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1.
Microb Cell Fact ; 21(1): 263, 2022 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-36529749

RESUMO

BACKGROUND: Phospholipase D (PLD) is highly valuable in the food and medicine industries, where it is used to convert low-cost phosphatidylcholine into high-value phospholipids (PLs). Despite being overexpressed in Streptomyces, PLD production requires expensive thiostrepton feeding during fermentation, limiting its industrialization. To address this issue, we propose a new thiostrepton-free system. RESULTS: We developed a system using a combinatorial strategy containing the constitutive promoter kasOp* and PLD G215S mutation fused to a signal peptide sigcin of Streptoverticillium cinnamoneum pld. To find a candidate vector, we first expressed PLD using the integrative vector pSET152 and then built three autonomously replicating vectors by substituting Streptomyces replicons to increase PLD expression. According to our findings, replicon 3 with stability gene (sta) inserted had an ideal result. The retention rate of the plasmid pOJ260-rep3-pld* was 99% after five passages under non-resistance conditions. In addition, the strain SK-3 harboring plasmid pOJ260-rep3-pld* produced 62 U/mL (3.48 mg/g) of PLD, which further improved to 86.8 U/mL (7.51 mg/g) at 32 °C in the optimized medium, which is the highest activity achieved in the PLD secretory expression to date. CONCLUSIONS: This is the first time that a thiostrepton-free PLD production system has been reported in Streptomyces. The new system produced stable PLD secretion and lays the groundwork for the production of PLs from fermentation stock. Meanwhile, in the Streptomyces expression system, we present a highly promising solution for producing other complex proteins.


Assuntos
Fosfolipase D , Streptomyces lividans , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo , Tioestreptona/metabolismo , Plasmídeos/genética
2.
Arch Microbiol ; 204(11): 687, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36324009

RESUMO

Antimicrobial proteins and peptides are an alternative to current antibiotics. Here, we report an antimicrobial activity in a low-molecular-weight protein secreted naturally by Streptomyces lividans TK24 when glucose or glycerol were used as carbon sources. The antimicrobial activity was demonstrated against Bacillus subtilis, Bacillus cereus, Kokuria rhizophila, Clostridium sporogenes and Clavibacter michiganensis, causal pathogen of tomato bacterial canker; one of the most destructive bacterial diseases of this crop. The protein fraction with antimicrobial activity was identified and quantified by LC-MS/MS. From a total of 155 proteins, 11 were found to be within the range of 11.3-13.9 kDa of which four proteins were selected by functional analysis as possibly responsible for the antimicrobial activity. Protein fractionation, correlation analysis between antimicrobial activity and abundance of selected proteins, as well as transcriptional expression analysis, indicate that 50S ribosomal protein L19 is the main candidate responsible for antimicrobial activity.


Assuntos
Anti-Infecciosos , Micrococcaceae , Streptomyces lividans , Cromatografia Líquida , Espectrometria de Massas em Tandem , Anti-Infecciosos/farmacologia
3.
J Gen Appl Microbiol ; 68(2): 109-116, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35831135

RESUMO

The aromatic compound 3-amino-4-hydroxybenzoic acid (3,4-AHBA) can be employed as a raw material for high-performance industrial plastics. The aim of this study is to produce 3,4-AHBA via a recombinant Streptomyces lividans strain containing griI and griH genes derived from Streptomyces griseus using culture medium with glucose and/or xylose, which are the main components in lignocellulosic biomass. Production of 3,4-AHBA by the recombinant S. lividans strain was successful, and the productivity was affected by the kind of sugar used as an additional carbon source. Metabolic profiles revealed that L aspartate-4-semialdehyde (ASA), a precursor of 3,4-AHBA, and coenzyme NADPH were supplied in greater amounts in xylose medium than in glucose medium. Moreover, cultivation in TSB medium with a mixed sugar (glucose/xylose) was found to be effective for 3,4-AHBA production, and optimal conditions for efficient production were designed by changing the ratio of glucose to xylose. The best productivity of 2.70 g/L was achieved using a sugar mixture of 25 g/L glucose and 25 g/L xylose, which was 1.5 times higher than the result using 50 g/L glucose alone. These results suggest that Streptomyces is a suitable candidate platform for 3,4-AHBA production from lignocellulosic biomass-derived sugars under appropriate culture conditions.


