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1.
Mol Biotechnol ; 65(2): 282-289, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36401710

RESUMO

Epothilones are a kind of 16-member macrolides with strong anticancer activity, which was produced by Sorangium cellulosum. Epothlione D shows better drug resistance and safety than taxol in clinical trials. However, the low yield of epothilone D in Sorangium cellulosum and thereof toxicity limited the application of epothilone D. In this study, the epoK gene in gene cluster for epothilone was firstly inactivated by the employment of TALEN gene knockout system. The qRT-PCR analysis and sequencing were performed to confirm the gene deletion of epoK, resulting in the epothilone D yield improvement by 34.9±1.6% and the decrease of epothilone B yield by 34.2±2.5%, which was demonstrated by LC-MS analysis. This study would lay a foundation for the yield improvement of epothilones D, B and thereof derivatives in S. cellulosum by genetic engineering, thus promoting the applications of epothilones in the field of anticancer.


Assuntos
Antineoplásicos , Epotilonas , Epotilonas/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Macrolídeos , Antineoplásicos/farmacologia
2.
Microb Cell Fact ; 17(1): 15, 2018 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-29378572

RESUMO

BACKGROUND: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. RESULTS: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. CONCLUSIONS: Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.


Assuntos
Sistemas CRISPR-Cas/genética , Epotilonas/biossíntese , Família Multigênica , Myxococcus xanthus/genética , Proteínas Recombinantes/genética , Transcrição Gênica , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Epotilonas/genética , Myxococcus xanthus/metabolismo , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/genética , Metabolismo Secundário , Ativação Transcricional
3.
World J Microbiol Biotechnol ; 33(7): 137, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28585173

RESUMO

Epothilones are cytotoxic macrolactones having auspicious anti-tumorous activities, but merely produced by rare Sorangium strains. Here, we have focused on the epothilone gene cluster from special niche bacterial strain, S. cellulosum So0157-2. Therefore, we have isolated a high pH tolerant S. cellulosum strain So0157-2 and characterized the epothilones gene cluster and its flanks by cosmid/fosmid libraries preparation and sequencing. The assembly spanned 94,459 bp and consisted of 56,019 bp core region. Remarkably, the core as well as upstream 420 bp and downstream 315 bp were highly conserved, while further neighboring regions varied extremely. Transposase traces were identified near the core of clusters, supporting that the transposon-mediated transgenesis is a naturally evolved strategy for the cluster's dissemination. A predicted neighboring esterase gene was identified as a potential epothilone-resistance gene preventing self-toxicity. Novel modification or regulatory genes, a multi-position-cyclo releasing gene and their relationship with corresponding analogs were identified in strain So0157-2. These findings open the door to discover additional, naturally evolved epothilone-related genes for significant applications in industrial as well as clinical sector.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Epotilonas/biossíntese , Myxococcales/isolamento & purificação , Epotilonas/genética , Esterases/genética , Evolução Molecular , Tamanho do Genoma , Biblioteca Genômica , Família Multigênica , Myxococcales/genética , Myxococcales/metabolismo , Análise de Sequência de DNA/métodos , Transposases/genética
4.
ACS Chem Biol ; 12(7): 1805-1812, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28467833

RESUMO

The cloning of microbial natural product biosynthetic gene clusters and their heterologous expression in a suitable host have proven to be a feasible approach to improve the yield of valuable natural products and to begin mining cryptic natural products in microorganisms. Myxobacteria are a prolific source of novel bioactive natural products with only limited choices of heterologous hosts that have been exploited. Here, we describe the use of Burkholderiales strain DSM 7029 as a potential heterologous host for the functional expression of myxobacterial secondary metabolites. Using a newly established electroporation procedure, the 56 kb epothilone biosynthetic gene cluster from the myxobacterium Sorangium cellulosum was introduced into the chromosome of strain DSM 7029 by transposition. Production of epothilones A, B, C, and D was detected despite their yields being low. Optimization of the medium, introduction of the exogenous methylmalonyl-CoA biosynthetic pathway, and overexpression of rare tRNA genes resulted in an approximately 75-fold increase in the total yields of epothilones to 307 µg L-1. These results show that strain DSM 7029 has the potential to produce epothilones with reasonable titers and might be a broadly applicable host for the heterologous expression of other myxobacterial polyketide synthases and nonribosomal peptide synthetases, expediting the process of genome mining.


