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1.
Molecules ; 27(11)2022 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-35684507

RESUMO

Laboratory cultures of two 'biosynthetically talented' bacterial strains harvested from tropical and temperate Pacific Ocean sediment habitats were examined for the production of new natural products. Cultures of the tropical Salinispora arenicola strain RJA3005, harvested from a PNG marine sediment, produced salinorcinol (3) and salinacetamide (4), which had previously been reported as products of engineered and mutated strains of Amycolatopsis mediterranei, but had not been found before as natural products. An S. arenicola strain RJA4486, harvested from marine sediment collected in the temperate ocean waters off British Columbia, produced the new aminoquinone polyketide salinisporamine (5). Natural products 3, 4, and 5 are putative shunt products of the widely distributed rifamycin biosynthetic pathway.


Assuntos
Actinomycetales , Produtos Biológicos , Micromonosporaceae , Produtos Biológicos/metabolismo , Sedimentos Geológicos/microbiologia , Micromonosporaceae/genética
2.
Microbiology (Reading) ; 168(4)2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35380529

RESUMO

Salinispora tropica was originally cultured from tropical marine sediments and described as the first obligate marine actinomycete genus. Soon after its discovery, it yielded the potent proteasome inhibitor salinosporamide A, a structurally novel natural product that is currently in phase III clinical trials for the treatment of cancer. If approved, it will be the first natural product derived from a cultured marine microbe to achieve clinical relevance. S. tropica produces many other biologically active natural products, including some linked to chemical defence, thus providing ecological context for their production. However, genomic analyses reveal that most natural product biosynthetic gene clusters remain orphan, suggesting that more compounds await discovery. The abundance of biosynthetic gene clusters in S. tropica supports the concept that the small molecules they encode serve important ecological functions, while their evolutionary histories suggest a potential role in promoting diversification. Better insights into the ecological functions of microbial natural products will help inform future discovery efforts.


Assuntos
Actinobacteria , Produtos Biológicos , Micromonosporaceae , Actinobacteria/genética , Micromonosporaceae/genética , Família Multigênica
3.
J Nat Prod ; 85(4): 980-986, 2022 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-35263117

RESUMO

A new manumycin-type natural product named pacificamide (1) and its candidate biosynthetic gene cluster (pac) were discovered from the marine actinobacterium Salinispora pacifica CNT-855. The structure of the compound was determined using NMR, electronic circular dichroism, and bioinformatic predictions. The pac gene cluster is unique to S. pacifica and found in only two of the 119 Salinispora genomes analyzed across nine species. Comparative analyses of biosynthetic gene clusters encoding the production of related manumycin-type compounds revealed genetic differences in accordance with the unique pacificamide structure. Further queries of manumycin-type gene clusters from public databases revealed their limited distribution across the phylum Actinobacteria and orphan diversity that suggests additional products remain to be discovered in this compound class. Production of the known metabolite triacsin D is also reported for the first time from the genus Salinispora. This study adds two classes of compounds to the natural product collective isolated from the genus Salinispora, which has proven to be a useful model for natural product research.


Assuntos
Produtos Biológicos , Micromonosporaceae , Produtos Biológicos/metabolismo , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Família Multigênica , Polienos , Alcamidas Poli-Insaturadas
4.
Syst Appl Microbiol ; 45(3): 126307, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35255274