Assuntos
Streptomyces lividans , Xilose , Aminobenzoatos , Fermentação , Glucose/metabolismo , Hidroxibenzoatos/metabolismo , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , Xilose/metabolismo
4.
PLoS One ; 17(7): e0270379, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35834474

RESUMO

Streptomyces lividans TK23 interacts with mycolic acid-containing bacteria (MACB), such as Tsukamurella pulmonis TP-B0596, and this direct cell contact activates its secondary metabolism (e.g., the production of undecylprodigiosin: RED). Here, we employed carbon (12C5+) ion beam-induced mutagenesis to investigate the signature of induced point mutations and further identify the gene(s) responsible for the production of secondary metabolites induced by T. pulmonis. We irradiated spores of the Streptomyces coelicolor strain JCM4020 with carbon ions to generate a mutant library. We screened the RED production-deficient mutants of S. coelicolor by mixing them with T. pulmonis TP-B0596 on agar plates, identifying the red/white phenotype of the growing colonies. Through this process, we selected 59 RED-deficient mutants from around 152,000 tested spores. We resequenced the genomes of 16 mutants and identified 44 point mutations, which revealed the signatures induced by 12C5+-irradiation. Via gene complementation experiments, we also revealed that two genes-glutamate synthase (gltB) and elongation factor G (fusA)-are responsible for the reduced production of RED.


Assuntos
Streptomyces coelicolor , Antibacterianos/metabolismo , Carbono/metabolismo , Íons/metabolismo , Mutagênese , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Streptomyces lividans/metabolismo
5.
Biosci Biotechnol Biochem ; 86(8): 1122-1127, 2022 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-35648472

RESUMO

We have previously reported a powerful promoter from the Streptomyces cinnamoneus TH-2 strain named "scmp" and created an expression vector of pTONA5a for expression using S. lividans. The full-length scmp promoter sequence consists of 424 bp upstream of a metalloendoprotease gene in the S. cinnamoneus TH-2 genome. The promoter works in the presence of inorganic phosphate and glucose. In this study, we present the essential region of the scmp promoter (promoter C), which lacks 358 bp of the 5' region of the full-length promoter. Promoter C was very short and contained only 63 bp. Using promoter C, we succeeded in the extracellular production of the Streptomyces enzymes of leucine aminopeptidase, ferulic acid esterase, and transglutaminase, which possessed signal peptides for secretion via the type II secretion pathway, at high levels.


Assuntos
Sinais Direcionadores de Proteínas , Streptomyces lividans , Regiões Promotoras Genéticas/genética , Sinais Direcionadores de Proteínas/genética , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , Transglutaminases/metabolismo
6.
J Antibiot (Tokyo) ; 74(9): 593-595, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34234285

RESUMO

A novel angucyclinone, 6,9-dihydroxytetrangulol, was isolated from Streptomyces lividans TK23 transformed with a kinanthraquinone biosynthetic gene cluster in which the kiqO gene was disrupted. The chemical structure was elucidated by spectroscopic analyses. It showed significant antibacterial activities with an IC50 value of 1.9 µM against Staphylococcus aureus and moderate anticancer activities against HL-60 cells.


Assuntos
Antraquinonas/farmacologia , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Streptomyces lividans/metabolismo , Antraquinonas/química , Antraquinonas/isolamento & purificação , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Células HL-60 , Humanos , Concentração Inibidora 50 , Família Multigênica , Staphylococcus aureus/efeitos dos fármacos , Streptomyces lividans/genética
7.
Commun Biol ; 4(1): 647, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059751

