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1.
NPJ Biofilms Microbiomes ; 7(1): 56, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215744

RESUMO

Aryl polyenes (APEs) are specialized polyunsaturated carboxylic acids that were identified in silico as the product of the most widespread family of bacterial biosynthetic gene clusters (BGCs). They are present in several Gram-negative host-associated bacteria, including multidrug-resistant human pathogens. Here, we characterize a biological function of APEs, focusing on the BGC from a uropathogenic Escherichia coli (UPEC) strain. We first perform a genetic deletion analysis to identify the essential genes required for APE biosynthesis. Next, we show that APEs function as fitness factors that increase protection from oxidative stress and contribute to biofilm formation. Together, our study highlights key steps in the APE biosynthesis pathway that can be explored as potential drug targets for complementary strategies to reduce fitness and prevent biofilm formation of multi-drug resistant pathogens.


Assuntos
Biofilmes , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Essenciais , Polienos/metabolismo , Biofilmes/crescimento & desenvolvimento , Transporte Biológico , Vias Biossintéticas , Regulação Bacteriana da Expressão Gênica , Estrutura Molecular , Mutação , Oxirredução , Fenótipo , Polienos/química
2.
Nat Commun ; 12(1): 4499, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301953

RESUMO

Aurantinins (ARTs) are antibacterial polyketides featuring a unique 6/7/8/5-fused tetracyclic ring system and a triene side chain with a carboxyl terminus. Here we identify the art gene cluster and dissect ART's C-methyl incorporation patterns to study its biosynthesis. During this process, an apparently redundant methyltransferase Art28 was characterized as a malonyl-acyl carrier protein O-methyltransferase, which represents an unusual on-line methyl esterification initiation strategy for polyketide biosynthesis. The methyl ester bond introduced by Art28 is kept until the last step of ART biosynthesis, in which it is hydrolyzed by Art9 to convert inactive ART 9B to active ART B. The cryptic reactions catalyzed by Art28 and Art9 represent a protecting group biosynthetic logic to render the ART carboxyl terminus inert to unwanted side reactions and to protect producing organisms from toxic ART intermediates. Further analyses revealed a wide distribution of this initiation strategy for polyketide biosynthesis in various bacteria.


Assuntos
Proteína de Transporte de Acila/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Metiltransferases/metabolismo , Policetídeos/metabolismo , Proteína de Transporte de Acila/genética , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Vias Biossintéticas/genética , Esterificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Metiltransferases/genética , Testes de Sensibilidade Microbiana/métodos , Modelos Químicos , Estrutura Molecular , Família Multigênica , Polienos/química , Polienos/metabolismo , Polienos/farmacologia , Policetídeos/química , Policetídeos/farmacologia
3.
Methods Mol Biol ; 2296: 333-350, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977457

RESUMO

Polyene antibiotics are macrolide antifungal compounds obtained by fermentation of producer Streptomyces strains. Here we describe commonly used methods for polyene production, detection, and their subsequent extraction and purification. While bioassays are used to detect these compounds based on their biological activity, quantification by spectrophotometry or high-performance liquid chromatography (HPLC ) relies on their physiochemical properties and is more reliable.


Assuntos
Antibacterianos/biossíntese , Antifúngicos/metabolismo , Macrolídeos/metabolismo , Polienos/metabolismo , Streptomyces/metabolismo , Bioensaio/métodos , Cromatografia Líquida de Alta Pressão/métodos , Fermentação/fisiologia
4.
Appl Environ Microbiol ; 87(12): e0295520, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-33837012