RESUMO

During a cultural diversity survey on hydrolytic bacteria in saline alkaline soils, a hydrolytic actinobacterium strain ACPA39T was enriched and isolated in pure culture from a soda solonchak soil in southwestern Siberia. It forms a substrate mycelium with rod-shaped sporangia containing 1-3 exospores. The isolate is obligately alkaliphilic, growing at pH 7.5-10.3 (optimum at 8.5-9.0) and moderately halophilic, tolerating up to 3 M total Na+ in the form of sodium carbonates. It is an obligately aerobic, organoheteroterophic, saccharolytic bacterium, utilizing various sugars and alpha/beta-glucans as growth substrates. According to the 16S rRNA gene-based phylogenetic analysis, strain ACPA39T forms a distinct branch within the family Micromonosporaceae, with the sequence identities below 94.5% with type strains of other genera. This is confirmed by phylogenomic analysis based on the 120 conserved single copy protein-based markers and genomic indexes (ANI, AAI). The cell-wall of ACPA39T contained meso-DAP, glycine, glutamic acid and alanine in a equimolar ratio, characteristic of the peptidoglycan type A1γ'. The whole-cell sugars include galactose and xylose. The major menaquinone is MK-10(H4). The identified polar lipids consist of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The polar lipid fatty acids were dominated by anteiso-C17:0, iso-C16:0, iso-C17:0, 10 Me-C18:0 and C18:1ω9. Based on the distinct phylogeny, the chemotaxonomy features and unique phenotypic properties, strain ACPA39T (DSM 106523T = VKM 2772T) is classified into a new genus and species in the family Micromonosporaceae for which the name Natronosporangium hydrolitycum gen. nov., sp. nov. is proposed.


Assuntos
Actinobacteria , Micromonosporaceae , Ásia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Graxos/química , Micromonosporaceae/genética , Fosfolipídeos/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Solo , Microbiologia do Solo , Açúcares , Vitamina K 2/química
5.
mBio ; 12(6): e0270021, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34809466

RESUMO

While specialized metabolites are thought to mediate ecological interactions, the evolutionary processes driving chemical diversification, particularly among closely related lineages, remain poorly understood. Here, we examine the evolutionary dynamics governing the distribution of natural product biosynthetic gene clusters (BGCs) among 118 strains representing all nine currently named species of the marine actinobacterial genus Salinispora. While much attention has been given to the role of horizontal gene transfer (HGT) in structuring BGC distributions, we find that vertical descent facilitates interspecies BGC diversification over evolutionary timescales. Moreover, we identified a distinct phylogenetic signal among Salinispora species at both the BGC and metabolite level, indicating that specialized metabolism represents a conserved phylogenetic trait. Using a combination of genomic analyses and liquid chromatography-high-resolution tandem mass spectrometry (LC-MS/MS) targeting nine experimentally characterized BGCs and their small molecule products, we identified gene gain/loss events, constrained interspecies recombination, and other evolutionary processes associated with vertical inheritance as major contributors to BGC diversification. These evolutionary dynamics had direct consequences for the compounds produced, as exemplified by species-level differences in salinosporamide production. Together, our results support the concept that specialized metabolites, and their cognate BGCs, can represent phylogenetically conserved functional traits with chemical diversification proceeding in species-specific patterns over evolutionary time frames. IMPORTANCE Microbial natural products are traditionally exploited for their pharmaceutical potential, yet our understanding of the evolutionary processes driving BGC evolution and compound diversification remain poorly developed. While HGT is recognized as an integral driver of BGC distributions, we find that the effects of vertical inheritance on BGC diversification had direct implications for species-level specialized metabolite production. As such, understanding the degree of genetic variation that corresponds to species delineations can enhance natural product discovery efforts. Resolving the evolutionary relationships between closely related strains and specialized metabolism can also facilitate our understanding of the ecological roles of small molecules in structuring the environmental distribution of microbes.


Assuntos
Transferência Genética Horizontal , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Família Multigênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Evolução Molecular , Genoma Bacteriano , Micromonosporaceae/classificação , Filogenia , Recombinação Genética , Metabolismo Secundário
6.
Chin J Nat Med ; 19(7): 536-539, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34247777

RESUMO

Over-expression of the pathway specific positive regulator gene is an effective way to activate silent gene cluster. In the curret study, the SARP family regulatory gene, vasR2, was over-expressed in strain Verrucosispora sp. NS0172 and the cryptic gene cluster responsible for the biosynthesis of pentaketide ansamycin was partially activated. Two tetraketides (1 and 2) and a triketide (3) ansamycins, together with five known compounds (4-8), were isolated and elucidated from strain NS0172OEvasR2. Their NMR data were completely assigned by analysis of their HR-ESI-MS and 1H, 13C NMR, HMQC, HMBC and 1H-1H COSY spectra.


