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1.
Nat Commun ; 12(1): 3381, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099669

RESUMO

Nutrient amendment diminished bacterial functional diversity, consolidating carbon flow through fewer bacterial taxa. Here, we show strong differences in the bacterial taxa responsible for respiration from four ecosystems, indicating the potential for taxon-specific control over soil carbon cycling. Trends in functional diversity, defined as the richness of bacteria contributing to carbon flux and their equitability of carbon use, paralleled trends in taxonomic diversity although functional diversity was lower overall. Among genera common to all ecosystems, Bradyrhizobium, the Acidobacteria genus RB41, and Streptomyces together composed 45-57% of carbon flow through bacterial productivity and respiration. Bacteria that utilized the most carbon amendment (glucose) were also those that utilized the most native soil carbon, suggesting that the behavior of key soil taxa may influence carbon balance. Mapping carbon flow through different microbial taxa as demonstrated here is crucial in developing taxon-sensitive soil carbon models that may reduce the uncertainty in climate change projections.


Assuntos
Ciclo do Carbono , Mudança Climática , Nutrientes/metabolismo , Microbiologia do Solo , Solo/química , Acidobacteria/genética , Acidobacteria/isolamento & purificação , Acidobacteria/metabolismo , Biodiversidade , Bradyrhizobium/genética , Bradyrhizobium/isolamento & purificação , Bradyrhizobium/metabolismo , Carbono/metabolismo , DNA Bacteriano/isolamento & purificação , Monitorização de Parâmetros Ecológicos/métodos , Previsões/métodos , Fósforo/metabolismo , RNA Ribossômico 16S/genética , Streptomyces/genética , Streptomyces/isolamento & purificação , Streptomyces/metabolismo
2.
Appl Microbiol Biotechnol ; 105(11): 4731-4741, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34021812

RESUMO

Genome sequencing has revealed that each Streptomyces contains a wide range of biosynthetic gene clusters (BGCs) and has the capability to produce more novel natural products than what is expected. However, most gene clusters for secondary metabolite biosynthesis are cryptic under normal growth conditions. In Streptomyces tsukubaensis, combining overexpression of the putative SARPs (Streptomyces antibiotic regulatory proteins) and bioactivity-guided screening, the silent gene cluster (tsu) was successfully activated and a novel bioactive anthracycline tsukubarubicin was further isolated and identified. Biological activity assays demonstrated that tsukubarubicin possessed much better antitumor bioactivities against various human cancer cell lines (especially the breast cancer cell lines) than clinically used doxorubicin. Moreover, the previously unreported gene cluster (tsu) for biosynthesis of tsukubarubicin was first characterized and detailed annotations of this gene cluster were also conducted. Our strategy presented in this work is broadly applicable in other Streptomyces and will assist in enriching the natural products for potential drug leads. KEY POINTS: • Generally scalable strategy to activate silent gene clusters by manipulating SARPs. • The novel anthracycline tsukubarubicin with potent antitumor bioactivities. • Identification and annotation of the previously uncharacterized tsu gene cluster.


Assuntos
Streptomyces , Antibacterianos/farmacologia , Humanos , Família Multigênica , Metabolismo Secundário , Streptomyces/genética
3.
Appl Microbiol Biotechnol ; 105(11): 4599-4607, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34043077