RESUMO

Streptomyces are one of the most important industrial microorganisms for the production of proteins and small-molecule drugs. Previously reported flow cytometry-based screening methods can only screen spores or protoplasts released from mycelium, which do not represent the filamentous stationary phase Streptomyces used in industrial cultivation. Here we show a droplet-based microfluidic platform to facilitate more relevant, reliable and rapid screening of Streptomyces mycelium, and achieved an enrichment ratio of up to 334.2. Using this platform, we rapidly characterized a series of native and heterologous constitutive promoters in Streptomyces lividans 66 in droplets, and efficiently screened out a set of engineered promoter variants with desired strengths from two synthetic promoter libraries. We also successfully screened out several hyperproducers of cellulases from a random S. lividans 66 mutant library, which had 69.2-111.4% greater cellulase production than the wild type. Our method provides a fast, simple, and powerful solution for the industrial engineering and screening of Streptomyces in more industry-relevant conditions.


Assuntos
Microfluídica/métodos , Micélio/metabolismo , Streptomyces/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Engenharia Metabólica/métodos , Micélio/fisiologia , Regiões Promotoras Genéticas/genética , Streptomyces/genética , Streptomyces lividans/genética , Streptomyces lividans/metabolismo
8.
Biotechnol Bioeng ; 118(8): 3076-3093, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33974270

RESUMO

Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met-bottromycin A2 up to 109 mg L-1 , the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L-1 talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re-balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of non-cyclized shunt by-products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13-fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline-type oxytetracycline, and the anthramycin-analogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone-type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and synthetic control. Given its simplicity and broad benefits, microparticle-supplementation appears as an enabling technology in natural product research of these most important microbes.


Assuntos
Antibacterianos/biossíntese , Engenharia Metabólica , Microrganismos Geneticamente Modificados , Peptídeos Cíclicos , Streptomyces lividans , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , Streptomyces lividans/genética , Streptomyces lividans/metabolismo
9.
ACS Synth Biol ; 10(5): 1053-1063, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33720688

RESUMO

CRISPR/Cas9-mediated base editors, based on cytidine deaminase or adenosine deaminase, are emerging genetic technologies that facilitate genomic manipulation in many organisms. Since base editing is free from DNA double-strand breaks (DSBs), it has certain advantages, such as a lower toxicity, compared to the traditional DSB-based genome engineering technologies. In terms of Streptomyces, a base editing method has been successfully applied in several model and non-model species, such as Streptomyces coelicolor and Streptomyces griseofuscus. In this study, we first proved that BE2 (rAPOBEC1-dCas9-UGI) and BE3 (rAPOBEC1-nCas9-UGI) were functional base editing tools in Streptomyces lividans 66, albeit with a much lower editing efficiency compared to that of S. coelicolor. Uracil generated in deamination is a key intermediate in the base editing process, and it can be hydrolyzed by uracil DNA glycosidase (UDG) involved in the intracellular base excision repair, resulting in a low base editing efficiency. By knocking out two endogenous UDGs (UDG1 and UDG2), we managed to improve the base editing efficiency by 3.4-67.4-fold among different loci. However, the inactivation of UDG is detrimental to the genome stability and future application of engineered strains. Therefore, we finally developed antisense RNA interference-enhanced CRISPR/Cas9 Base Editing method (asRNA-BE) to transiently disrupt the expression of uracil DNA glycosidases during base editing, leading to a 2.8-65.8-fold enhanced editing efficiency and better genome stability. Our results demonstrate that asRNA-BE is a much better editing tool for base editing in S. lividans 66 and might be beneficial for improving the base editing efficiency and genome stability in other Streptomyces strains.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Interferência de RNA , RNA Antissenso/genética , Streptomyces lividans/enzimologia , Streptomyces lividans/genética , Reparo do DNA/genética , Regulação para Baixo/genética , Técnicas de Inativação de Genes , Loci Gênicos , Genoma Bacteriano , Instabilidade Genômica/genética , Microrganismos Geneticamente Modificados , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Uracila/metabolismo , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo
10.
Appl Microbiol Biotechnol ; 105(5): 2123-2137, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33564923