Assuntos
Produtos Biológicos/metabolismo , Epotilonas/biossíntese , Microbiologia Industrial/métodos , Myxococcales/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroporação , Epotilonas/química , Epotilonas/genética , Estrutura Molecular , Myxococcales/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Regulação para Cima
5.
Microb Cell Fact ; 14: 105, 2015 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-26194479

RESUMO

BACKGROUND: Exotic genes, especially clustered multiple-genes for a complex pathway, are normally integrated into chromosome for heterologous expression. The influences of insertion sites on heterologous expression and allotropic expressions of exotic genes on host remain mostly unclear. RESULTS: We compared the integration and expression efficiencies of single and multiple exotic genes that were inserted into Myxococcus xanthus genome by transposition and attB-site-directed recombination. While the site-directed integration had a rather stable chloramphenicol acetyl transferase (CAT) activity, the transposition produced varied CAT enzyme activities. We attempted to integrate the 56-kb gene cluster for the biosynthesis of antitumor polyketides epothilones into M. xanthus genome by site-direction but failed, which was determined to be due to the insertion size limitation at the attB site. The transposition technique produced many recombinants with varied production capabilities of epothilones, which, however, were not paralleled to the transcriptional characteristics of the local sites where the genes were integrated. Comparative transcriptomics analysis demonstrated that the allopatric integrations caused selective changes of host transcriptomes, leading to varied expressions of epothilone genes in different mutants. CONCLUSIONS: With the increase of insertion fragment size, transposition is a more practicable integration method for the expression of exotic genes. Allopatric integrations selectively change host transcriptomes, which lead to varied expression efficiencies of exotic genes.


Assuntos
Myxococcus xanthus/genética , Transcriptoma , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Epotilonas/genética , Epotilonas/metabolismo , Expressão Gênica , Mutagênese Insercional , Myxococcus xanthus/metabolismo
6.
ACS Synth Biol ; 3(10): 759-72, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-23654254

RESUMO

Natural products of microbial origin continue to be an important source of pharmaceuticals and agrochemicals exhibiting potent activities and often novel modes of action. Due to their inherent structural complexity chemical synthesis is often hardly possible, leaving fermentation as the only viable production route. In addition, the pharmaceutical properties of natural products often need to be optimized for application by sophisticated medicinal chemistry and/or biosynthetic engineering. The latter requires a detailed understanding of the biosynthetic process and genetic tools to modify the producing organism that are often unavailable. Consequently, heterologous expression of complex natural product pathways has been in the focus of development over recent years. However, piecing together existing DNA cloned from natural sources and achieving efficient expression in heterologous circuits represent several limitations that can be addressed by synthetic biology. In this work we have redesigned and reassembled the 56 kb epothilone biosynthetic gene cluster from Sorangium cellulosum for expression in the high GC host Myxococcus xanthus. The codon composition was adapted to a modified codon table for M. xanthus, and unique restriction sites were simultaneously introduced and others eliminated from the sequence in order to permit pathway assembly and future interchangeability of modular building blocks from the epothilone megasynthetase. The functionality of the artificial pathway was demonstrated by successful heterologous epothilone production in M. xanthus at significant yields that have to be improved in upcoming work. Our study sets the stage for future engineering of epothilone biosynthesis and production optimization using a highly flexible assembly strategy.


Assuntos
Epotilonas/biossíntese , Genes Sintéticos , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Algoritmos , Vias Biossintéticas/genética , Biotecnologia , Códon/genética , Epotilonas/química , Epotilonas/genética , Engenharia Genética , Família Multigênica , Myxococcales/genética , Myxococcales/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Policetídeos/metabolismo , Biologia Sintética
7.
Appl Microbiol Biotechnol ; 97(15): 6857-66, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23549746