RESUMO

Avermectin, produced by Streptomyces avermitilis, is an active compound protective against nematodes, insects, and mites. However, its potential usage is limited by its low aqueous solubility. The uridine diphosphate (UDP)-glycosyltransferase (BLC) from Bacillus licheniformis synthesizes avermectin glycosides with improved water solubility and in vitro antinematodal activity. However, enzymatic glycosylation of avermectin by BLC is limited due to the low conversion rate of this reaction. Thus, improving BLC enzyme activity is necessary for mass production of avermectin glycosides for field application. In this study, the catalytic activity of BLC toward avermectin was enhanced via directed evolution. Three mutants from the BLC mutant library (R57H, V227A, and D252V) had specific glucosylation activity for avermectin 2.0-, 1.8-, and 1.5-fold higher, respectively, than wild-type BLC. Generation of combined mutations via site-directed mutagenesis led to even further enhancement of activity. The triple mutant, R57H/V227A/D252V, had the highest activity, 2.8-fold higher than that of wild-type BLC. The catalytic efficiencies (Kcat/Km) of the best mutant (R57H/V227A/D252V) toward the substrates avermectin and UDP-glucose were improved by 2.71- and 2.29-fold, respectively, compared to those of wild-type BLC. Structural modeling analysis revealed that the free energy of the mutants was - 1.1 to - 7.1 kcal/mol lower than that of wild-type BLC, which was correlated with their improved activity. KEY POINTS: • Directed evolution improved the glucosylation activity of BLC toward avermectin. • Combinatorial site-directed mutagenesis led to further enhanced activity. • The mutants exhibited lower free energy values than wild-type BLC.


Assuntos
Glicosiltransferases , Streptomyces , Glicosiltransferases/genética , Ivermectina/análogos & derivados , Streptomyces/genética
4.
Appl Microbiol Biotechnol ; 105(10): 4177-4187, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33944982

RESUMO

Bioconversion using microorganisms and their enzymes is an important tool in many industrial fields. The discovery of useful new microbial enzymes contributes to the development of industries utilizing bioprocesses. Streptomyces sp. EAS-AB2608, isolated from a soil sample collected in Japan, can convert the tetrahydrobenzotriazole CPD-1 (a selective positive allosteric modulator of metabotropic glutamate receptor 5) to its hydroxylated form at the C4-(R) position. The current study was performed to identify the genes encoding the enzymes involved in CPD-1 bioconversion and to verify their function. To identify gene products responsible for the conversion of CPD-1, we used RNA sequencing to analyze EAS-AB2608; from its 8333 coding sequences, we selected two genes, one encoding cytochrome P450 (easab2608_00800) and the other encoding ferredoxin (easab2608_00799), as encoding desirable gene products involved in the bioconversion of CPD-1. The validity of this selection was tested by using a heterologous expression approach. A bioconversion assay using genetically engineered Streptomyces avermitilis SUKA24 ∆saverm3882 ∆saverm7246 co-expressing the two selected genes (strain ES_SUKA_63) confirmed that these gene products had hydroxylation activity with respect to CPD-1, indicating that they are responsible for the conversion of CPD-1. Strain ES_SUKA_63 also showed oxidative activity toward other compounds and therefore might be useful not only for bioconversion of CPD-1 but also as a tool for synthesis of drug metabolites and in optimization studies of various pharmaceutical lead compounds. We expect that this approach will be useful for bridging the gap between the latest enzyme optimization technologies and conventional enzyme screening using microorganisms. KEY POINTS: • Genes easab2608_00800 (cyp) and easab2608_00799 (fdx) were selected by RNA-Seq. • Selection validity was evaluated by an engineered S. avermitilis expression system. • Strain ES_SUKA_63 showed oxidative activity toward CPD-1 and other compounds.


Assuntos
Ferredoxinas , Streptomyces , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Japão , Streptomyces/genética , Streptomyces/metabolismo
5.
Artigo em Inglês | MEDLINE | ID: mdl-34029177

RESUMO

We studied the taxonomic relationship between Streptomyces cinnamonensis and Streptomyces virginiae. These type strains shared the same 16S rRNA gene sequence. Phylogenomic analysis supported them being closely related. Digital DNA-DNA relatedness and average nucleotide identity using whole genome sequences indicated that the two species represent the same genomospecies. They shared similar phenotypic characteristics and harboured the same set of secondary metabolite-biosynthetic gene clusters for polyketides and nonribosomal peptides in the genomes. Therefore, according to Rule 24b of the Bacteriological Code, S. cinnamonensis Okami 1952, 572AL (Approved Lists 1980) should be reclassified as a later heterotypic synonym of S. virginiae Grundy et al. 1952, 399AL (Approved Lists 1980) emend. Nouioui et al. 2018. Although 16S rRNA gene sequences were identical among type strains of Streptomyces xanthophaeus, Streptomyces spororaveus and Streptomyces nojiriensis and between those of Streptomyces vinaceus and Streptomyces cirratus, respectively, digital DNA-DNA relatedness indicated that these species are not synonymous.