RESUMO

The bacteria of the genus Streptomyces are among the most important producers of biologically active secondary metabolites. Moreover, recent genomic sequence data have shown their enormous genetic potential for new natural products, although many new biosynthetic gene clusters (BGCs) are silent. Therefore, efficient and stable genome modification techniques are needed to activate their production or to manipulate their biosynthesis towards increased production or improved properties. We have recently developed an efficient markerless genome modification system for streptomycetes based on positive blue/white selection of double crossovers using the bpsA gene from indigoidine biosynthesis, which has been successfully applied for markerless deletions of genes and BGCs. In the present study, we optimized this system for markerless insertion of large BGCs. In a pilot test experiment, we successfully inserted a part of the landomycin BGC (lanFABCDL) under the control of the ermEp* promoter in place of the actinorhodin BGC (act) of Streptomyces lividans TK24 and RedStrep 1.3. The resulting strains correctly produced UWM6 and rabelomycin in twice the yield compared to S. lividans strains with the same construct inserted using the PhiBT1 phage-based integration vector system. Moreover, the system was more stable. Subsequently, using the same strategy, we effectively inserted the entire BGC for mithramycin (MTM) in place of the calcium-dependent antibiotic BGC (cda) of S. lividans RedStrep 1.3 without antibiotic-resistant markers. The resulting strain produced similar levels of MTM when compared to the previously described S. lividans RedStrep 1.3 strain with the VWB phage-based integration plasmid pMTMF. The system was also more stable. KEY POINTS: • Optimized genome editing system for markerless insertion of BGCs into Streptomyces genomes • Efficient heterologous production of MTM in the stable engineered S. lividans strain.


Assuntos
Streptomyces , Cromossomos , Família Multigênica , Plasmídeos/genética , Streptomyces/genética , Streptomyces lividans/genética
11.
Biosci Biotechnol Biochem ; 85(3): 714-721, 2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33590007

RESUMO

Recent advances in genome sequencing have revealed a variety of secondary metabolite biosynthetic gene clusters in actinomycetes. Understanding the biosynthetic mechanism controlling secondary metabolite production is important for utilizing these gene clusters. In this study, we focused on the kinanthraquinone biosynthetic gene cluster, which has not been identified yet in Streptomyces sp. SN-593. Based on chemical structure, 5 type II polyketide synthase gene clusters were listed from the genome sequence of Streptomyces sp. SN-593. Among them, a candidate gene cluster was selected by comparing the gene organization with grincamycin, which is synthesized through an intermediate similar to kinanthraquinone. We initially utilized a BAC library for subcloning the kiq gene cluster, performed heterologous expression in Streptomyces lividans TK23, and identified the production of kinanthraquinone and kinanthraquinone B. We also found that heterologous expression of kiqA, which belongs to the DNA-binding response regulator OmpR family, dramatically enhanced the production of kinanthraquinones.


Assuntos
Antraquinonas/metabolismo , Streptomyces lividans/genética , Antraquinonas/farmacologia , Cromossomos Artificiais Bacterianos , Família Multigênica , Policetídeo Sintases/genética , Regiões Promotoras Genéticas , Streptomyces lividans/metabolismo
12.
Extremophiles ; 25(2): 115-128, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33515353

RESUMO

A gene (estA', 804 bp) from Streptomyces lividans TK24 was artificially synthesized and successfully overexpressed as a 6His-tagged fusion protein in Escherichia coli. It encoded a carboxylesterase (EstA) that composed of 267 amino acids with a predicted molecular weight of 28.56 kDa. Multiple sequence alignment indicated that EstA has typical characteristics of esterases, including a catalytic triad (Ser93-Asp194-His224) and a conserved pentapeptide motif (Gly91-Leu92-Ser93-Met94-Gly95). Simultaneously, phylogenetic analysis indicated that EstA belongs to family VI. Biochemical characterization displayed its optimum enzyme activity was at 55 â„ƒ and pH 8.5. Additionally, EstA exhibited higher activity towards short carbon substrates and showed the outstanding catalytic efficiency for pNPA2 with kcat/Km of 2296.14 ± 10.35 s-1 mM-1. Notably, EstA has hyper-thermostability and good alkali stability. The activity of EstA did not change obviously when incubated at 50 and 100 â„ƒ for 337 and 1 h, independently. Besides, by incubating at 100 â„ƒ for 6 h, EstA remained about half of its initial activity. Moreover, EstA showed stability at pH ranging from 8.0 to 11.0, and about 90% residual enzyme activity was reserved by being treated at pH 8.0 or 9.0 for 80 h, especially. Such multiple features prepare EstA for a potential candidate in the field of biological catalysis of some industrial applications under harsh conditions.