RESUMO

Biofilms are the predominant bacterial lifestyle and can protect microorganisms from environmental stresses. Multispecies biofilms can affect the survival of enteric pathogens that contaminate food products, and thus, investigating the underlying mechanisms of multispecies biofilms is essential for food safety and human health. In this study, we investigated the ability of the natural isolate Bacillus subtilis PS-216 to restrain Campylobacter jejuni biofilm formation and adhesion to abiotic surfaces as well as to disrupt preestablished C. jejuni biofilms. Using confocal laser scanning microscopy and colony counts, we demonstrate that the presence of B. subtilis PS-216 prevents C. jejuni biofilm formation, decreases growth of the pathogen by 4.2 log10, and disperses 26-h-old preestablished C. jejuni biofilms. Furthermore, the coinoculation of B. subtilis and C. jejuni interferes with the adhesion of C. jejuni to abiotic surfaces, reducing it by 2.4 log10. We also show that contact-independent mechanisms contribute to the inhibitory effect of B. subtilis PS-216 on C. jejuni biofilm. Using B. subtilis mutants in genes coding for nonribosomal peptides and polyketides revealed that bacillaene significantly contributes to the inhibitory effect of B. subtilis PS-216. In summary, we show a strong potential for the use of B. subtilis PS-216 against C. jejuni biofilm formation and adhesion to abiotic surfaces. Our research could bring forward novel applications of B. subtilis in animal production and thus contribute to food safety. IMPORTANCE Campylobacter jejuni is an intestinal commensal in animals (including broiler chickens) but also the most frequent cause of bacterial foodborne infection in humans. This pathogen forms biofilms which enhance survival of C. jejuni in food processing and thus threaten human health. Probiotic bacteria represent a potential alternative in the prevention and control of foodborne infections. The beneficial bacterium Bacillus subtilis has an excellent probiotic potential to reduce C. jejuni in the animal gastrointestinal tract. However, data on the effect of B. subtilis on C. jejuni biofilms are scarce. Our study shows that the B. subtilis natural isolate PS-216 prevents adhesion to the abiotic surfaces and the development of submerged C. jejuni biofilm during coculture and destroys the preestablished C. jejuni biofilm. These insights are important for development of novel applications of B. subtilis that will reduce the use of antibiotics in human and animal health and increase productivity in animal breeding.


Assuntos
Bacillus subtilis , Biofilmes , Agentes de Controle Biológico , Campylobacter jejuni/fisiologia , Polienos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Aderência Bacteriana , Mutação , Poliestirenos
5.
ACS Chem Biol ; 16(3): 539-547, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33625847

RESUMO

Hitachimycin is a macrolactam antibiotic with an (S)-ß-phenylalanine (ß-Phe) at the starter position of its polyketide skeleton. (S)-ß-Phe is formed from l-α-phenylalanine by the phenylananine-2,3-aminomutase HitA in the hitachimycin biosynthetic pathway. In this study, we produced new hitachimycin analogs via mutasynthesis by feeding various (S)-ß-Phe analogs to a ΔhitA strain. We obtained six hitachimycin analogs with F at the ortho, meta, or para position and Cl, Br, or a CH3 group at the meta position of the phenyl moiety, as well as two hitachimycin analogs with thienyl substitutions. Furthermore, we carried out a biochemical and structural analysis of HitB, a ß-amino acid-selective adenylation enzyme that introduces (S)-ß-Phe into the hitachimycin biosynthetic pathway. The KM values of the incorporated (S)-ß-Phe analogs and natural (S)-ß-Phe were similar. However, the KM values of unincorporated (S)-ß-Phe analogs with Br and a CH3 group at the ortho or para position of the phenyl moiety were high, indicating that HitB functions as a gatekeeper to select macrolactam starter units during mutasynthesis. The crystal structure of HitB in complex with (S)-ß-3-Br-phenylalanine sulfamoyladenosine (ß-m-Br-Phe-SA) revealed that the bulky meta-Br group is accommodated by the conformational flexibility around Phe328, whose side chain is close to the meta position. The aromatic group of ß-m-Br-Phe-SA is surrounded by hydrophobic and aromatic residues, which appears to confer the conformational flexibility that enables HitB to accommodate the meta-substituted (S)-ß-Phe. The new hitachimycin analogs exhibited different levels of biological activity in HeLa cells and multidrug-sensitive budding yeast, suggesting that they may target different molecules.


Assuntos
Adenilato Quinase/química , Fenilalanina/química , Policetídeos/química , Proteínas Recombinantes/química , Adenilato Quinase/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas , Halogênios/química , Células HeLa , Humanos , Cinética , Metano/química , Modelos Moleculares , Conformação Molecular , Mutação , Fenilalanina/metabolismo , Polienos/química , Polienos/metabolismo , Policetídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
6.
ACS Synth Biol ; 10(2): 228-235, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33535752