Assuntos
Streptomyces/classificação , Sequência de Bases , DNA Bacteriano/genética , Família Multigênica , Fenótipo , Filogenia , RNA Ribossômico 16S/genética , Streptomyces/genética
6.
J Agric Food Chem ; 69(17): 5096-5104, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-33826316

RESUMO

Natural and modified versions of the 5-enolpyruvylshikimate-3-phosphate synthase (epsps) gene have been used to confer tolerance to the broad-spectrum herbicide glyphosate in a variety of commercial crops. The most widely utilized trait was obtained from the Agrobacterium tumefaciens strain CP4 and has been commercialized in several glyphosate-tolerant crops. The EPSPS gene products are enzymes that have been divided into three classes based on sequence similarity, sensitivity to glyphosate, and steady-state catalytic parameters. Herein, we describe the informatics-guided identification and biochemical and structural characterization of a novel EPSPS from Streptomyces sviceus (DGT-28 EPSPS). The data suggest DGT-28 EPSPS and other closely related homologues exemplify a distinct new class (Class IV) of EPSPS enzymes that display intrinsic tolerance to high concentrations of glyphosate (Ki ≥ 5000 µM). We further demonstrate that dgt-28 epsps, when transformed into stable plants, provides robust (≥4× field rates) vegetative/reproductive herbicide tolerance and has utility in weed-control systems comparable to that of commercialized events.


Assuntos
Herbicidas , Streptomyces , 3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Glicina/análogos & derivados , Glicina/farmacologia , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Streptomyces/genética
7.
BMC Genomics ; 22(1): 247, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33827425

RESUMO

BACKGROUND: Antibiotic-producing Streptomyces bacteria are ubiquitous in nature, yet most studies of its diversity have focused on free-living strains inhabiting diverse soil environments and those in symbiotic relationship with invertebrates. RESULTS: We studied the draft genomes of 73 Streptomyces isolates sampled from the skin (wing and tail membranes) and fur surfaces of bats collected in Arizona and New Mexico. We uncovered large genomic variation and biosynthetic potential, even among closely related strains. The isolates, which were initially identified as three distinct species based on sequence variation in the 16S rRNA locus, could be distinguished as 41 different species based on genome-wide average nucleotide identity. Of the 32 biosynthetic gene cluster (BGC) classes detected, non-ribosomal peptide synthetases, siderophores, and terpenes were present in all genomes. On average, Streptomyces genomes carried 14 distinct classes of BGCs (range = 9-20). Results also revealed large inter- and intra-species variation in gene content (single nucleotide polymorphisms, accessory genes and singletons) and BGCs, further contributing to the overall genetic diversity present in bat-associated Streptomyces. Finally, we show that genome-wide recombination has partly contributed to the large genomic variation among strains of the same species. CONCLUSIONS: Our study provides an initial genomic assessment of bat-associated Streptomyces that will be critical to prioritizing those strains with the greatest ability to produce novel antibiotics. It also highlights the need to recognize within-species variation as an important factor in genetic manipulation studies, diversity estimates and drug discovery efforts in Streptomyces.


