacyl-acyl carrier protein synthase III, and a protein of unknown function, all of which collectively allow heterologous production of 2-hexyl-5-propylresorcinol in Escherichia coli. Two regulatory genes exhibiting similarity to members of the AraC family of transcriptional regulators are also present in the identified gene cluster. Based on the deduced functions of the proteins encoded by the gene cluster and the observed incorporation of labeled carbons from octanoic acid into 2-hexyl-5-propylresorcinol, we propose that dialkylresorcinols are derived from medium-chain-length fatty acids by an unusual head-to-head condensation of beta-ketoacyl thioester intermediates. Genomic evidence suggests that there is a similar pathway for the biosynthesis of the flexirubin-type pigments in certain bacteria belonging to the order Cytophagales.