Assuntos
Micromonosporaceae , Policetídeos , Rifabutina/metabolismo , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Família Multigênica , Policetídeos/metabolismo
7.
Mar Drugs ; 19(6)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073764

RESUMO

Abyssomicins represent a new family of polycyclic macrolactones. The first described compounds of the abyssomicin family were abyssomicin B, C, atrop-C, and D, produced by the marine actinomycete strain Verrucosispora maris AB-18-032, which was isolated from a sediment collected in the Sea of Japan. Among the described abyssomicins, only abyssomicin C and atrop-abyssomicin C show a high antibiotic activity against Gram-positive bacteria, including multi-resistant and vancomycin-resistant strains. The inhibitory activity is caused by a selective inhibition of the enzyme 4-amino-4-deoxychorismate synthase, which catalyzes the transformation of chorismate to para-aminobenzoic acid, an intermediate in the folic acid pathway.


Assuntos
Antibacterianos , Compostos Bicíclicos Heterocíclicos com Pontes , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Fermentação , Humanos , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Micromonosporaceae/ultraestrutura
8.
Nat Prod Res ; 35(22): 4211-4217, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31729260

RESUMO

The actinomycete strain FIM06-0036 was isolated from marine sponge sample collected from the East China Sea and identified as Verrucosispora sp. based upon the results of 16S rRNA sequence analysis. One new alkaloid, 2-ethylhexyl 1H-imidazole-4-carboxylate (1), together with a known alkaloid butyl 1H-imidazole-4-carboxylate (2) was obtained from the fermentation products of this strain, the structures of compounds 1 and 2 were determined by their detailed analysis of 1 D, 2 D NMR and HR-ESI-MS data, along with literature data analysis. Compounds 1 and 2 were evaluated for their antimicrobial activity with MIC (minimum inhibitory concentration) values ranging from 8 to 256 µg · mL-1 against Helicobacter pylori, Klebsiella Pneumonia, Staphylococcus aureus and Enterococcus faecalis.


Assuntos
Alcaloides , Anti-Infecciosos , Micromonosporaceae , Alcaloides/farmacologia , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Micromonosporaceae/genética , RNA Ribossômico 16S/genética
9.
Microb Biotechnol ; 14(1): 291-306, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33280260

RESUMO

Filamentous members of the phylum Actinobacteria are a remarkable source of natural products with pharmaceutical potential. The discovery of novel molecules from these organisms is, however, hindered because most of the biosynthetic gene clusters (BGCs) encoding these secondary metabolites are cryptic or silent and are referred to as orphan BGCs. While co-culture has proven to be a promising approach to unlock the biosynthetic potential of many microorganisms by activating the expression of these orphan BGCs, it still remains an underexplored technique. The marine actinobacterium Salinispora tropica, for instance, produces valuable compounds such as the anti-cancer molecule salinosporamide but half of its putative BGCs are still orphan. Although previous studies have used marine heterotrophs to induce orphan BGCs in Salinispora, its co-culture with marine phototrophs has yet to be investigated. Following the observation of an antimicrobial activity against a range of phytoplankton by S. tropica, we here report that the photosynthate released by photosynthetic primary producers influences its biosynthetic capacities with production of cryptic molecules and the activation of orphan BGCs. Our work, using an approach combining metabolomics and proteomics, pioneers the use of phototrophs as a promising strategy to accelerate the discovery of novel natural products from marine actinobacteria.