Assuntos
Quirópteros , Streptomyces , Animais , Arizona , Quirópteros/genética , Genômica , Família Multigênica , New Mexico , Filogenia , RNA Ribossômico 16S/genética , Streptomyces/genética
8.
Molecules ; 26(8)2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917975

RESUMO

Rhizosphere microorganisms play important ecological roles in promoting herb growth and producing abundant secondary metabolites. Studies on the rhizosphere microbes of traditional Chinese medicines (TCMs) are limited, especially on the genomic and metabolic levels. In this study, we reported the isolation and characterization of a Steptomyces netropsis WLXQSS-4 strain from the rhizospheric soil of Clematis manshurica Rupr. Genomic sequencing revealed an impressive total of 40 predicted biosynthetic gene clusters (BGCs), whereas metabolomic profiling revealed 13 secondary metabolites under current laboratory conditions. Particularly, medium screening activated the production of alloaureothin, whereas brominated and chlorinated pimprinine derivatives were identified through precursor-directed feeding. Moreover, antiproliferative activities against Hela and A549 cancer cell lines were observed for five compounds, of which two also elicited potent growth inhibition in Enterococcus faecalis and Staphylococcus aureus, respectively. Our results demonstrated the robust secondary metabolism of S. netropsis WLXQSS-4, which may serve as a biocontrol agent upon further investigation.


Assuntos
Genômica , Medicina Tradicional Chinesa , Metabolômica , Rizosfera , Streptomyces/genética , Streptomyces/metabolismo , Vias Biossintéticas/genética , Linhagem Celular Tumoral , Cromossomos Bacterianos/genética , Humanos , Metaboloma , Anotação de Sequência Molecular , Família Multigênica , Fenótipo , Filogenia , RNA Ribossômico 16S/genética , Metabolismo Secundário , Análise de Sequência de DNA , Streptomyces/isolamento & purificação , Streptomyces/ultraestrutura
9.
Appl Microbiol Biotechnol ; 105(7): 2737-2745, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33738551

RESUMO

Microbial transglutaminase (MTG) has been used extensively in academic research and the food industry through cross-linking or posttranslational modification of proteins. In our previous paper, the activity-increased MTG mutants were obtained by means of rational mutagenesis and random mutagenesis coupled with the newly developed screening system. In addition, the improvement of heat resistance of MTG is needed to expand further its industrial applications. Here, a structure-based rational enzyme engineering approach was applied to improve the thermostability of MTG by introducing an artificial disulfide bridge. As a result of narrowing down candidates using a rational approach, we successfully engineered a disulfide bridge into the N-terminal region of MTG by substituting Thr-7 and Glu-58 with cysteine. The T7C/E58C mutant was observed to have a de novo disulfide bridge and showed an increased melting temperature (Tm value) of 4.3 °C with retained enzymatic activity. To address the benefit-gained reason, we focused on the Cß temperature factor of the amino-acid residues that might form a disulfide bridge in MTG. Introducing the disulfide bridge had no remarkable effect on the mutant aiming to stabilize the high temperature factor. On the other hand, the mutation was effective on the relatively stable region. The introduction of a disulfide bridge may therefore be effective to stabilize further the relatively stable part. This finding is considered to be useful for the rational design of mutants aiming at heat resistance of proteins.Key Points• Microbial transglutaminase (MTG) is used as a binder in the food industry.• MTG has the potential for use in the manufacturing of various commercial materials.• Enhanced thermostability was observed for the disulfide bridge mutant, T7C/G58C.


Assuntos
Streptomyces , Transglutaminases , Dissulfetos , Estabilidade Enzimática , Mutagênese , Streptomyces/genética , Streptomyces/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismo
10.
J Agric Food Chem ; 69(10): 3144-3153, 2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33651593

RESUMO

Streptomyces transglutaminase (TGase) is widely used to improve food texture properties. In this study, random mutagenesis and site-directed genetic modification were used to improve the production of TGase in Streptomyces mobaraensis. First, S. mobaraensis DSM40587 (smWT) was subjected to atmospheric and room-temperature plasma mutagenesis, and then a mutant (smY2019) with a 5.5-fold increase in TGase yield was screened from approximately 3000 × 25 (round) mutants. Compared to smWT, smY2019 exhibits a 3.2-fold higher TGase mRNA level and two site mutations within the -10 region of the TGase promoter. The recombinant expression analysis in the TGase-deficient S. mobaraensis suggests that the mutated TGase promoter is more robust than the wild-type one. Finally, we integrated two additional TGase expression cassettes into the smY2019 genome, yielding the recombinant strain smY2019-3C with a 103% increase in TGase production compared to smY2019. The smY2019-3C strain with 40 U/mL of TGase yield could be a suitable candidate for the industrial production of TGase.


