RESUMEN
Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.
Asunto(s)
Sistema Libre de Células , Regiones Promotoras Genéticas , Streptomyces griseus/enzimología , Streptomyces griseus/genética , Streptomyces griseus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Familia de Multigenes , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Codón/genética , AciltransferasasRESUMEN
Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.
Asunto(s)
Antibiosis , Proteínas Bacterianas , Toxinas Bacterianas , Streptomyces , Antibacterianos/biosíntesis , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antibiosis/efectos de los fármacos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Proteínas Bacterianas/ultraestructura , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacología , Microscopía por Crioelectrón , Lectinas/química , Lectinas/genética , Lectinas/metabolismo , Lectinas/ultraestructura , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Streptomyces/química , Streptomyces/efectos de los fármacos , Streptomyces/genética , Streptomyces/crecimiento & desarrollo , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Streptomyces griseus/efectos de los fármacos , Streptomyces griseus/genética , Streptomyces griseus/crecimiento & desarrollo , Streptomyces griseus/metabolismoRESUMEN
Tetrapetalones make up a unique class of pentaketide ansamycins that feature a tetracyclic skeleton and exhibit potent inhibitory activities against soybean lipoxygenase. However, a detailed biosynthetic route to tetrapetalones has not been published. Herein we report the activation of the tetrapetalones' biosynthetic gene cluster (tpt) in Streptomyces sp. S10 by promoter engineering along with constitutive expression of pathway-specific regulator genes, leading to the discovery of seven new derivatives, tetrapetalones E-K (2-8), and the known tetrapetalone A (1). In vivo gene deletion experiments and heterologous expression of the minimized tpt cluster in Streptomyces albus J1074 suggest that the tetracyclic system of tetrapetalones is probably formed spontaneously, and the regioselective glycosylation of tetrapetalones at the C-9 hydroxy group with d-rhamnose or d-rhodinose was catalyzed by the glycosyltransferase Tpt14.
Asunto(s)
Streptomyces griseus , Streptomyces griseus/genética , Familia de Multigenes , Glicosilación , Glycine maxRESUMEN
Fidaxomicin (Dificid) is a commercial macrolide antibiotic for treating Clostridium difficile infection. Total synthesis of fidaxomicin and its aglycone had been achieved through different synthetic schemes. In this study, an alternative biological route to afford the unique 18-membered macrolactone aglycone of fidaxomicin was developed. The promoter refactored fidaxomicin biosynthetic gene cluster from Dactylosporangium aurantiacum was expressed in the commonly used host Streptomyces albus J1074, thereby delivering five structurally diverse fidaxomicin aglycones with the corresponding titers ranging from 4.9 to 15.0 mg L-1. In general, these results validated a biological strategy to construct and diversify fidaxomicin aglycones on the basis of promoter refactoring and heterologous expression.
Asunto(s)
Antibacterianos , Streptomyces griseus , Fidaxomicina , Macrólidos/metabolismo , Streptomyces griseus/genética , Familia de Multigenes , AminoglicósidosRESUMEN
Septacidin is an adenine nucleoside antibiotic with antifungal and antitumor activities. During the efforts to construct a better septacidin producer, we obtained a high yield strain S. albus 1597 by putting the biosynthetic gene cluster (BGC) of septacidin under the control of the constitutive strong promoter ermE*. S. albus 1597 could produce new septacidin congeners SEP-538 and SEP-552 with shorter fatty acyl chains. Moreover, SEP-624 with an unprecedented hydroxylated fatty acyl chain was also isolated from this titre improved strain, enriching the diversity of septacidins. SEP-552 showed moderate inhibitory effects against Epidermophyton floccosum 57312 with MIC value 62.5 µM, while SEP-538 and SEP-624 only exhibited weak antifungal activities. The structure-activity relationship investigation revealed that the antifungal activity of septacidins is significantly influenced by the length of and the decoration on their fatty acyl chains.