RESUMO

Conjugated alkenes such as dienes and polyenes have a range of applications as pharmaceutical agents and valuable building blocks in the polymer industry. Development of a renewable route to these compounds provides an alternative to fossil fuel derived production. The enzyme family of the UbiD decarboxylases offers substantial scope for alkene production, readily converting poly unsaturated acids. However, biochemical pathways producing the required substrates are poorly characterized, and UbiD-application has hitherto been limited to biological styrene production. Herein, we present a proof-of-principle study for microbial production of polyenes using a bioinspired strategy employing a polyketide synthase (PKS) in combination with a UbiD-enzyme. Deconstructing a bacterial iterative type II PKS enabled repurposing the broad-spectrum antibiotic andrimid biosynthesis pathway to access the metabolic intermediate 2,4,6-octatrienoic acid, a valuable chemical for material and pharmaceutical industry. Combination with the fungal ferulic acid decarboxylase (Fdc1) led to a biocatalytic cascade-type reaction for the production of hepta-1,3,5-triene in vivo. Our approach provides a novel route to generate unsaturated hydrocarbons and related chemicals and provides a blue-print for future development and application.


Assuntos
Antibacterianos/metabolismo , Aspergillus niger/enzimologia , Carboxiliases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas Fúngicas/metabolismo , Engenharia Metabólica/métodos , Biocatálise , Escherichia coli/genética , Ácidos Graxos Insaturados/metabolismo , Polienos/metabolismo , Policetídeo Sintases/metabolismo , Pirróis/metabolismo
7.
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
8.
Sci Rep ; 10(1): 15323, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948786

RESUMO

Complex polyketides of bacterial origin are biosynthesised by giant assembly-line like megaenzymes of the type 1 modular polyketide synthase (PKS) class. The trans-AT family of modular PKSs, whose biosynthetic frameworks diverge significantly from those of the archetypal cis-AT type systems represent a new paradigm in natural product enzymology. One of the most distinctive enzymatic features common to trans-AT PKSs is their ability to introduce methyl groups at positions ß to the thiol ester in the growing polyketide chain. This activity is achieved through the action of a five protein HCS cassette, comprising a ketosynthase, a 3-hydroxy-3-methylglutaryl-CoA synthase, a dehydratase, a decarboxylase and a dedicated acyl carrier protein. Here we report a molecular level description, achieved using a combination of X-ray crystallography, in vitro enzyme assays and site-directed mutagenesis, of the bacillaene synthase dehydratase/decarboxylase enzyme couple PksH/PksI, responsible for the final two steps in ß-methyl branch installation in this trans-AT PKS. Our work provides detailed mechanistic insight into this biosynthetic peculiarity and establishes a molecular framework for HCS cassette enzyme exploitation and manipulation, which has future potential value in guiding efforts in the targeted synthesis of functionally optimised 'non-natural' natural products.


Assuntos
Carboxiliases/metabolismo , Hidroliases/metabolismo , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Polienos/metabolismo , Policetídeo Sintases/genética , Conformação Proteica
9.
J Antibiot (Tokyo) ; 73(10): 711-720, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32820242

RESUMO

The conspicuous bright golden to orange-reddish coloration of species of the basidiomycete genus Laetiporus is a hallmark feature of their fruiting bodies, known among mushroom hunters as the "chicken of the woods". This report describes the identification of an eight-domain mono-modular highly reducing polyketide synthase as sole enzyme necessary for laetiporic acid biosynthesis. Heterologous pathway reconstitution in both Aspergillus nidulans and Aspergillus niger verified that LpaA functions as a multi-chain length polyene synthase, which produces a cocktail of laetiporic acids with a methyl-branched C26-C32 main chain. Laetiporic acids show a marked antifungal activity on Aspergillus protoplasts. Given the multiple products of a single biosynthesis enzyme, our work underscores the diversity-oriented character of basidiomycete natural product biosynthesis.


Assuntos
Antifúngicos/metabolismo , Polienos/metabolismo , Policetídeo Sintases/metabolismo , Polyporales/enzimologia , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Aspergillus nidulans/efeitos dos fármacos , Aspergillus niger/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Polienos/isolamento & purificação , Polienos/farmacologia , Policetídeo Sintases/genética , Polyporales/química , Polyporales/genética , Polyporales/metabolismo
10.
Enzyme Microb Technol ; 137: 109532, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32423669