Assuntos
Streptomyces , Transglutaminases , Mutagênese , Regiões Promotoras Genéticas , Streptomyces/genética , Streptomyces/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismo
11.
Appl Environ Microbiol ; 87(10)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33712427

RESUMO

ε-Poly-l-lysine is a potent antimicrobial produced through fermentation of Streptomyces and used in many Asian countries as a food preservative. It is synthesized and excreted by a special nonribosomal peptide synthetase (NRPS)-like enzyme called Pls. In this study, we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the Pls from Streptomyces in terms of domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus Pls promoter, we confirmed that its product is indeed ε-poly-l-lysine. A comprehensive sequence analysis suggested that Pls genes are widely spread among coryneform actinobacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain it in their genomes. This finding raises the possibility that ε-poly-l-lysine as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems.IMPORTANCE Every year, microbial contamination causes billions of tons of food wasted and millions of cases of illness. ε-Poly-l-lysine has potent, wide-spectrum inhibitory activity and is heat stable and biodegradable. It has been approved for food preservation by an increasing number of countries. ε-Poly-l-lysine is produced from soil bacteria of the genus Streptomyces, also producers of various antibiotic drugs and toxins and not considered to be a naturally occurring food component. The frequent finding of pls in cheese and skin bacteria suggests that ε-poly-l-lysine may naturally exist in cheese and on our skin, and ε-poly-l-lysine producers are not limited to filamentous actinobacteria.


Assuntos
Proteínas de Bactérias/genética , Corynebacterium/enzimologia , Peptídeo Sintases/genética , Queijo/microbiologia , Clonagem Molecular , Corynebacterium/genética , Humanos , Polilisina/metabolismo , Pele/microbiologia , Streptomyces/genética , Streptomyces coelicolor/genética
12.
Chemistry ; 27(29): 7923-7929, 2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-33769623

RESUMO

The dauc-8-en-11-ol synthase from Streptomyces venezuelae was investigated for its catalytic activity towards alternative terpene precursors, specifically designed to enable new cyclisation pathways. Exchange of aromatic amino acid residues at the enzyme surface by site-directed mutagenesis led to a 4-fold increase of the yield in preparative scale incubations, which likely results from an increased enzyme stability instead of improved enzyme kinetics.


Assuntos
Streptomyces , Ciclização , Mutagênese Sítio-Dirigida , Streptomyces/genética , Terpenos
13.
Appl Environ Microbiol ; 87(11)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33741615