Asunto(s)
Streptomyces/genética , Antibacterianos/biosíntesis , Epidermophyton/genética , Familia de Multigenes/genética , Nucleósidos de Purina/genética , Streptomyces griseus/genéticaRESUMEN
Indolizidine alkaloids, which have versatile bioactivities, are produced by various organisms. Although the biosynthesis of some indolizidine alkaloids has been studied, the enzymatic machinery for their biosynthesis in Streptomyces remains elusive. Here, we report the identification and analysis of the biosynthetic gene cluster for iminimycin, an indolizidine alkaloid with a 6-5-3 tricyclic system containing an iminium cation from Streptomyces griseus. The gene cluster has 22 genes, including four genes encoding polyketide synthases (PKSs), which consist of eight modules in total. Inâ vitro analysis of the first module revealed that its acyltransferase domain selects malonyl-CoA, although predicted to select methylmalonyl-CoA. Inactivation of seven tailoring enzyme-encoding genes and structural elucidation of four compounds accumulated in mutants provided important insights into iminimycin biosynthesis, although some of these compounds appeared to be shunt products. This study expands our knowledge of the biosynthetic machinery of indolizidine alkaloids and the enzymatic chemistry of PKS.
Asunto(s)
Alcaloides/biosíntesis , Familia de Multigenes , Streptomyces griseus/química , Streptomyces griseus/genética , Alcaloides/química , Indolicidinas/química , Conformación Molecular , Streptomyces griseus/metabolismoRESUMEN
In the present study, α-amylase from Streptomyces griseus TBG19NRA1 was amplified, cloned and successfully expressed in E. coli BL21/DE3. Sequence analysis of S. griseus α-amylase (SGAmy) revealed the presence of four domains (A, B, C and E). Alpha-amylases with E domain (also known as carbohydrate binding module 20 (CBM20)) are capable of degrading raw starch and this property holds great potential for application in starch processing industries. Though α-amylase is a well-studied and characterized enzyme, there is no experimental structure available for this four domain-containing α-amylases. To gain more insight about SGAmy structure and function, homology modelling was performed using a multi-template method. The template α-amylase from Pseudoalteromonas haloplanktis (PDB ID 1AQH) and E domain of Cyclodextrin glucanotransferase from Bacillus circulans (PDB ID 1CGY) was found to have significant similarity with the complete target sequence of SGAmy. Therefore, homology model for SGAmy was generated from the crystal structure of 1AQH and 1CGY and the resulting structure was subjected to 10 ns molecular dynamics (MD) simulation. Remarkably, CBM20 domain of SGAmy showed greater flexibility in MD simulation than other three domains. This observation is highly rational as this part of SGAmy is strongly implicated in substrate (raw starch) binding. Thus, conformational plasticity at CBM20 is functionally beneficial.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Streptomyces griseus , alfa-Amilasas , Secuencia de Aminoácidos , Bacillus , Clonación Molecular , Escherichia coli/genética , Simulación de Dinámica Molecular , Pseudoalteromonas , Streptomyces griseus/genética , Streptomyces griseus/metabolismo , alfa-Amilasas/genética , alfa-Amilasas/metabolismoRESUMEN
Lytic polysaccharide monooxygenases (LPMOs) are redox-active enzymes that cleave insoluble polysaccharides by an oxidative reaction. In the present study, we have characterized four recombinant putative chitin-active LPMOs from Streptomyces griseus (SgLPMO10B, -C, -D, and -F) and evaluated their potential in enhancing hydrolysis of α- and ß-chitin by three families of 18 chitinases of Serratia marcescens, SmChiA, -B, and -C. All four recombinant SgLPMO10s showed oxidative activity toward both α- and ß-chitin but exhibited different abilities to promote the release of chitobiose from chitin by chitinases depending on both the chitinase and the chitin type. These effects were observed under conditions where the amount of LPMO in the reaction was not rate-limiting, showing that the observed functional differences relate to different abilities of the LPMOs to interact with and act on the substrate. These results show that four seemingly similar LPMOs carrying out the same reaction, cleavage of chitin by C1 oxidation, may have different roles in natural chitin conversion, which provides a rationale for the multiplicity of these enzymes within the same organism. The ability of the LPMOs to act on more natural substrates was demonstrated by showing that SgLPMO10B improved chitin solubilization in dried powdered shrimp shells.