RESUMO

aurJ3M(GenBank:EU697915.1), a 579 bp gene, whose deduced product (192 amino acid) was found to have amino acid sequence homology with some bacterial regulatory proteins. Computer-assisted analysis showed that AurJ3M is PAS-LuxR family regulatory protein, it combines a PAS domain with a helix-turn-helix (HTH) motif of the LuxR type. Gene deletion of aurJ3M from the S.aureofuscus SYAU0709 through gene replacement with the apramycin resistance gene aac (3) IV and the DNA fragment of the replication initiation site sequence OriT resulted in complete loss of aureofuscin production, indicating that AurJ3M is a positive regulator during the aureofuscin biosynthesis. Gene expression analyses in S.aureofuscus SYAU0709 and S.aureofuscus ΔaurJ3M by reverse transcriptase PCR (RT-PCR) indicated there no transcripts for the genes B、C、G、F、S0、S1、D in S.aureofuscus ΔaurJ3M, maintains some transcription of these genes although the levels is low. Transcription was also significantly reduced for gene A, and no difference in the transcription pattern was observed for the genes R、E and H, a similar transcription pattern was also observed for AurJ3M, A and B showed a different transcription pattern, while transcription of gene A in S.aureofuscus ΔaurJ3M when compared to the parental strain, no transcripts could be detected for gene B in the mutant; This result indicates that the deletion of aurJ3M gene doesn't have a polar effect on the transcription of genes located downstream from aurJ3M. In the ΔaurJ3M knockout mutant, except for the transcription of E、H、M and R, the transcription of other genes in the cluster was down-regulated, HPLC shows there no secondary metabolites was produced,aurJ3M has a pathway-specific regulation effect on the biosynthesis of aureofuscin. Aureofuscin had broad application prospects in food preservation, and enriched the resource pool of anti-fungal antibiotics of tetraphenyl macrolide in China.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/genética , Streptomyces/genética , Transativadores/genética , Simulação por Computador , Computadores Moleculares , Deleção de Genes , Família Multigênica , Polienos/metabolismo , Streptomyces/metabolismo , Fatores de Transcrição/genética
11.
J Chem Ecol ; 46(5-6): 461-474, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32300913

RESUMO

Congeneric species that live in sympatry may have evolved various mechanisms that maintain reproductive isolation among species. However, with the spread of invasive organisms owing to increased global human activity, some species that evolved in allopatry can now be found outside their native range and may have the opportunity to interact, in the absence of mechanisms for reproductive isolation. In South Florida, where the Asian subterranean termite, Coptotermes gestroi (Wamann), and the Formosan subterranean termite, Coptotermes formosanus Shiraki (Blattodea: Rhinotermitidae) are invasive, the two species can engage in heterospecific mating behavior as their distribution range and their dispersal flight season both overlap. Termites rely on semiochemicals for many of their activities, including finding a mate after a dispersal flight. In this study, we showed that females of both species produce (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (DTE) from their tergal glands as a shared sex pheromone. We suggest that both species primarily rely on an inundative dispersal flight strategy to find a mate, and that DTE is used as a short distance pheromone or contact pheromone to initiate and maintain the tandem between males and females. The preference of C. gestroi males for C. formosanus females during tandem resulted from the relatively high amount of DTE produced by tergal glands of C. formosanus females, when compared with those of C. gestroi females. This results in confusion of mating in the field during simultaneous dispersal flights, with a potential for hybridization. Such observations imply that no prezygotic barriers emerged while the two species evolved in allopatry for ~18 Ma.


Assuntos
Isópteros/fisiologia , Polienos/metabolismo , Atrativos Sexuais/metabolismo , Comportamento Sexual Animal , Distribuição Animal , Animais , Evolução Biológica , Corte , Feminino , Florida , Espécies Introduzidas , Masculino , Estações do Ano
12.
Appl Microbiol Biotechnol ; 104(10): 4445-4455, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32221690

RESUMO

The polyene macrolide rimocidin, produced by Streptomyces rimosus M527, is highly effective against a broad range of fungal plant pathogens, but at low yields. Elicitation is an effective method of stimulating the yield of bioactive secondary metabolites. In this study, the biomass and filtrate of a culture broth of Escherichia coli JM109, Bacillus subtilis WB600, Saccharomyces cerevisiae, and Fusarium oxysporum f. sp. cucumerinum were employed as elicitors to promote rimocidin production in S. rimosus M527. Adding culture broth and biomass of S. cerevisiae (A3) and F. oxysporum f. sp. cucumerinum (B4) resulted in an increase of rimocidin production by 51.2% and 68.3% respectively compared with the production under normal conditions in 5-l fermentor. In addition, quantitative RT-PCR analysis revealed that the transcriptions of ten genes (rimA to rimK) located in the gene cluster involved in rimocidin biosynthesis in A3 or B4 elicitation experimental group were all higher than those of a control group. Using a ß-glucuronidase (GUS) reporter system, GUS enzyme activity assay, and Western blot analysis, we discovered that elicitation of A3 or B4 increased protein synthesis in S. rimosus M527. These results demonstrate that the addition of elicitors is a useful approach to improve rimocidin production.Key Points • An effective strategy for enhancing rimocidin production in S. rimosus M527 is demonstrated. • Overproduction of rimocidin is a result of higher expressed structural genes followed by an increase in protein synthesis.