RESUMO

Cyclodipeptide synthases (CDPSs) catalyze the formation of cyclodipeptides using aminoacylated tRNAs as the substrates and have great potential in the production of diverse 2,5-diketopiperazines (2,5-DKPs). Genome mining of Streptomyces leeuwenhoekii NRRL B-24963 revealed a two-gene locus, saz, encoding CDPS SazA and a unique fused enzyme (SazB) harboring two domains: phytoene synthase-like prenyltransferase (PT) and methyltransferase (MT). Heterologous expression of the saz gene(s) in Streptomyces albus J1074 led to the production of four prenylated indole alkaloids, among which streptoazines A to C (compounds 3 to 5) are new compounds. Expression of different gene combinations showed that the SazA catalyzes the formation of cyclo(l-Trp-l-Trp) (cWW; compound 1), followed by consecutive prenylation and methylation by SazB. Biochemical assays demonstrated that SazB is a bifunctional enzyme, catalyzing sequential C-3/C-3' prenylation(s) by SazB-PT and N-1/N-1' methylation(s) by SazB-MT. Of note, the substrate selectivity of SazB-PT and SazB-MT was probed, revealing the stringent specificity of SazB-PT but relative flexibility of SazB-MT.IMPORTANCE Natural products with a 2,5-diketopiperazine (2,5-DKP) skeleton have long sparked interest in drug discovery and development. Recent advances in microbial genome sequencing have revealed that the potential of cyclodipeptide synthase (CDPS)-dependent pathways encoding new 2,5-DKPs are underexplored. In this study, we report the genome mining of a new CDPS-encoding two-gene operon and activation of this cryptic gene cluster through heterologous expression, leading to the discovery of four indole 2,5-DKP alkaloids. The cyclo(l-Trp-l-Trp) (cWW)-synthesizing CDPS SazA and the unusual prenyltransferase (PT)-methyltransferase (MT) fused enzyme SazB were characterized. Our results expand the repertoire of CDPSs and associated tailoring enzymes, setting the stage for accessing diverse prenylated alkaloids using synthetic biology strategies.


Assuntos
Proteínas de Bactérias/metabolismo , Alcaloides Indólicos/metabolismo , Peptídeo Sintases/metabolismo , Streptomyces/metabolismo , Redes e Vias Metabólicas , Microrganismos Geneticamente Modificados/metabolismo , Prenilação , Streptomyces/enzimologia , Streptomyces/genética
14.
Appl Microbiol Biotechnol ; 105(5): 1875-1887, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33564920

RESUMO

Milbemycins are used commercially as insect repellents and acaricides; however, their high cost remains a significant challenge to commercial production. Hence, improving the titer of milbemycins for commercial application is an urgent priority. The present study aimed to effectively increase the titer of milbemycins using a combination of genome re-sequencing and metabolic engineering. First, 133 mutation sites were identified by genome re-sequencing in the mutagenized high-yielding strain BC04. Among them, three modifiable candidate genes (sbi_04868 encoding citrate synthase, sbi_06921 and sbi_06922 encoding alpha and beta subunits of acetyl-CoA carboxylase, and sbi_04683 encoding carbon uptake system gluconate transporter) related to primary metabolism were screened and identified. Next, the DNase-deactivated Cpf1-based integrative CRISPRi system was used in S. bingchenggensis to downregulate the transcription level of gene sbi_04868. Then, overexpression of the potential targets sbi_06921-06922 and sbi_04683 further facilitated milbemycin biosynthesis. Finally, those candidate genes were engineered to produce strains with combinatorial downregulation and overexpression, which resulted in the titer of milbemycin A3/A4 increased by 27.6% to 3164.5 mg/L. Our research not only identified three genes in S. bingchenggensis that are closely related to the production of milbemycins, but also offered an efficient engineering strategy to improve the titer of milbemycins using genome re-sequencing. KEY POINTS: • We compared the genomes of two strains with different titers of milbemycins. • We found three genes belonging to primary metabolism influence milbemycin production. • We improved titer of milbemycins by a combinatorial engineering of three targets.


Assuntos
Macrolídeos , Streptomyces , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Streptomyces/genética
15.
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
16.
Nat Commun ; 12(1): 1171, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33608525