Assuntos
Carboxilesterase , Streptomyces lividans , Álcalis , Sequência de Aminoácidos , Carboxilesterase/genética , Clonagem Molecular , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Filogenia , Streptomyces lividans/genética , Especificidade por Substrato
13.
Biochim Biophys Acta Biomembr ; 1863(3): 183491, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33065136

RESUMO

The membrane environment, including specific lipid characteristics, plays important roles in the folding, stability, and gating of the prokaryotic potassium channel KcsA. Here we study the effect of membrane composition on the population of various functional states of KcsA. The spectra provide support for the previous observation of copurifying phospholipids with phosphoglycerol headgroups. Additional, exogenously added anionic lipids do not appear to be required to stabilize the open conductive conformation of KcsA, which was previously thought to be the case. On the contrary, NMR-based binding studies indicate that including anionic lipids in proteoliposomes at acidic pH leads to a weaker potassium ion affinity at the selectivity filter. Since K+ ion loss leads to channel inactivation, these results suggest that anionic lipids promote channel inactivation.


Assuntos
Proteínas de Bactérias/química , Lipídeos/química , Ressonância Magnética Nuclear Biomolecular , Canais de Potássio/química , Streptomyces lividans/química , Transporte de Íons , Potássio/química
14.
Nat Commun ; 11(1): 5168, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33057011

RESUMO

The potassium ion (K+) channel plays a fundamental role in controlling K+ permeation across the cell membrane and regulating cellular excitabilities. Mutations in the transmembrane pore reportedly affect the gating transitions of K+ channels, and are associated with the onset of neural disorders. However, due to the lack of structural and dynamic insights into the functions of K+ channels, the structural mechanism by which these mutations cause K+ channel dysfunctions remains elusive. Here, we used nuclear magnetic resonance spectroscopy to investigate the structural mechanism underlying the decreased K+-permeation caused by disease-related mutations, using the prokaryotic K+ channel KcsA. We demonstrated that the conformational equilibrium in the transmembrane region is shifted toward the non-conductive state with the closed intracellular K+-gate in the disease-related mutant. We also demonstrated that this equilibrium shift is attributable to the additional steric contacts in the open-conductive structure, which are evoked by the increased side-chain bulkiness of the residues lining the transmembrane helix. Our results suggest that the alteration in the conformational equilibrium of the intracellular K+-gate is one of the fundamental mechanisms underlying the dysfunctions of K+ channels caused by disease-related mutations.


Assuntos
Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico/genética , Canais de Potássio/metabolismo , Potássio/metabolismo , Alanina/genética , Ataxia/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Epilepsia/genética , Humanos , Síndrome do QT Longo/genética , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Canais de Potássio/genética , Canais de Potássio/isolamento & purificação , Conformação Proteica em alfa-Hélice/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Streptomyces lividans , Valina/genética
15.
J Chem Phys ; 153(14): 141103, 2020 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-33086825

RESUMO

Many membrane proteins are modulated by external stimuli, such as small molecule binding or change in pH, transmembrane voltage, or temperature. This modulation typically occurs at sites that are structurally distant from the functional site. Revealing the communication, known as allostery, between these two sites is key to understanding the mechanistic details of these proteins. Residue interaction networks of isolated proteins are commonly used to this end. Membrane proteins, however, are embedded in a lipid bilayer, which may contribute to allosteric communication. The fast diffusion of lipids hinders direct use of standard residue interaction networks. Here, we present an extension that includes cofactors such as lipids and small molecules in the network. The novel framework is applied to three membrane proteins: a voltage-gated ion channel (KCNQ1), a G-protein coupled receptor (GPCR-ß2 adrenergic receptor), and a pH-gated ion channel (KcsA). Through systematic analysis of the obtained networks and their components, we demonstrate the importance of lipids for membrane protein allostery. Finally, we reveal how small molecules may stabilize different protein states by allosterically coupling and decoupling the protein from the membrane.