Assuntos
Família Multigênica , Streptomyces rimosus/metabolismo , Bacillus subtilis , Biomassa , Vias Biossintéticas , Meios de Cultura/farmacologia , Escherichia coli , Fusarium , Polienos/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae , Metabolismo Secundário/efeitos dos fármacos , Streptomyces rimosus/efeitos dos fármacos
13.
J Biotechnol ; 311: 25-34, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32057784

RESUMO

Myxococcus xanthus DK1622 is known as a proficient producer of different kinds of secondary metabolites (SM) with various biological activities, including myxovirescin A, myxalamide A, myxochromide A and DKxanthene. Low production of SM in the wild type bacteria makes searching for production optimization methods highly desirable. Identification and induction of endogenous key molecular feature(s) regulating the production level of the metabolites remain promising, while heterologous expression of the biosynthetic genes is not always efficient because of various complicating factors including codon usage bias. This study established proteomic and molecular approaches to elucidate the regulatory roles of the ROK regulatory protein in the modification of secondary metabolite biosynthesis. Interestingly, the results revealed that rok inactivation significantly reduced the production of the SM and also changed the motility in the bacteria. Electrophoretic mobility shift assay using purified ROK protein indicated a direct enhancement of the promoters encoding transcription of the DKxanthene, myxochelin A, and myxalamide A biosynthesis machinery. Comparative proteomic analysis by two-dimensional fluorescence difference in-gel electrophoresis (2D-DIGE) was employed to identify the protein profiles of the wild type and rok mutant strains during early and late logarithmic growth phases of the bacterial culture. Resulting data demonstrated overall 130 differently altered proteins by the effect of the rok gene mutation, including putative proteins suspected to be involved in transcriptional regulation, carbohydrate metabolism, development, spore formation, and motility. Except for a slight induction seen in the production of myxovirescin A in a rok over-expression background, no changes were found in the formation of the other SM. From the outcome of our investigation, it is possible to conclude that ROK acts as a pleiotropic regulator of secondary metabolite formation and development in M. xanthus, while its direct effects still remain speculative. More experiments are required to elucidate in detail the variable regulation effects of the protein and to explore applicable approaches for generating valuable SM in this bacterium.


Assuntos
Proteínas de Bactérias/metabolismo , Myxococcus xanthus/metabolismo , Proteínas de Bactérias/genética , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Lisina/análogos & derivados , Lisina/metabolismo , Myxococcus xanthus/genética , Polienos/metabolismo , Regiões Promotoras Genéticas/genética , Proteômica/métodos , Metabolismo Secundário/genética , Metabolismo Secundário/fisiologia
14.
J Biosci Bioeng ; 129(5): 558-564, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31924510

RESUMO

ε-Poly-l-lysine (ε-PL) produced as a secondary metabolite of Streptomyces albulus has long been used as a natural food preservative in a number of countries, including Japan, the United States, South Korea, and China. To date, numerous studies employing classical biotechnological approaches have been carried out to improve its productivity. Here we report a modern and rational genetic approach to enhancing metabolic flux toward ε-PL biosynthesis. Based on in silico genome analyses, we revealed that S. albulus NBRC14147 produces five antifungal polyene antibiotics-tetramycin A and B, tetrin A and B, and a trace amount of nystatin A1-concomitantly with antimicrobial ε-PL. Targeted inactivation of the biosynthetic gene cluster for tetramycins and tetrins in a nystatin A1 production-deficient mutant completely abolished the production of polyene macrolides, which in turn led to an approximately 20% improvement in ε-PL production that closely correlated with the polyene defects. The biosynthetic flux for ε-PL was thus successfully enhanced by inactivation of the concomitant secondary metabolite biosynthetic pathways. Since this elimination of concomitantly produced metabolites also allows for simpler purification after fermentation production of ε-PL, the rational strain engineering strategy we show here will improve its industrial production.