RESUMO

Direct cloning represents the most efficient strategy to access the vast number of uncharacterized natural product biosynthetic gene clusters (BGCs) for the discovery of novel bioactive compounds. However, due to their large size, repetitive nature, or high GC-content, large-scale cloning of these BGCs remains an overwhelming challenge. Here, we report a scalable direct cloning method named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) which consists of Cas12a digestion, a DNA assembly approach termed T4 polymerase exo + fill-in DNA assembly, and Cre-lox in vivo DNA circularization. We apply this method to clone 47 BGCs ranging from 10 to 113 kb from both Actinomycetes and Bacilli with ~100% efficiency. Heterologous expression of cloned BGCs leads to the discovery of 15 previously uncharacterized natural products including six cyclic head-to-tail heterodimers with a unique 5/6/6/6/5 pentacyclic carbon skeleton, designated as bipentaromycins A-F. Four of the bipentaromycins show strong antimicrobial activity to both Gram-positive and Gram-negative bacteria such as methicillin-resistant Staphylococcus aureus, vancomycinresistant Enterococcus faecium, and bioweapon Bacillus anthracis. Due to its robustness and efficiency, our direct cloning method coupled with heterologous expression provides an effective strategy for large-scale discovery of novel natural products.


Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Clonagem Molecular/métodos , Endodesoxirribonucleases/genética , Integrases/genética , Recombinação Genética , Actinobacteria/genética , Actinobacteria/metabolismo , Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , DNA Bacteriano , Enterococcus faecium/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Família Multigênica , Streptomyces/genética
17.
Curr Microbiol ; 78(4): 1245-1255, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33629120

RESUMO

From acid corroded iron plates five different types of actinobacteria were isolated. Among the five, JMCACA3 strain was selected for the present study. In ISP media, JMCACA3 strain showed well-developed aerial and substrate mycelia were observed. This strain showed good growth in 12 different carbon and 4 different nitrogen sources. The 16S rRNA sequence of phylogenetic analysis by neighbor-joining method identified the studied strain belongs to Streptomyces sp. The biodegradation activity of the strain analyzed by UV and FTIR analysis, which revealed that the various concentrations of Benzimidazole inhibitor with JMCACA3 culture showed slightly varied results. For weight loss method, mild steel coupons incubated with JMCACA3 culture, Benzimidazole inhibitor + JMCACA3 culture and mixed sample showed that JMCACA3 strain utilized the inhibitor as their energy source and the weight the coupons were slightly varied, evidenced by XRD spectra and showed Fe2O3 corrosion products. Our study concluded that the JMCACA3 strain, an iron-reducing actinobacteria which utilizes and converted the corrosion inhibitor Benzimidazole as their energy source. So, it is very urgent to develop more powerful corrosion inhibitor from green biocide or microbial-based biocide and their analog which incorporated into the pre-existing Benzimidazole to increase the corrosion inhibitor level against the biofilm of actinobacterial influenced corrosion.


Assuntos
Ferro , Streptomyces , Biofilmes , Corrosão , Filogenia , RNA Ribossômico 16S/genética , Streptomyces/genética
18.
Appl Environ Microbiol ; 87(9)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33608297

RESUMO

Diseases caused by soilborne fungal pathogens result in significant crop yield losses and quality reduction. Streptomyces albidoflavus strain W68 is effective in controlling several soilborne fungal diseases. To identify antifungal substances critical for biocontrol activity of W68, the genome of W68 was sequenced and a linear chromosome of 6.80 Mb was assembled. A total of 21 secondary metabolite biosynthesis gene clusters (BGCs), accounting for 12.27% of the genome, were identified. Core gene deletion mutants for each of all 8 BGCs for nonribosomal peptide synthetases and polyketide synthases were created. Among them, only the mutant lacking ctg1-5755 (the gene was renamed as fscD W68) in BGC 19, which shares 100% sequence similarity with the BGC for candicidin synthesis, showed obvious reduction in antifungal activity. A pot experiment revealed that biocontrol effects of the ΔfscD W68 mutant in Rhizoctonia rot of cucumber were also significantly compromised relative to W68. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that W68 but not the ΔfscD W68 mutant can produce candicidin isomers, indicating that the production of candicidin isomers is key for antifungal activity and biocontrol activity of S. albidoflavus W68.IMPORTANCE This study reports that candicidin-like secondary metabolites produced by microbial cells in natural soil environments can effectively control soilborne fungal diseases, revealing a novel mechanism of microbial biocontrol agents. We demonstrated that the main antifungal activity and biocontrol activity of Streptomyces albidoflavus strain W68 are attributable to the production of candicidin isomers, suggesting that gene clusters for candicidin-like compound biosynthesis might be used as molecular markers to screen and breed microbial strains for biocontrol agent development.