Assuntos
Actinobacteria , Micromonosporaceae , Actinobacteria/genética , Micromonosporaceae/genética , Família Multigênica , Fitoplâncton
10.
Mol Genet Genomics ; 295(6): 1529-1535, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32894358

RESUMO

Lanthipeptides are a subgroup of ribosomally encoded and post-translationally modified peptides (RiPPs) which frequently possess potent biological activity. Here we provide the first comprehensive bioinformatic analysis of the lanthipeptide-producing capability of the Salinispora genus, a marine actinomycete. One hundred twenty-two Salinispora arenicola, tropica, and pacifica genomic sequences were analyzed for lanthipeptide gene clusters, and the resulting 182 clusters were divided into seven groups based on sequence similarities. Group boundaries were defined based on LanB and LanM sequences with greater than 80% similarity within groups. Of the seven groups, six are predicted to encode class I lanthipeptides while only one group is predicted to encode class II lanthipeptides. Leader and core peptides were predicted for each cluster along with the number of possible lanthionine bridges. Notably, all of the predicted products of these clusters would represent novel lanthipeptide scaffolds. Of the 122 Salinispora genomes analyzed in this study, 92% contained at least one lanthipeptide gene cluster suggesting that Salinispora is a rich, yet untapped, source of lanthipeptides.


Assuntos
Alanina/análogos & derivados , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Micromonosporaceae/metabolismo , Fragmentos de Peptídeos/metabolismo , Sulfetos/metabolismo , Alanina/isolamento & purificação , Alanina/metabolismo , Proteínas de Bactérias/genética , Genômica , Micromonosporaceae/genética , Micromonosporaceae/crescimento & desenvolvimento , Fragmentos de Peptídeos/isolamento & purificação , Sulfetos/isolamento & purificação
11.
Appl Microbiol Biotechnol ; 104(21): 9283-9294, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32989516

RESUMO

Actinoplanes sp. SE50/110 is the industrially relevant producer of acarbose, which is used in the treatment of diabetes mellitus. Recent studies elucidated the expression dynamics in Actinoplanes sp. SE50/110 during growth. From these data, we obtained a large genomic region (ACSP50_3900 to ACSP50_3950) containing 51 genes, of which 39 are transcribed in the same manner. These co-regulated genes were found to be stronger transcribed on maltose compared with glucose as a carbon source. The transcriptional regulator MalT was identified as an activator of this maltose-regulated large genomic region (MRLGR). Since most of the genes are poorly annotated, the function of this region is farther unclear. However, comprehensive BLAST analyses indicate similarities to enzymes involved in amino acid metabolism. We determined a conserved binding motif of MalT overlapping the -35 promoter region of 17 transcription start sites inside the MRLGR. The corresponding sequence motif 5'-TCATCC-5nt-GGATGA-3' displays high similarities to reported MalT binding sites in Escherichia coli and Klebsiella pneumoniae, in which MalT is the activator of mal genes. A malT deletion and an overexpression mutant were constructed. Differential transcriptome analyses revealed an activating effect of MalT on 40 of the 51 genes. Surprisingly, no gene of the maltose metabolism is affected. In contrast to many other bacteria, MalT is not the activator of mal genes in Actinoplanes sp. SE50/110. Finally, the MRLGR was found partly in other closely related bacteria of the family Micromonosporaceae. Even the conserved MalT binding site was found upstream of several genes inside of the corresponding regions. KEY POINTS : • MalT is the maltose-dependent activator of a large genomic region in ACSP50_WT. • The consensus binding motif is similar to MalT binding sites in other bacteria. • MalT is not the regulator of genes involved in maltose metabolism in ACSP50_WT.