Assuntos
Macrolídeos/metabolismo , Polienos/metabolismo , Polilisina/biossíntese , Streptomyces/metabolismo , Fermentação , Conservantes de Alimentos/metabolismo , Macrolídeos/química , Polienos/química , Streptomyces/química , Streptomyces/genética
15.
Org Lett ; 22(2): 729-733, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31891272

RESUMO

Disruption of an aminotransferase family gene dtlA activated the production of a novel dimeric benzoic polyene acids (BPAs), named youssoufene A1 (1), along with four new (2-5, youssoufenes B1-B4) and a known (6) monomeric BPA in the marine-derived Streptomyces youssoufiensis OUC6819. The structures of 1-5 were elucidated by extensive spectroscopic and computational approaches. Youssoufene A1 (1) exhibited notably increased growth inhibition (MIC = 12.5 µg/mL) against multidrug resistant Enterococcus faecalis compared to monomeric structures (2-6).


Assuntos
Polienos/química , Streptomyces/enzimologia , Transaminases/metabolismo , Estrutura Molecular , Polienos/metabolismo , Estereoisomerismo , Transaminases/genética
16.
Chem Commun (Camb) ; 56(5): 822-825, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31848534

RESUMO

Produced by a newly isolated Streptomycetes strain, meijiemycin is a gigantic linear polyene-polyol that exhibits structural features not seen in other members of the polyene-polyol family. We propose a biosynthetic mechanism and demonstrate that meijiemycin inhibits hyphal growth by inducing the aggregation of ergosterol and restructuring of the fungal plasma membrane.


Assuntos
Antifúngicos/farmacologia , Álcoois Graxos/farmacologia , Polienos/farmacologia , Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Candida albicans/efeitos dos fármacos , Descoberta de Drogas , Álcoois Graxos/isolamento & purificação , Álcoois Graxos/metabolismo , Genes Bacterianos , Genômica , Testes de Sensibilidade Microbiana , Família Multigênica , Polienos/isolamento & purificação , Polienos/metabolismo , Policetídeo Sintases/genética , Streptomyces/química
17.
Biomacromolecules ; 21(2): 743-752, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-31790208

RESUMO

Two homopolyesters and a series of novel random copolyesters were synthesized from two bio-based diacid esters, dimethyl 2,5-furandicarboxylate, a well-known renewable monomer, and dimethyl 2,2'-bifuran-5,5'-dicarboxylate, a more uncommon diacid based on biochemical furfural. Compared to homopolyesters poly(butylene furanoate) (PBF) and poly(butylene bifuranoate) (PBBf), their random copolyesters differed dramatically in that their melting temperatures were either lowered significantly or they showed no crystallinity at all. However, the thermal stabilities of the homopolyesters and the copolyesters were comparable. Based on tensile tests from amorphous film specimens, it was concluded that the elastic moduli, tensile strengths, and elongation at break values for all copolyesters were similar as well, irrespective of the furan:bifuran molar ratio. Tensile moduli of approximately 2 GPa and tensile strengths up to 66 MPa were observed for amorphous film specimens prepared from the copolyesters. However, copolymerizing bifuran units into PBF allowed the glass transition temperature to be increased by increasing the amount of bifuran units. Besides enhancing the glass transition temperatures, the bifuran units also conferred the copolyesters with significant UV absorbance. This combined with the highly amorphous nature of the copolyesters allowed them to be melt-pressed into highly transparent films with very low ultraviolet light transmission. It was also found that furan-bifuran copolyesters could be as effective, or better, oxygen barrier materials as neat PBF or PBBf, which themselves were found superior to common barrier polyesters such as PET.