Assuntos
Agentes de Controle Biológico/metabolismo , Candicidina/metabolismo , Cucumis sativus/microbiologia , Doenças das Plantas/prevenção & controle , Rhizoctonia , Streptomyces/metabolismo , Agentes de Controle Biológico/química , Candicidina/química , Isomerismo , Família Multigênica , Metabolismo Secundário , Microbiologia do Solo , Streptomyces/genética
19.
Appl Environ Microbiol ; 87(10)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33637575

RESUMO

A group of polyene macrolides mainly composed of two constituents was isolated from the fermentation broth of Streptomyces roseoflavus Men-myco-93-63, which was isolated from soil where potato scabs were repressed naturally. One of these macrolides was roflamycoin, which was first reported in 1968, and the other was a novel compound named Men-myco-A, which had one methylene unit more than roflamycoin. Together, they were designated RM. This group of antibiotics exhibited broad-spectrum antifungal activities in vitro against 17 plant-pathogenic fungi, with 50% effective concentrations (EC50) of 2.05 to 7.09 µg/ml and 90% effective concentrations (EC90) of 4.32 to 54.45 µg/ml, which indicates their potential use in plant disease control. Furthermore, their biosynthetic gene cluster was identified, and the associated biosynthetic assembly line was proposed based on a module and domain analysis of polyketide synthases (PKSs), supported by findings from gene inactivation experiments.IMPORTANCE Streptomyces roseoflavus Men-myco-93-63 is a biocontrol strain that has been studied in our laboratory for many years and exhibits a good inhibitory effect in many crop diseases. Therefore, the identification of antimicrobial metabolites is necessary and our main objective. In this work, chemical, bioinformatic, and molecular biological methods were combined to identify the structures and biosynthesis of the active metabolites. This work provides a new alternative agent for the biological control of plant diseases and is helpful for improving both the properties and yield of the antibiotics via genetic engineering.


Assuntos
Agentes de Controle Biológico , Macrolídeos/metabolismo , Polienos/metabolismo , Streptomyces/metabolismo , Ascomicetos/efeitos dos fármacos , Ascomicetos/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Vias Biossintéticas/genética , Macrolídeos/farmacologia , Família Multigênica , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Doenças das Plantas/prevenção & controle , Polienos/farmacologia , Streptomyces/genética
20.
Nat Commun ; 12(1): 173, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420084

RESUMO

Bio-based production of many chemicals is not yet possible due to the unknown biosynthetic pathways. Here, we report a strategy combining retrobiosynthesis and precursor selection step to design biosynthetic pathways for multiple short-chain primary amines (SCPAs) that have a wide range of applications in chemical industries. Using direct precursors of 15 target SCPAs determined by the above strategy, Streptomyces viridifaciens vlmD encoding valine decarboxylase is examined as a proof-of-concept promiscuous enzyme both in vitro and in vivo for generating SCPAs from their precursors. Escherichia coli expressing the heterologous vlmD produces 10 SCPAs by feeding their direct precursors. Furthermore, metabolically engineered E. coli strains are developed to produce representative SCPAs from glucose, including the one producing 10.67 g L-1 of iso-butylamine by fed-batch culture. This study presents the strategy of systematically designing biosynthetic pathways for the production of a group of related chemicals as demonstrated by multiple SCPAs as examples.


Assuntos
Aminas/química , Aminas/metabolismo , Vias Biossintéticas , Engenharia de Proteínas , Vias Biossintéticas/genética , Carboxiliases/genética , Carboxiliases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Glucose/metabolismo , Microbiologia Industrial , Engenharia Metabólica , Simulação de Acoplamento Molecular , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismo
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