Assuntos
Regulação Alostérica , Membrana Celular/metabolismo , Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Camelídeos Americanos , Membrana Celular/química , Escherichia coli/química , Canal de Potássio KCNQ1/química , Camundongos , Simulação de Dinâmica Molecular , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Receptores Adrenérgicos beta 2/química , Streptomyces lividans/química
16.
ACS Chem Biol ; 15(9): 2551-2557, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32786260

RESUMO

The ever-growing drug resistance problem worldwide highlights the urgency to discover and develop new drugs. Microbial natural products are a prolific source of drugs. Genome sequencing has revealed a tremendous amount of uncharacterized natural product biosynthetic gene clusters (BGCs) encoded within microbial genomes, most of which are cryptic or express at very low levels under standard culture conditions. Therefore, developing effective strategies to awaken these cryptic BGCs is of great interest for natural product discovery. In this study, we designed and validated a Transcription-Translation in One (TTO) approach for activation of cryptic BGCs. This approach aims to alter the metabolite profiles of target strains by directly overexpressing exogenous rpsL (encoding ribosomal protein S12) and rpoB (encoding RNA polymerase ß subunit) genes containing beneficial mutations for natural product production using a plug-and-play plasmid system. As a result, this approach bypasses the tedious screening work and overcomes the false positive problem in the traditional ribosome engineering approach. In this work, the TTO approach was successfully applied to activating cryptic BGCs in three Streptomyces strains, leading to the discovery of two aromatic polyketide antibiotics, piloquinone and homopiloquinone. We further identified a single BGC responsible for the biosynthesis of both piloquinone and homopiloquinone, which features an unusual starter unit incorporation step. This powerful strategy can be further exploited for BGC activation in strains even beyond streptomycetes, thus facilitating natural product discovery research in the future.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Metaboloma/genética , Família Multigênica , Proteínas Ribossômicas/metabolismo , Antraquinonas/metabolismo , Produtos Biológicos/metabolismo , Técnicas Genéticas , Biossíntese de Proteínas , Streptomyces lividans/genética , Transcrição Genética
17.
Angew Chem Int Ed Engl ; 59(48): 21656-21662, 2020 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-32780931

RESUMO

Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxide to form the catalytic intermediate compound I (FeIV =O and a porphyrin cation radical). Using serial femtosecond X-ray crystallography (SFX), we have determined the pristine structures of the FeIII and FeIV =O redox states of a B-type DyP. These structures reveal a water-free distal heme site that, together with the presence of an asparagine, imply the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.


Assuntos
Arginina/metabolismo , Corantes/metabolismo , Heme/metabolismo , Compostos de Ferro/metabolismo , Oxigênio/metabolismo , Peroxidase/metabolismo , Arginina/química , Biocatálise , Corantes/química , Cristalografia por Raios X , Heme/química , Compostos de Ferro/química , Modelos Moleculares , Oxirredução , Oxigênio/química , Peroxidase/química , Streptomyces lividans/enzimologia
18.
Curr Microbiol ; 77(10): 2933-2939, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32681314

RESUMO

Activating the genetic potential of Streptomyces strains to produce secondary metabolites can improve the production of useful biologically active compounds and facilitate the discovery of novel biologically active compounds. In this study, we found that Streptomyces lividans carrying the R440H mutation in rpoB, encoding the RNA polymerase beta subunit, grown in the presence of lincomycin at concentrations below the minimum inhibitory concentration (MIC) produced abundant amounts of actinorhodin and certain cryptic secondary metabolites despite culture conditions that restrict their production by the wild-type strain. The results indicate that lincomycin at concentrations below the MIC may strongly potentiate secondary metabolite production by Streptomyces strains carrying a specific rpoB mutation. In this study, we report an interesting phenomenon induced by combining the positive effects of certain rpoB mutations and concentration-dependent responses to lincomycin on secondary metabolism in S. lividans 66 and discuss the mechanisms and their applicability in exploring cryptic secondary metabolite production in streptomycetes.