Assuntos
Materiais Biocompatíveis/síntese química , Furaldeído/síntese química , Polienos/síntese química , Poliésteres/síntese química , Materiais Biocompatíveis/metabolismo , Biomassa , Butileno Glicóis/síntese química , Butileno Glicóis/metabolismo , Furaldeído/metabolismo , Polienos/metabolismo , Poliésteres/metabolismo , Polímeros/síntese química , Polímeros/metabolismo
18.
ACS Chem Biol ; 15(5): 1195-1203, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31816232

RESUMO

Burkholderia thailandensis has emerged as a model organism for investigating the production and regulation of diverse secondary metabolites. Most of the biosynthetic gene clusters encoded in B. thailandensis are silent, motivating the development of new methods for accessing their products. In the current work, we add to the canon of available approaches using phenotype-guided transposon mutagenesis to characterize a silent biosynthetic gene cluster. Because secondary metabolite biosynthesis is often associated with phenotypic changes, we carried out random transposon mutagenesis followed by phenotypic inspection of the resulting colonies. Several mutants exhibited intense pigmentation and enhanced expression of an iterative type I polyketide synthase cluster that we term org. Disruptions of orgA, orgB, and orgC abolished the biosynthesis of the diffusible pigment, thus linking it to the org operon. Isolation and structural elucidation by HR-MS and 1D/2D NMR spectroscopy revealed three novel, cryptic metabolites, thailandene A-C. Thailandenes are linear formylated or acidic polyenes containing a combination of cis and trans double bonds. Variants A and B exhibited potent antibiotic activity against Staphylococcus aureus and Saccharomyces cerevisiae but not against Escherichia coli. One of the transposon mutants that exhibited an enhanced expression of org contained an insertion upstream of a σ54-dependent transcription factor. Closer inspection of the org operon uncovered a σ54 promoter consensus sequence upstream of orgA, providing clues regarding its regulation. Our results showcase the utility of phenotype-guided transposon mutagenesis in uncovering cryptic metabolites encoded in bacterial genomes.


Assuntos
Antibacterianos/biossíntese , Produtos Biológicos/química , Burkholderia/genética , Polienos/metabolismo , Antibacterianos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Burkholderia/química , Elementos de DNA Transponíveis , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genoma Bacteriano , Família Multigênica , Mutagênese , Fenótipo , Polienos/isolamento & purificação , Policetídeo Sintases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Metabolismo Secundário , Fatores de Transcrição/metabolismo
19.
Org Lett ; 22(1): 126-129, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31829019

RESUMO

Leaves of Leucosceptrum canum harbor abundant toxic aromatic abietanoids, and they are rarely attacked by insect herbivores, except for the larvae of Nacna malachitis. The excrements of the insect that fed on L. canum leaves were investigated, leading to the isolation and identification of two unprecedented 11,12-seco-abietane diterpene polyenes: nacnabietanins A (1) and B (2). This discovery heralds a unique detoxification mechanism of plant aromatic abietanoids by insects through enzymatic cleavage of stable benzene rings into more easily degraded polyenes.


Assuntos
Abietanos/metabolismo , Diterpenos/metabolismo , Lamiaceae/química , Polienos/metabolismo , Spodoptera/metabolismo , Abietanos/química , Abietanos/isolamento & purificação , Animais , Diterpenos/química , Lamiaceae/metabolismo , Estrutura Molecular , Folhas de Planta/química , Polienos/química , Spodoptera/química
20.
Antonie Van Leeuwenhoek ; 113(1): 1-12, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31721032

RESUMO

Bacillus subtilis currently encompasses four subspecies, Bacillus subtilis subsp. subtilis, Bacillus subtilis subsp. inaquosorum, Bacillus subtilis subsp. spizizenii and Bacillus subtilis subsp. stercoris. Several studies based on genomic comparisons have suggested these subspecies should be promoted to species status. Previously, one of the main reasons for leaving them as subspecies was the lack of distinguishing phenotypes. In this study, we used comparative genomics to determine the genes unique to each subspecies and used these to lead us to the unique phenotypes. The results show that one difference among the subspecies is they produce different bioactive secondary metabolites. B. subtilis subsp. spizizenii is shown conserve the genes to produce mycosubtilin, bacillaene and 3,3'-neotrehalosadiamine. B. subtilis subsp. inaquosorum is shown conserve the genes to produce bacillomycin F, fengycin and an unknown PKS/NRPS cluster. B. subtilis subsp. stercoris is shown conserve the genes to produce fengycin and an unknown PKS/NRPS cluster. While B. subtilis subsp. subtilis is shown to conserve the genes to produce 3,3'-neotrehalosadiamine. In addition, we update the chemotaxonomy and phenotyping to support their promotion to species status.


Assuntos
Bacillus subtilis/classificação , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Genoma Bacteriano/genética , Lipopeptídeos/metabolismo , Lipoproteínas/metabolismo , Peptídeos Cíclicos/metabolismo , Polienos/metabolismo
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