RESUMO

The epothilones, compounds with anticancer mechanisms similar to that of paclitaxel (Taxol), are produced by strains of the myxobacterium Sorangium cellulosum, and the gene cluster responsible for epothilone biosynthesis is organised as a large operon. In this work, we showed that the 440-bp promoter regions of the operons from eight S. cellulosum strains have 94.27 % DNA sequence identity and 50 % variability in promoter activity in Escherichia coli. A primer extension analysis revealed two transcriptional start sites (TSSs) at 246 (TSS1) and 193 bp (TSS2) upstream of the translation start site (TLS), respectively. Promoter truncation determined that the basal promoter from the So0157-2 strain is located within a 264-bp region containing weak promoter activity; whereas in the 38-bp region upstream, the 264-bp promoter was required for the strong promoter activity, which was dramatically increased by 11-fold in average. There was a conserved stem-loop structure between TSS2 and the TLS, which was identified in E. coli as a negative regulatory element. In addition, the upstream non-conserved 357-bp non-coding region contributes to the promoter activity, increasing it by 1.5-fold. In conclusion, the expression of the epothilone operon non-coding region in E. coli is regulated by a double promoter (with -35 and -10 regions and two distinct TSSs), a stem-loop structure, and a distal non-coding region.


Assuntos
Epotilonas/genética , Escherichia coli/genética , Myxococcales/genética , Óperon , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Primers do DNA , Epotilonas/química , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Transcrição Gênica
8.
Sci Rep ; 1: 141, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22355658

RESUMO

We describe a site-specific recombination-based tandem assembly (SSRTA) method for reconstruction of biological parts in synthetic biology. The system was catalyzed by Streptomyces phage φBT1 integrase, which belongs to the large serine recombinase subfamily. This one-step approach was efficient and accurate, and able to join multiple DNA molecules in vitro in a defined order. Thus, it could have applications in constructing metabolic pathways and genetic networks.


Assuntos
Epotilonas/biossíntese , Epotilonas/genética , Família Multigênica , Recombinação Genética , Bacteriófagos/enzimologia , Redes Reguladoras de Genes , Integrases/metabolismo , Redes e Vias Metabólicas , Streptomyces/virologia , Biologia Sintética/métodos
9.
J Microbiol Biotechnol ; 18(8): 1416-22, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18756102

RESUMO

Epothilone and its analogs are a potent new class of anticancer compounds produced by myxobacteria. Thus, in an effort to identify new myxobacterial strains producing epothilone and its analogs, cellulose-degrading myxobacteria were isolated from Korean soils, and 13 strains carrying epothilone biosynthetic gene homologs were screened using a polymerase chain reaction. A migration assay revealed that Sorangium cellulosum KYC3013, 3016, 3017, and 3018 all produced microtubule-stabilizing compounds, and an LCMS/ MS analysis showed that S. cellulosum KYC3013 synthesized epothilone A.


Assuntos
Epotilonas/genética , Myxococcales/genética , Myxococcales/isolamento & purificação , Microbiologia do Solo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Celulose/metabolismo , Cromatografia Líquida , DNA Bacteriano/química , DNA Bacteriano/genética , Epotilonas/biossíntese , Humanos , Microscopia de Interferência , Dados de Sequência Molecular , Myxococcales/metabolismo , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Alinhamento de Sequência , Espectrometria de Massas em Tandem , Moduladores de Tubulina/isolamento & purificação , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacologia
10.
Chem Biol ; 11(11): 1533-42, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15556004

RESUMO

Epothilone (Epo) D, an antitumor agent currently in clinical trials, is a hybrid natural product produced by the combined action of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). In the epothilone biosynthetic pathway, EpoB, a 165 kDa NRPS is inserted into an otherwise entirely PKS assembly line, forming two hybrid NRPS-PKS interfaces. In light of the terminal linker effect previously identified in PKS, the N- and C-terminal sequences of EpoB were examined for their roles in propagating the incipient natural product. Eight amino acid residues at EpoB C terminus, in which six are positively charged, were found to be a key component of the C-terminal linker effect. A minimal sequence of 56 residues at EpoB N terminus was required for elongating the acetyl group from the acyl carrier protein (ACP) of EpoA to form methylthiazolyl-S-EpoB.