Asunto(s)
Quitina , Oxigenasas de Función Mixta , Streptomyces griseus , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Polisacáridos , Streptomyces griseus/genética , Especificidad por SustratoRESUMEN
Frigocyclinone is a novel antibiotic with antibacterial and anticancer activities. It is produced by both Antarctica-derived Streptomyces griseus NTK 97 and marine sponge-associated Streptomyces sp. M7_15. Here, we first report the biosynthetic gene cluster of frigocyclinone in the S. griseus NTK 97. The frigocyclinone gene cluster spans a DNA region of 33-kb which consists of 30 open reading frames (ORFs), encoding minimal type II polyketide synthase, aromatase and cyclase, redox tailoring enzymes, sugar biosynthesis-related enzymes, C-glycosyltransferase, a resistance protein, and three regulatory proteins. Based on the bioinformatic analysis, a biosynthetic pathway for frigocyclinone was proposed. Second, to verify the cloned gene cluster, CRISPR-Cpf1 mediated gene disruption was conducted. Mutant with the disruption of beta-ketoacyl synthase encoding gene frig20 fully loses the ability of producing frigocyclinone, while inactivating the glycosyltransferase gene frig1 leads to the production of key intermediate of anti-MRSA anthraquinone tetrangomycin.
Asunto(s)
Antraquinonas/metabolismo , Familia de Multigenes/genética , Streptomyces griseus/genética , Streptomyces griseus/metabolismo , Clonación Molecular , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Streptomyces griseus/enzimologíaRESUMEN
Here, we investigate an unusual antiviral mechanism developed in the bacterium Streptomyces griseus SgrAI is a type II restriction endonuclease that forms run-on oligomer filaments when activated and possesses both accelerated DNA cleavage activity and expanded DNA sequence specificity. Mutations disrupting the run-on oligomer filament eliminate the robust antiphage activity of wild-type SgrAI, and the observation that even relatively modest disruptions completely abolish this anti-viral activity shows that the greater speed imparted by the run-on oligomer filament mechanism is critical to its biological function. Simulations of DNA cleavage by SgrAI uncover the origins of the kinetic advantage of this newly described mechanism of enzyme regulation over more conventional mechanisms, as well as the origin of the sequestering effect responsible for the protection of the host genome against damaging DNA cleavage activity of activated SgrAI.IMPORTANCE This work is motivated by an interest in understanding the characteristics and advantages of a relatively newly discovered enzyme mechanism involving filament formation. SgrAI is an enzyme responsible for protecting against viral infections in its host bacterium and was one of the first such enzymes shown to utilize such a mechanism. In this work, filament formation by SgrAI is disrupted, and the effects on the speed of the purified enzyme as well as its function in cells are measured. It was found that even small disruptions, which weaken but do not destroy filament formation, eliminate the ability of SgrAI to protect cells from viral infection, its normal biological function. Simulations of enzyme activity were also performed and show how filament formation can greatly speed up an enzyme's activation compared to that of other known mechanisms, as well as to better localize its action to molecules of interest, such as invading phage DNA.
Asunto(s)
Bacteriófagos/genética , ADN Viral/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Streptomyces griseus/virología , Bacteriófagos/crecimiento & desarrollo , Secuencia de Bases/genética , ADN Viral/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Activación Enzimática , Streptomyces griseus/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Diazofluorene compounds such as kinamycin and lomaiviticin feature unique molecular structures and compelling medicinal bioactivities. However, a complete understanding of the biosynthetic details for this family of natural products has yet to be fully elucidated. In addition, a lack of genetically and technically amenable production hosts has limited access to the full medicinal potential of these compounds. Here, we report the capture of the complete kinamycin gene cluster from Streptomyces galtieri Sgt26 by bacterial artificial chromosome cloning, confirmed by successful production of kinamycin in the heterologous host Streptomyces albus J1074. Sequence analysis and a series of gene deletion experiments revealed the boundary of the cluster, which spans 75 kb DNA. To probe the last step in biosynthesis, acetylation of kinamcyin F to kinamycin D, gene knockout, and complementation experiments identified a single gene product involved with final acetylation conversions. This study provides full genetic information for the kinamycin gene cluster from S. galtieri Sgt26 and establishes heterologous biosynthesis as a production platform for continued mechanistic assessment of compound formation and utilization.