Assuntos
Lincomicina , Streptomyces lividans , Antibacterianos , RNA Polimerases Dirigidas por DNA , Mutação , Metabolismo Secundário , Streptomyces lividans/genética
19.
ACS Chem Biol ; 15(3): 766-773, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32118401

RESUMO

Totopotensamide A (TPM A, 1) is a polyketide-peptide glycoside featuring a nonproteinogenic amino acid 4-chloro-6-methyl-5,7-dihydroxyphenylglycine (ClMeDPG). The biosynthetic gene cluster (BGC) of totopotensamides (tot) was previously activated by manipulating transcription regulators in marine-derived Streptomyces pactum SCSIO 02999. Herein, we report the heterologous expression of the tot BGC in Streptomyces lividans TK64, and the production improvement of TPM A via in-frame deletion of two negative regulators totR5 and totR3. The formation of ClMeDPG was proposed to require six enzymes, including four enzymes TotC1C2C3C4 for 3,5-dihydroxyphenylglycine (DPG) biosynthesis and two modifying enzymes TotH (halogenase) and TotM (methyltransferase). Heterologous expression of the four-gene cassette totC1C2C3C4 led to production of 3,5-dihydroxyphenylglyoxylate (DPGX). The aminotransferase TotC4 was biochemically characterized to convert DPGX to S-DPG. Inactivation of totH led to a mutant accumulated a deschloro derivative TPM H1, and the ΔtotHi/ΔtotMi double mutant afforded two deschloro-desmethyl products TPMs HM1 and HM2. A hydrolysis experiment demonstrated that the DPG moiety in TPM HM2 was S-DPG, consistent with that of the TotC4 enzymatic product. These results confirmed that TotH and TotM were responsible for ClMeDPG biosynthesis. Bioinformatics analysis indicated that both TotH and TotM might act on thiolation domain-tethered substrates. This study provided evidence for deciphering enzymes leading to ClMeDPG in TPM A, and unambiguously determined its absolute configuration as S.


Assuntos
Aminoácidos/química , Glicosídeos/biossíntese , Glicosídeos/genética , Metoxi-Hidroxifenilglicol/análogos & derivados , Metiltransferases/metabolismo , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , Regulação da Expressão Gênica , Hidrólise , Metoxi-Hidroxifenilglicol/química , Metilação , Estrutura Molecular , Família Multigênica , Mutação , Recombinação Genética , Streptomyces lividans/genética , Compostos de Sulfidrila/química , Transaminases/metabolismo
20.
Biotechnol Appl Biochem ; 67(4): 563-573, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32134142

RESUMO

We report a new artificial hydrogenase made by covalent anchoring of the iron Knölker's complex to a xylanase S212C variant. This artificial metalloenzyme was found to be able to catalyze efficiently the transfer hydrogenation of the benchmark substrate trifluoroacetophenone by sodium formate in water, yielding the corresponding secondary alcohol as a racemic. The reaction proceeded more than threefold faster with the XlnS212CK biohybrid than with the Knölker's complex alone. In addition, efficient conversion of trifluoroacetophenone to its corresponding alcohol was reached within 60 H with XlnS212CK, whereas a ≈2.5-fold lower conversion was observed with Knölker's complex alone as catalyst. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggested that the Knölker's complex was most likely flexible and could experience free rotational reorientation within the active-site pocket of Xln A, allowing it to access the subsite pocket populated by trifluoroacetophenone.


Assuntos
Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Streptomyces lividans/enzimologia , Proteínas de Bactérias/genética , Catálise , Endo-1,4-beta-Xilanases/genética , Hidrogenase/genética , Hidrogenação , Proteínas Ferro-Enxofre/genética , Streptomyces lividans/genética , Água
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