Assuntos
Actinoplanes , Micromonosporaceae , Acarbose , Proteínas de Bactérias/genética , Genômica , Maltose , Micromonosporaceae/genética
12.
Pol J Microbiol ; 69(3): 321-330, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33574861

RESUMO

Marine obligate actinobacteria produce a wide variety of secondary metabolites with biological activity, notably those with antibiotic activity urgently needed against multi-drug-resistant bacteria. Seventy-five marine actinobacteria were isolated from a marine sediment sample collected in Punta Arena de La Ventana, Baja California Sur, Mexico. The 16S rRNA gene identification, Multi Locus Sequence Analysis, and the marine salt requirement for growth assigned seventy-one isolates as members of the genus Salinispora, grouped apart but related to the main Salinispora arenicola species clade. The ability of salinisporae to inhibit bacterial growth of Staphylococcus epidermidis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacer baumannii, Pseudomonas aeruginosa, and Enterobacter spp. was evaluated by cross-streaking plate and supernatant inhibition tests. Ten supernatants inhibited the growth of eight strains of S. epidermidis from patients suffering from ocular infections, two out of the eight showed growth inhibition on ten S. epidermidis strains from prosthetic joint infections. Also, it inhibited the growth of the remaining six multi-drug-resistant bacteria tested. These results showed that some Salinispora strains could produce antibacterial compounds to combat bacteria of clinical importance and prove that studying different geographical sites uncovers untapped microorganisms with metabolic potential.


Assuntos
Bactérias/crescimento & desenvolvimento , Farmacorresistência Bacteriana Múltipla , Sedimentos Geológicos/microbiologia , Micromonosporaceae/fisiologia , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Actinobacteria/fisiologia , Antibacterianos/farmacologia , Antibiose , Bactérias/efeitos dos fármacos , Bactérias/patogenicidade , Proteínas de Bactérias/genética , Meios de Cultivo Condicionados/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Humanos , México , Micromonosporaceae/classificação , Micromonosporaceae/genética , Micromonosporaceae/isolamento & purificação , Filogenia , RNA Ribossômico 16S/genética , Água do Mar/microbiologia
13.
Nat Prod Res ; 34(21): 3082-3088, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31075998

RESUMO

Using the draft genome sequence of Verrucosispora sp. FIM060022, we have identified a new desferrioxamine-like siderophore, FW0622. This is the first chemically characterized siderophore obtained from Verrucosispora. The structure was elucidated by extensive spectral analyses. The biosynthetic pathway of FW0622 was proposed to occur via the non-ribosomal peptide synthetase (NRPS)-independent (NIS) synthetase pathway based on the putative biosynthetic siderophore gene cluster in FIM060022. The results demonstrate that marine-derived Verrucosispora species deserve recognition as an important source of new natural products. Furthermore, this study verified that genome mining is an effective way to identify compounds that may be overlooked by traditional methods.


Assuntos
Anti-Infecciosos/farmacologia , Micromonosporaceae/genética , Sideróforos/química , Sideróforos/metabolismo , Anti-Infecciosos/química , Vias Biossintéticas , Genoma Bacteriano , Genômica , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Micromonosporaceae/metabolismo , Estrutura Molecular , Família Multigênica , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Metabolismo Secundário/genética , Sideróforos/genética , Sideróforos/farmacologia
14.
Microb Cell Fact ; 18(1): 114, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253141