Asunto(s)
Productos Biológicos/metabolismo , Streptomyces griseus/genética , Eliminación de Gen , Familia de Multigenes/genéticaRESUMEN
BACKGROUND/AIMS: A-factor, a γ-butyrolactone autoregulator, in Streptomyces griseus is involved in the regulation of differentiation and antibiotic production. Here we studied the S. griseus B2682-AFN (A-factor negative) bald mutant that harbors a nonsense mutation in the afsR gene encoding a pleiotropic regulator. Our aim was to prove that this mutation is the cause of the A-factor deficiency in AFN. We also studied whether AfsR regulates A-factor production by AfsA, which is supposed to be the only specific key enzyme in A-factor biosynthesis. METHODS: Wild afsR was cloned to the pHJL401 shuttle vector and was transformed to the S. griseus AFN and B2682 strains. During phenotypic characterization, sporulation, antibiotic, protease, A-factor, and AfsA protein production were studied. RESULTS: Transformation of AFN by a wild afsR restored its phenotype including sporulation, antibiotic, extracellular protease, and A-factor production. Introduction of afsR to the B2682 wild-type strain resulted in antibiotic and extracellular protease overproduction that was accompanied with an elevated A-factor level. AfsA was detected both in AFN and B2682. CONCLUSIONS: AfsR has an effect on the regulation of A-factor production in S. griseus. The presence of AfsA is not sufficient for normal A-factor production. AfsR regulates A-factor biosynthesis independently of AfsA.
Asunto(s)
4-Butirolactona/análogos & derivados , Proteínas Bacterianas/genética , Mutación , Streptomyces griseus/genética , Streptomyces griseus/metabolismo , 4-Butirolactona/biosíntesis , Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos/genética , Péptido Hidrolasas/metabolismo , Fenotipo , Streptomyces griseus/crecimiento & desarrollo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transformación BacterianaRESUMEN
Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven to be a powerful analytical tool for the characterization of modified ribonucleic acids (RNAs). The typical approach for analyzing modified nucleosides within RNA sequences by mass spectrometry involves ribonuclease digestion followed by LC-MS/MS analysis and data interpretation. Here we describe a new software tool, RNAModMapper (RAMM), to assist in the interpretation of LC-MS/MS data. RAMM is a stand-alone package that requires user-submitted DNA or RNA sequences to create a local database against which collision-induced dissociation (CID) data of modified oligonucleotides can be compared. RAMM can interpret MS/MS data containing modified nucleosides in two modes: fixed and variable. In addition, RAMM can also utilize interpreted MS/MS data for RNA modification mapping back against the input sequence(s). The applicability of RAMM was first tested using total tRNA isolated from Escherichia coli. It was then applied to map modifications found in 16S and 23S rRNA from Streptomyces griseus.
Asunto(s)
ARN/análisis , Programas Informáticos , Espectrometría de Masas en Tándem , Área Bajo la Curva , Cromatografía Líquida de Alta Presión , Bases de Datos Factuales , Nucleósidos/química , ARN/metabolismo , ARN Ribosómico 16S/análisis , ARN Ribosómico 16S/metabolismo , ARN Ribosómico 23S/análisis , ARN Ribosómico 23S/metabolismo , Curva ROC , Ribonucleasa T1/metabolismo , Streptomyces griseus/genéticaRESUMEN
Streptomyces griseus S4-7 was originally isolated from the strawberry rhizosphere as a microbial agent responsible for Fusarium wilt suppressive soils. S. griseus S4-7 shows specific and pronounced antifungal activity against Fusarium oxysporum f. sp. fragariae. In the Streptomyces genus, the whi transcription factors are regulators of sporulation, cell differentiation, septation, and secondary metabolites production. wblE2 function as a regulator has emerged as a new group in whi transcription factors. In this study, we reveal the involvement of the wblE2 transcription factor in the plant-protection by S. griseus S4-7. We generated ΔwblE, ΔwblE2, ΔwhiH, and ΔwhmD gene knock-out mutants, which showed less antifungal activity both in vitro and in planta. Among the mutants, wblE2 mutant failed to protect the strawberry against the Fusarium wilt pathogen. Transcriptome analyses revealed major differences in the regulation of phenylalanine metabolism, polyketide and siderophore biosynthesis between the S4-7 and the wblE2 mutant. The results contribute to our understanding of the role of streptomycetes wblE2 genes in a natural disease suppressing system.