Assuntos
Epotilonas/biossíntese , Peptídeo Sintases/biossíntese , Policetídeo Sintases/biossíntese , Proteína de Transporte de Acila/genética , Sequência de Aminoácidos , Antineoplásicos/química , Antineoplásicos/metabolismo , Sequência de Bases , Epotilonas/química , Epotilonas/classificação , Epotilonas/genética , Células Híbridas/química , Células Híbridas/metabolismo , Dados de Sequência Molecular , Mutação , Elongação Traducional da Cadeia Peptídica , Peptídeo Sintases/metabolismo , Policetídeo Sintases/metabolismo , Deleção de Sequência
11.
J Am Chem Soc ; 126(24): 7436-7, 2004 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-15198579

RESUMO

Engineered biosynthetic pathways provide a powerful method for generating complex molecules. Precursor-directed biosynthesis, which combines chemical synthesis and enzymatic transformations, allows non-native starting materials to be incorporated into biosynthetic pathways. Using this approach, we achieved the production of the anticancer agent epothilone C in Escherichia coli. An E. coli strain was engineered to express the last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF) and the substrate required to complement the biosynthetic enzymes was obtained by chemical synthesis. Under high-density cell culture conditions, the E. coli strain processed exogenously fed synthetic substrate into epothilone C at levels comparable to the native host (1 mg/L) and at higher levels than other heterologous hosts. Importantly, this precursor-directed approach will allow chemical modifications to be introduced into the polyketide backbone and may ultimately provide access to epothilone analogues with improved pharmacological properties in quantities sufficient for clinical development.


Assuntos
Epotilonas/biossíntese , Escherichia coli/metabolismo , Complexos Multienzimáticos/biossíntese , Precursores de Proteínas/fisiologia , Antibióticos Antineoplásicos/análise , Antibióticos Antineoplásicos/biossíntese , Epotilonas/análise , Epotilonas/genética , Escherichia coli/genética , Estrutura Molecular , Complexos Multienzimáticos/genética
12.
J Bacteriol ; 185(23): 6870-82, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14617651

RESUMO

Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy. Recent work has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer. To search for the genetic basis of virulence in M. ulcerans, we took advantage of the close genetic relationship between M. ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M. ulcerans with M. marinum. We identified several DNA fragments specific to M. ulcerans, in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement. We postulate that this locus is responsible for the synthesis of mycolactone in M. ulcerans.


Assuntos
Epotilonas/genética , Complexos Multienzimáticos/genética , Mycobacterium/genética , Sequência de Aminoácidos , Toxinas Bacterianas , Southern Blotting , Epotilonas/análise , Humanos , Macrolídeos , Dados de Sequência Molecular , Complexos Multienzimáticos/análise , Mycobacterium/enzimologia , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase , Alinhamento de Sequência
13.
Front Biosci ; 8: c1-13, 2003 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-12456305

RESUMO

Modular polyketide synthases (PKS) are large multifunctional enzymes that synthesize complex polyketides, a therapeutically important class of natural products. The linear order and composition of catalytic sites that comprise the PKS represent a "code" that determines the identity of the polyketide product. By re-programming the PKS through genetic engineering, it is possible to alter the code in a predictable manner to create specific structural modifications of polyketides and to produce new libraries of these natural products.


Assuntos
Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Engenharia de Proteínas/métodos , Sequência de Carboidratos/genética , Epotilonas/química , Epotilonas/genética , Eritromicina/química , Eritromicina/metabolismo , Estrutura Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/fisiologia , Peptídeos/química , Peptídeos/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Sirolimo/química , Sirolimo/metabolismo
14.
Antimicrob Agents Chemother ; 46(9): 2772-8, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12183227

RESUMO

Epothilones are potential anticancer drugs that stabilize microtubules in a manner similar to paclitaxel (Taxol). Epothilones are produced from the myxobacterium Sorangium cellulosum, which has a 16-h doubling time and produces only milligram-per-liter amounts of epothilone A and epothilone B. Furthermore, genetic manipulation of S. cellulosum is difficult. To produce epothilones in a more genetically amenable and rapidly growing host, we chose the closely related and best-characterized myxobacteria Myxococcus xanthus. We inserted 65.4 kb of S. cellulosum DNA that encompassed the entire epothilone gene cluster into the chromosome of M. xanthus by a series of homologous recombination events. The resulting strain produced epothilones A and B. Construction of a strain that contained a mutation in epoK, the P450 epoxidase, resulted in production of epothilones C and D.


Assuntos
Epotilonas/biossíntese , Epotilonas/genética , Regulação Bacteriana da Expressão Gênica/genética , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , Meios de Cultura , Sistema Enzimático do Citocromo P-450/genética , DNA Bacteriano/genética , Escherichia coli/genética , Família Multigênica/genética , Mutação/genética , Oxirredutases/genética , Plasmídeos/genética , Engenharia de Proteínas
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