RESUMO

BACKGROUND: Actinoplanes sp. SE50/110 is a natural producer of acarbose. It has been extensively studied in the last decades, which has led to the comprehensive analysis of the whole genome, transcriptome and proteome. First genetic and microbial techniques have been successfully established allowing targeted genome editing by CRISPR/Cas9 and conjugal transfer. Still, a suitable system for the overexpression of singular genes does not exist for Actinoplanes sp. SE50/110. Here, we discuss, test and analyze different strategies by the example of the acarbose biosynthesis gene acbC. RESULTS: The integrative φC31-based vector pSET152 was chosen for the development of an expression system, as for the replicative pSG5-based vector pKC1139 unwanted vector integration by homologous recombination was observed. Since simple gene duplication by pSET152 integration under control of native promoters appeared to be insufficient for overexpression, a promoter screening experiment was carried out. We analyzed promoter strengths of five native and seven heterologous promoters using transcriptional fusion with the gusA gene and glucuronidase assays as well as reverse transcription quantitative PCR (RT-qPCR). Additionally, we mapped transcription starts and identified the promoter sequence motifs by 5'-RNAseq experiments. Promoters with medium to strong expression were included into the pSET152-system, leading to an overexpression of the acbC gene. AcbC catalyzes the first step of acarbose biosynthesis and connects primary to secondary metabolism. By overexpression, the acarbose formation was not enhanced, but slightly reduced in case of strongest overexpression. We assume either disturbance of substrate channeling or a negative feed-back inhibition by one of the intermediates, which accumulates in the acbC-overexpression mutant. According to LC-MS-analysis, we conclude, that this intermediate is valienol-7P. This points to a bottleneck in later steps of acarbose biosynthesis. CONCLUSION: Development of an overexpression system for Actinoplanes sp. SE50/110 is an important step for future metabolic engineering. This system will help altering transcript amounts of singular genes, that can be used to unclench metabolic bottlenecks and to redirect metabolic resources. Furthermore, an essential tool is provided, that can be transferred to other subspecies of Actinoplanes and industrially relevant derivatives.


Assuntos
Acarbose/metabolismo , Proteínas de Bactérias/genética , Técnicas Genéticas , Vetores Genéticos/genética , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Proteínas de Bactérias/metabolismo , Edição de Genes , Vetores Genéticos/metabolismo , Genoma Bacteriano , Proteoma , Transcriptoma
15.
Methods Enzymol ; 621: 87-110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31128791

RESUMO

Heterologous expression of natural product biosynthetic gene clusters (BGCs) is a robust approach not only to decipher biosynthetic logic behind natural product (NP) biosynthesis, but also to discover new chemicals from uncharacterized BGCs. This approach largely relies on techniques used for cloning large BGCs into suitable expression vectors. Recently, several whole-pathway direct cloning approaches, including full-length RecE-mediated recombination in Escherichia coli, Cas9-assisted in vitro assembly, and transformation-associated recombination (TAR) in Saccharomyces cerevisiae, have been developed to accelerate BGC isolation. In this chapter, we summarize a protocol for TAR cloning large NP BGCs, detailing the process of choosing TAR plasmids, designing pathway-specific TAR vectors, generating yeast spheroplasts, performing yeast transformation, and heterologously expressing BGCs in various host strains. We believe that the established platforms can accelerate the process of discovering new NPs, understanding NP biosynthetic logic, and engineering biosynthetic pathways.


Assuntos
Bactérias/genética , Clonagem Molecular/métodos , Família Multigênica , Recombinação Genética , Saccharomyces cerevisiae/genética , Bactérias/metabolismo , Produtos Biológicos/metabolismo , Vias Biossintéticas , Firmicutes/genética , Firmicutes/metabolismo , Engenharia Genética/métodos , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Proteobactérias/genética , Proteobactérias/metabolismo , Saccharomyces cerevisiae/metabolismo , Streptomyces/genética , Streptomyces/metabolismo
16.
Appl Microbiol Biotechnol ; 103(10): 4089-4102, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30937499

RESUMO

Teicoplanin is a frontline glycopeptide antibiotic produced by Actinoplanes teichomyceticus. It is used to treat complicated cases of infection, including pediatric ones, caused by Gram-positive pathogens. There is a steady interest in elucidating the genetic mechanisms determining teicoplanin production, as they would help overproduce known teicoplanins and discover novel glycopeptides. Herein, we investigate the transcriptional organization of the tei biosynthetic gene cluster and the roles of the cluster-situated regulatory genes in controlling teicoplanin production and self-resistance in A. teichomyceticus. We demonstrate that the tei cluster is organized into nine polygenic and nine monogenic transcriptional units. Most of tei biosynthetic genes are subjected to StrR-like Tei15* control, which, in turn, appears to be regulated by LuxR-type Tei16*. Expression of the genes conferring teicoplanin self-resistance in A. teichomyceticus is not co-regulated with antibiotic production. The gene tei31*, coding for a putative DNA binding protein, is not expressed under teicoplanin producing conditions and is dispensable for antibiotic production. Finally, phylogenesis reconstruction of the glycopeptide cluster-encoded regulators reveals two main clades of StrR-like regulators. Tei15* and close orthologues form one of these clades; the second clade is composed by orthologues of Bbr and Dbv4, governing the biosynthesis of balhimycin and teicoplanin-like A40926, respectively. In addition, the LuxR-type Tei16* appears unrelated to the LuxR-like Dbv3, which is controlling A40926 biosynthesis. Our results shed new light on teicoplanin biosynthesis regulation and on the evolution of novel and old glycopeptide biosynthetic gene clusters.


Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Teicoplanina/biossíntese , Farmacorresistência Bacteriana , Perfilação da Expressão Gênica , Ordem dos Genes , Óperon
17.
Biotechnol Bioeng ; 116(1): 99-109, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30102770

RESUMO

trans-4-Hydroxy- l-proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti-inflammatory drugs in the pharmaceutical industry. Proline 4-hydroxylase (P4H) can catalyze the conversion of l-proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp. strain RH1. In silico model-based simulation showed that α-ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl-CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD-encoding the succinyl-CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine-tuning of plasmid-borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp-7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L -1 ·hr -1 in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose-minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products.


Assuntos
Ciclo do Ácido Cítrico/genética , Corynebacterium glutamicum/metabolismo , Hidroxiprolina/metabolismo , Engenharia Metabólica/métodos , Simulação por Computador , Corynebacterium glutamicum/genética , Fermentação , Glucose/metabolismo , Análise do Fluxo Metabólico , Micromonosporaceae/enzimologia , Micromonosporaceae/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
Int J Syst Evol Microbiol ; 68(10): 3345-3350, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30168792

RESUMO

A novel actinomycete, strain PLAI 1-1T, which formed spiny single spore directly on substrate mycelium was isolated from root tissue of Zingiber montanum. The isolate contained meso-diaminopimelic acid and 3-hydroxydiaminopimelic acid in the cell-wall peptidoglycan. The acyl type of the cell-wall muramic acid was glycolyl. The whole-cell sugars of strain PLAI 1-1T were glucose, arabinose, xylose, ribose and a trace amount of mannose. The membrane phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. The major menaquinone was MK-9 (H4). The main cellular fatty acids were iso-C15 : 0 and C17 : 1ω8c. The G+C content of the genomic DNA was 70.6 mol%. 16S rRNA gene sequence analysis revealed that strain PLAI 1-1T was a member of the genus Jishengella and had the highest 16S rRNA gene sequence similarity to Jishengella endophytica DSM 45430T (99.2 %). Based on the data of physiological and biochemical tests, including the result of DNA-DNA hybridization, strain PLAI 1-1T represents a novel species of the genus Jishengella, for which the name Jishengellazingiberis sp. nov. is proposed. The type strain is PLAI 1-1T (=TBRC 7644T=NBRC 113144T).


Assuntos
Micromonosporaceae/classificação , Filogenia , Microbiologia do Solo , Zingiberaceae/microbiologia , Actinobacteria/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , Parede Celular/química , DNA Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Graxos/química , Micromonosporaceae/genética , Micromonosporaceae/isolamento & purificação , Hibridização de Ácido Nucleico , Peptidoglicano/química , Fosfolipídeos/química , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Tailândia , Vitamina K 2/análogos & derivados , Vitamina K 2/química
19.
Appl Microbiol Biotechnol ; 102(19): 8437-8446, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30105571

RESUMO

The marine actinomycete genus Salinispora is a remarkably prolific source of structurally diverse and biologically active secondary metabolites. Herein, we select the model organism Salinispora tropica CNB-440 for development as a heterologous host for the expression of biosynthetic gene clusters (BGCs) to complement well-established Streptomyces host strains. In order to create an integratable host with a clean background of secondary metabolism, we replaced three genes (salA-C) essential for salinosporamide biosynthesis with a cassette containing the Streptomyces coelicolor ΦC31 phage attachment site attB to generate the mutant S. tropica CNB-4401 via double-crossover recombination. This mutagenesis not only knocks-in the attachment site attB in the genome of S. tropica CNB-440 but also abolishes production of the salinosporamides, thereby simplifying the strain's chemical background. We validated this new heterologous host with the successful integration and expression of the thiolactomycin BGC that we recently identified in several S. pacifica strains. When compared to the extensively engineered superhost S. coelicolor M1152, the production of thiolactomycins from S. tropica CNB-4401 was approximately 3-fold higher. To the best of our knowledge, this is the first example of using a marine actinomycete as a heterologous host for natural product BGC expression. The established heterologous host may provide a useful platform to accelerate the discovery of novel natural products and engineer biosynthetic pathways.


Assuntos
Produtos Biológicos/metabolismo , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Família Multigênica/genética , Actinobacteria/genética , Sítios de Ligação Microbiológicos/genética , Vias Biossintéticas/genética , Metabolismo Secundário/genética , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Tiofenos/metabolismo
20.
Antonie Van Leeuwenhoek ; 111(12): 2303-2310, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29974366

RESUMO

A novel actinomycete, designated strain YIM CF22T, was isolated from a desert soil sample collected from Turpan in Xinjiang Uyghur Autonomous Region, north-western China. The taxonomic position of the strain YIM CF22T is described based on a polyphasic approach. Strain YIM CF22T was found to form irregular sporangia on agar media. It contains meso-diaminopimelic acid in the cell wall peptidoglycan. The major menaquinone was identified as MK-9(H4); the polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, two unidentified phospholipids and two unidentified glycolipids. The whole cell sugars were found to be ribose, mannose, galactose, glucose and xylose. The major cellular fatty acids were found to be (> 5%) iso-C16:0 (43.5%), anteiso-C17:0 (10.2%), iso-C15:0 (7.1%), C17:1 ω8c (6.3%) and iso H-C16:1 (5.9%). The G+C content was determined to be 70.8%. 16S rRNA gene sequence analysis of strain YIM CF22T showed high similarity (97.0%) to Actinoplanes rishiriensis NBRC 108556T. The strain also showed high 16S rRNA gene sequence similarities to Verrucosispora sediminis CGMCC 4.3550T (96.9%) and Micromonospora tulbaghiae DSM 45142T (96.8%). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain YIM CF22T clusters with A. rishiriensis NBRC 108556T, Actinoplanes globisporus JCM 3186T and Actinoplanes rhizophilus NEAU-A-2T. Based on the differential phenotypic characteristics and the results of DNA-DNA relatedness and phylogenetic analysis, it is proposed that strain YIM CF22T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes deserti sp. nov. is proposed. The type strain is YIM CF22T (= KCTC 39543T = CCTCC AB2018113T).


Assuntos
Parede Celular/química , Micromonosporaceae/genética , Peptidoglicano/química , Filogenia , RNA Ribossômico 16S/genética , Composição de Bases , Parede Celular/metabolismo , China , Clima Desértico , Ácido Diaminopimélico/metabolismo , Ácidos Graxos/metabolismo , Galactose/metabolismo , Glucose/metabolismo , Glicolipídeos/metabolismo , Manose/metabolismo , Micromonosporaceae/classificação , Micromonosporaceae/isolamento & purificação , Peptidoglicano/metabolismo , Fosfolipídeos/metabolismo , Ribose/metabolismo , Microbiologia do Solo , Esporos Bacterianos/química , Esporos Bacterianos/crescimento & desenvolvimento , Vitamina K 2/metabolismo , Xilose/metabolismo
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