Asunto(s)
Antibiosis , Proteínas Bacterianas , Enfermedades de las Plantas/microbiología , Plantas/microbiología , Streptomyces griseus/genética , Streptomyces griseus/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Antiinfecciosos/metabolismo , Análisis por Conglomerados , Biología Computacional/métodos , Resistencia a la Enfermedad , Expresión Génica , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Genoma Bacteriano , Mutación , Fenotipo , Raíces de Plantas/microbiología , Plásmidos/genética , Streptomyces griseus/crecimiento & desarrollo , Streptomyces griseus/ultraestructura , TranscriptomaRESUMEN
In order to produce enantiomerically pure epoxides for the synthesis of value-added chemicals, a novel putative epoxide hydrolase (EH) sgeh was cloned and overexpressed in pET28a/Escherichia coli BL21(DE3). The 1047 bp sgeh gene was mined from Streptomyces griseus NBRC 13350 genome sequence. The recombinant hexahistidyl-tagged SGEH was purified (16.6-fold) by immobilized metal-affinity chromatography, with 90% yield as a homodimer of 100 kDa. The recombinant E. coli whole cells overexpressing SGEH could kinetically resolve racemic phenyl glycidyl ether (PGE) into (R)-PGE with 98% ee, 40% yield, and enantiomeric ratio (E) of 20. This was achieved under the optimized reaction conditions i.e. cell/substrate ratio of 20:1 (w/w) at pH 7.5 and 20 °C in 10% (v/v) dimethylformamide (DMF) in a 10 h reaction. 99% enantiopure (R)-PGE was obtained when the reaction time was prolonged to 12 h with a yield of 34%. In conclusion, an economically viable and environment friendly green process for the production of enantiopure (R)-PGE was developed by using wet cells of E. coli expressing recombinant SGEH.
Asunto(s)
Epóxido Hidrolasas/metabolismo , Éteres Fenílicos/metabolismo , Streptomyces griseus/enzimología , Cromatografía en Gel , Clonación Molecular , Epóxido Hidrolasas/genética , Escherichia coli/genética , Cinética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Streptomyces griseus/genética , Especificidad por SustratoRESUMEN
A novel ß-glucosidase from Streptomyces griseus was cloned and overexpressed in E. coli. The purified ß-glucosidase (44 kDa) had a Km of 8.6 ± 0.5 mM and a Vmax of 217 ± 5.0 µmoles-1min-1mg at 37 °C, pH 7.2 with p-nitrophenyl-ß-D glucopyranoside as substrate. The enzyme was characterised in terms of pH optimum (pH 6.9), temperature optimum (69 °C) and the influence of solvents and effectors. Purified S. griseus ß-glucosidase was successfully immobilised, by simple absorption, onto zinc oxide (ZnO) nanoparticles without covalent modification. It remained tightly bound even after extensive washing and could be reused up to ten times without significant loss of activity. The immobilised enzyme had a higher optimum temperature and greater thermostability than the free enzyme. In immobilised form the enzyme readily catalysed the synthesis of alkyl glucosides.
Asunto(s)
Proteínas Bacterianas , Glucósidos/síntesis química , Nanopartículas/química , Streptomyces griseus/genética , beta-Glucosidasa , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Glucósidos/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Streptomyces griseus/enzimología , beta-Glucosidasa/biosíntesis , beta-Glucosidasa/química , beta-Glucosidasa/genética , beta-Glucosidasa/aislamiento & purificaciónRESUMEN
AmfS, a class III lantipeptide serves as a morphogen in Streptomyces griseus. Here, we constructed a high production system of AmfS in S. griseus. We isolated S. griseus Grd1 strain defective in glucose repression of aerial mycelium formation and found it suitable for the overproduction of AmfS. Two expression vectors carrying the strong and constitutive ermE2 promoter were constructed using a multicopy number plasmid, pIJ702. The use of the Grd1 strain combined with the expression vectors enabled high production of AmfS by S. griseus into its culture broth. The expression system was also effective for the generation of abundant AmfS derived from Streptomyces avermitilis. In addition, site-directed mutagenesis revealed the amino acid residues essential for the morphogen activity of AmfS. These results indicate that the constructed system enables efficient production of class III lantipeptides by Streptomyces.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Ingeniería Genética , Streptomyces griseus/genética , Streptomyces griseus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vectores Genéticos/genética , Familia de Multigenes/genética , MutaciónRESUMEN
The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of enzymes in reaction, economic, ecological issues. Many carbonyl reductases for producing chiral alcohols have been reported but there is still a lack of good catalytic efficacies. Herein, five carbonyl reductases from different Streptomyces were discovered by the strategy of genome mining. These reductases were overexpressed, and we chose SgCR for further study as it owned better enzyme activity. This protein was purified to apparent homogeneity, and its amino acid sequence was analyzed in comparison with that of the reported SDRs. The biocatalytic properties of SgCR were investigated, and this enzyme was confirmed to have the ability to convert various prochiral ketones into highly optically active alcohols. SgCR exhibited the highest activity towards ethyl 4-chloro-3-oxobutanoate (COBE) and the corresponding product ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) was obtained with high yield and excellent e.e. value by optimizing the biphasic system. Eventually, using isopropanol as the co-substrate for NADH recycling in the substrate-coupled reaction, the yield and enantioselectivity of (S)-CHBE were obtained at the values of 90% and 99%, respectively. These results indicate that SgCR is a promising boicatalyst for the synthesis of chiral alcohols in industry.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Alcoholes/metabolismo , Proteínas Bacterianas/metabolismo , Streptomyces griseus/enzimología , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Alcoholes/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Minería de Datos , Estabilidad de Enzimas , Genoma Bacteriano , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Estereoisomerismo , Streptomyces griseus/genética , Especificidad por SustratoRESUMEN
We screened for a gene that inhibits streptomycin production in Streptomyces griseus when it is introduced on a high-copy-number plasmid pIJ702, and obtained a plasmid pKM545. The introduction of pKM545 abolished streptomycin production on all media tested including YMP-sugar and Nutrient broth. S1 protection analysis demonstrated that the introduction of this plasmid downregulated the transcriptional activity of the promoter preceding strR, the pathway-specific transcriptional regulator for streptomycin biosynthesis. The 2.8-kb BamHI fragment cloned onto pKM545 contained two coding sequences SGR_5442 and 5443. These coding sequences and the two downstream ones (SGR_5444 and 5445) constituted a possible operon structure designated to be rspABCD (regulation of streptomycin production). RspB and RspC exhibited a marked similarity with an ATP-binding domain and a membrane-associating domain of an ABC-2 type transporter, respectively, suggesting that the Rsp proteins comprise a membrane exporter. The gene cluster consisting of the rsp operon and the upstream divergent small coding sequence (SGR_5441) was widely distributed to Streptomyces genome. An rspB mutant of S. griseus produced 3-fold streptomycin of the parental strain in YMP liquid medium. The evidence implies that the Rsp translocator is involved in the export of a substance that specifies the expression level of streptomycin biosynthesis genes in S. griseus.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Streptomyces griseus/genética , Streptomyces griseus/metabolismo , Estreptomicina/biosíntesis , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Orden Génico , Sitios Genéticos , Mutación , Plásmidos/genética , Dominios y Motivos de Interacción de ProteínasRESUMEN
Isoleucyl-tRNA synthetase (IleRS) is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity. Alongside the typical bacterial IleRS (such as Escherichia coli IleRS), some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. Our phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. We present the analysis of evolutionary constraints of the synthetic and editing reactions in eukaryotic/eukaryote-like IleRSs, which share a common origin but diverged through adaptation to different cell environments. The enzyme from the yeast cytosol exhibits tRNA-dependent pre-transfer editing analogous to E. coli IleRS. This argues for the presence of this proofreading in the common ancestor of both IleRS types and an ancient origin of the synthetic site-based quality control step. Yet surprisingly, the eukaryote-like enzyme from Streptomyces griseus IleRS lacks this capacity; at the same time, its synthetic site displays the 10(3)-fold drop in sensitivity to antibiotic mupirocin relative to the yeast enzyme. The discovery that pre-transfer editing is optional in IleRSs lends support to the notion that the conserved post-transfer editing domain is the main checkpoint in these enzymes. We substantiated this by showing that under error-prone conditions S. griseus IleRS is able to rescue the growth of an E. coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability.