Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 111
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Plant J ; 101(1): 18-36, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31454118

RESUMO

The plant flavonoid dogma proposes that labile plant flavonoid carbocations (PFCs) play vital roles in the biosynthesis of proanthocyanidins (PAs). However, whether PFCs exist in plants and how PFCs function remain unclear. Here, we report the use of an integrative strategy including enzymatic assays, mutant analysis, metabolic engineering, isotope labeling and metabolic profiling to capture PFCs and demonstrate their functions. In anthocyanidin reductase (ANR) assays, an (-)-epicatechin conjugate was captured in protic polar nucleophilic methanol alone or methanol-HCl extracts. Tandem mass spectrum (MS/MS) analysis characterized this compound as an (-)-epicatechin-4-O-methyl (EOM) ether, which resulted from (-)-epicatechin carbocation and the methyl group of methanol. Acid-based catalysis of procyanidin B2 and B3 produced four compounds, which were annotated as two EOM and two (+)-catechin-4-O-methyl (COM) ethers. Metabolic profiling of seven PA pathway mutants showed an absence or reduction of two EOM ether isomers in seeds. Camellia sinensis ANRa (CsANRa), leucoanthocyanidin reductase c (CsLARc), and CsMYB5b (a transcription factor) were independently overexpressed for successful PA engineering in tobacco. The EOM ether was remarkably increased in CsANRa and CsMYB5b transgenic flowers. Further metabolic profiling for eight green tea tissues revealed two EOM and two COM ethers associated with PA biosynthesis. Moreover, an incubation of (-)-epicatechin or (+)-catechin with epicatechin carbocation in CsANRa transgenic flower extracts formed dimeric procyanidin B1 or B2, demonstrating the role of flavan-3-ol carbocation in the formation of PAs. Taken together, these findings indicated that flavan-3-ol carbocations exist in extracts and are involved in the biosynthesis of PAs of plants.

2.
Sci China Life Sci ; 62(12): 1638-1654, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31820200

RESUMO

Staurosporine, belonging to indolocarbazole compounds, is regarded as an excellent lead compound for synthesizing antitumor agents as a potent inhibitor against various protein kinases. In this study, two separate clusters (cluster A and cluster B), corresponding to biosyntheses of K-252c (staurosporine aglycone) and sugar moiety, were identified in Streptomyces fradiae CGMCC 4.576 and heterologously expressed in Streptomyces coelicolor M1146 separately or together. StaR, a cluster-situated LAL family regulator, activates staurosporine biosynthesis by binding to the promoter regions of staO-staC and staG-staN. The conserved sequences GGGGG and GCGCG were found through gradually truncating promoters of staO and staG, and further determined by mutational experiments. Overexpression of staR with the supplementation of 0.01 g L-1 FeSO4 increased staurosporine production to 5.2-fold compared with that of the parental strain Streptomyces fradiae CGMCC 4.576 in GYM medium. Our results provided an approach for improvement of staurosporine production mediated by a positive regulator and established the basis for dissecting the regulatory mechanisms of other indolocarbazole compounds with clinical application value.

3.
J Antibiot (Tokyo) ; 72(12): 906-912, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31501499

RESUMO

Antimicrobial resistance is a major threat for public health worldwide. Novel antimicrobial drugs are urgently needed to combat the growing prevalence of antimicrobial resistance. Nucleoside antibiotics represent a unique class of microbial natural products with distinctive structural features and diverse biological activities. We herein summarize recent findings on the biosynthesis of representative nucleoside antibiotics, and highlight recent advances in the discovery and rational generation of nucleoside antibiotics for the development of novel antimicrobial agents.

4.
Appl Microbiol Biotechnol ; 103(5): 2263-2275, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30685809

RESUMO

Neomycin, an aminoglycoside antibiotic, is widely used in the livestock husbandry due to its higher antimicrobial activity and availability of feed additives in animals. However, its production yield is relatively low and cannot meet the needs of developing market and clinical application. Here, the entire natural neo cluster was cloned from Streptomyces fradiae CGMCC 4.576 by φBT1 integrase-mediated site-specific recombination. Then, the rational reconstruction of the neo cluster was performed by using λ-Red-mediated PCR targeting for improving neomycin production. In order to coordinate with this attempt, the supplementation of suitable precursors was carried out. The constructed recombinant strain Sf/pKCZ03 has multi-copy of the neo cluster modified by disrupting the negative regulatory gene neoI and replacing the native promoter of the neoE-D with PkasO*. Compared to the yield (1282 mg/L) of Streptomyces fradiae CGMCC 4.576, the engineered strain Sf/pKCZ03 had a 36% enhancement of neomycin production. Quantitative real-time PCR analysis revealed the increased transcription of structural genes (neoE, neoB, neoL, aacC8) and regulatory genes (neoR, neoH) in Sf/pKCZ03. Additionally, under the supplementation of 1 g/L N-acetyl-D-glucosamine and 5 g/L L-glutamine, the yield of engineered strain Sf/pKCZ03 showed 62% and 107% improvements compared to that of the wild-type strain in the original medium, respectively. These findings demonstrated that engineering the antibiotic gene cluster in combination with precursors feeding was an effective approach for strain improvement, and would be potentially extended to other Streptomyces for large-scale production of commercialized antibiotics.


Assuntos
Antibacterianos/biossíntese , Neomicina/biossíntese , Engenharia de Proteínas/métodos , Streptomyces/genética , Streptomyces/metabolismo , Clonagem Molecular/métodos , Testes de Sensibilidade Microbiana , Família Multigênica/genética , Staphylococcus aureus/efeitos dos fármacos
6.
Synth Syst Biotechnol ; 3(4): 261-267, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30417142

RESUMO

Streptomyces are the soil-dwelling bacteria with a complex lifecycle and a considerable ability to produce a variety of secondary metabolites. Osmoregulation is important for their lifecycle in nature. In the genome of Streptomyces coelicolor M145, SCO3128 (encodes a putative fatty acid desaturase), SCO3129 (encodes a putative TetR family regulator) and SCO3130 (encodes a putative l-carnitine dehydratase) constitute a transcriptional unit, and its transcript was found to be in response to osmotic stress. Disruption of SCO3130 led to a bald phenotype on MMG medium and the mycelia lysis on the edge of the colony when KCl/NaCl was added to the medium. These results indicated that SCO3130 is important for the osmotic stress resistance in S. coelicolor. Transcriptional analysis and electrophoretic mobility shift assays (EMSA) demonstrated that SCO3129 repressed the transcription of SCO3128-3130 operon through directly binding to the promoter region of SCO3128, indicating that SCO3129 regulates the transcription of SCO3128-3130 in response to osmotic stress.

7.
J Biol Chem ; 293(52): 20029-20040, 2018 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-30355730

RESUMO

Butenolides are an emerging family of signaling molecules in Streptomyces. They control complex physiological traits, such as morphological differentiation and antibiotic production. However, how butenolides regulate these processes is poorly investigated because of obstacles in obtaining these signaling molecules. This study reports the identification of a butenolide-type signaling system for nikkomycin biosynthesis in Streptomyces ansochromogenes with distinct features. We identified a gene cluster, sab, consisting of three genes, sabAPD, for butenolide biosynthesis and two regulator genes, sabR1 and sabR2, and characterized three butenolides (SAB1, -2, and -3) by heterologous expression of sabAPD. sabA disruption abolished nikkomycin production, which could be restored by the addition of SABs or by deletion of sabR1 in ΔsabA. Electrophoretic mobility-shift assays and transcriptional analyses indicated that SabR1 indirectly represses the transcription of nikkomycin biosynthetic genes, but directly represses sabA and sabR1 In the presence of SABs, the SabR1 transcriptional regulator dissociated from its target genes, verifying that SabR1 is the cognate receptor of SABs. Genome-wide scanning with the conserved SabR1-binding sequence revealed another SabR1 target gene, cprC, whose transcription was strongly repressed by SabR1. Intriguingly, CprC positively regulated the pleiotropic regulatory gene adpA by binding to its promoter and, in turn, activated nikkomycin biosynthesis. This is the first report that butenolide-type signaling molecules and their cognate receptor SabR1 can regulate adpA via a newly identified activator, CprC, to control nikkomycin production. These findings pave the way for further studies seeking to unravel the regulatory mechanism and functions of the butenolide signaling system in Streptomyces.


Assuntos
4-Butirolactona/análogos & derivados , Aminoglicosídeos/metabolismo , Vias Biossintéticas , Streptomyces/metabolismo , 4-Butirolactona/genética , 4-Butirolactona/metabolismo , Aminoglicosídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Família Multigênica , Streptomyces/genética , Ativação Transcricional
8.
Cell Chem Biol ; 25(2): 143-153.e4, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29198568

RESUMO

Nosiheptide, an archetypal member of thiopeptide antibiotics, arises from post-translational modifications of a ribosomally synthesized precursor peptide that contains an N-terminal leader peptide (LP) sequence and a C-terminal core peptide (CP) sequence. Despite extensive efforts concerning the biosynthesis of thiopeptide antibiotics, the regulatory mechanisms in this process remain poorly understood. Using the nosiheptide-producing Streptomyces actuosus strain as a model system, we report here that NosP, a Streptomyces antibiotic regulatory protein, serves as the only cluster-situated regulator and activates the transcription of all structural genes, which are organized into two divergently transcribed operons in the nos cluster, by binding to their intergenic region. NocP, the counterpart of NosP in Nocardia sp., regulates the production of structurally related nocathiacin I in a similar manner. NosP activity senses the nosiheptide biosynthetic process by interactions with both peptidyl and small-molecule ligands that result from the LP and CP parts of the precursor peptide, respectively.


Assuntos
Proteínas de Bactérias/metabolismo , Peptídeos/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Ligantes , Conformação Molecular , Peptídeos/química , Bibliotecas de Moléculas Pequenas/química , Streptomyces/química , Streptomyces/metabolismo , Tiazóis/metabolismo
9.
J Biol Chem ; 292(48): 19708-19720, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28972184

RESUMO

Genome sequencing analysis has revealed at least 35 clusters of likely biosynthetic genes for secondary metabolites in Streptomyces ansochromogenes. Disruption of adpA encoding a global regulator (AdpA) resulted in the failure of nikkomycin production, whereas other antibacterial activities against Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis were observed with the fermentation broth of ΔadpA but not with that of the wild-type strain. Transcriptional analysis showed that a cryptic gene cluster (pks7), which shows high identity with an oviedomycin biosynthetic gene cluster (ovm), was activated in ΔadpA. The corresponding product of pks7 was characterized as oviedomycin by MS and NMR spectroscopy. To understand the molecular mechanism of ovm activation, the roles of six regulatory genes situated in the ovm cluster were investigated. Among them, proteins encoded by co-transcribed genes ovmZ and ovmW are positive regulators of ovm AdpA directly represses the transcription of ovmZ and ovmW Co-overexpression of ovmZ and ovmW can relieve the repression of AdpA on ovm transcription and effectively activate oviedomycin biosynthesis. The promoter of ovmOI-ovmH is identified as the direct target of OvmZ and OvmW. This is the first report that AdpA can simultaneously activate nikkomycin biosynthesis but repress oviedomycin biosynthesis in one strain. Our findings provide an effective strategy that is able to activate cryptic secondary metabolite gene clusters by genetic manipulation of global regulatory genes.


Assuntos
Aminoglicosídeos/genética , Genes Bacterianos , Genes Reguladores , Família Multigênica , Streptomyces/genética , Aminoglicosídeos/biossíntese , Éteres Cíclicos
11.
Sci China Life Sci ; 60(9): 939-947, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28785949

RESUMO

There is an urgent need for new antifungal agents to treat or combat fungal infection in humans and plants. Antifungal nucleoside antibiotics are an important family of natural products with distinctive structural features. Understanding their biosynthetic machinery is of great importance for the improvement of antibiotics titers. More importantly, it is a requisite for combinatorial biosynthesis to create hybrid nucleoside antibiotics. We herein focus on findings on the natural and designed biosynthesis of this important family of nucleoside antibiotics.


Assuntos
Antifúngicos/metabolismo , Vias Biossintéticas , Nucleosídeos/biossíntese , Antifúngicos/química , Engenharia Metabólica , Nucleosídeos/química , Nucleosídeos/genética
12.
Sci China Life Sci ; 60(9): 968-979, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28840532

RESUMO

Genetic modification of large DNA fragments (gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity. In this study, we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction (PCR) targeting combined with Gibson assembly. In this strategy, the biosynthetic genes for peptidyl moieties (HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid (CPOAA) from the polyoxin biosynthetic gene cluster to generate a ~40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette. The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic, polynik A, was obtained and verified. This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.


Assuntos
Aminoglicosídeos/genética , Engenharia Genética/métodos , Família Multigênica/genética , Streptomyces/genética , Antifúngicos/metabolismo , Vias Biossintéticas/genética , Ácido Oxâmico/análogos & derivados , Nucleosídeos de Pirimidina/genética , Streptomyces/metabolismo
13.
Sci China Life Sci ; 60(9): 980-991, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28812297

RESUMO

Neomycins are a group of aminoglycoside antibiotics with both clinical and agricultural applications. To elucidate the regulatory mechanism of neomycin biosynthesis, we completed draft genome sequencing of a neomycin producer Streptomyces fradiae CGMCC 4.7387 from marine sediments, and the neomycin biosynthesis gene cluster was identified. Inactivation of the afsA-g gene encoding a γ-butyrolactone (GBL) synthase in S. fradiae CGMCC 4.7387 resulted in a significant decrease of neomycin production. Quantitative RT-PCR analysis revealed that the transcriptional level of neoR and the aphA-neoGH operon were reduced in the afsA-g::aac(3)IV mutant. Interestingly, a conserved binding site of AdpA, a key activator in the GBL regulatory cascade, was discovered upstream of neoR, a putative regulatory gene encoding a protein with an ATPase domain and a tetratricopeptide repeat domain. When neoR was inactivated, the neomycin production was reduced about 40% in comparison with the WT strain. Quantitative RT-PCR analysis revealed that the transcriptional levels of genes in the aphA-neoGH operon were reduced clearly in the neoR::aac(3)IV mutant. Finally, the titers of neomycin were improved considerably by overexpression of afsA-g and neoR in S. fradiae CGMCC 4.7387.


Assuntos
Proteínas de Bactérias/genética , Família Multigênica/genética , Neomicina/biossíntese , Streptomyces/genética , DNA Bacteriano/análise , DNA Bacteriano/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Óperon/genética , Reação em Cadeia da Polimerase em Tempo Real , Streptomyces/metabolismo
14.
Wei Sheng Wu Xue Bao ; 56(3): 406-17, 2016 Mar 04.
Artigo em Chinês | MEDLINE | ID: mdl-27382784

RESUMO

OBJECTIVE: To determine the functions of gouC and gouD in gougerotin biosynthesis, disruption of these two genes was performed. As gougerotin producing strain Streptomyces graminearus lacks efficient genetic manipulation system, the gene cluster for gougerotin biosynthesis was heterologously expressed in Streptomyces coelicolor M1146 to facilitate genetic manipulations of gouC and gouD. METHODS: By using fosmid D6-4H containing the complete gougerotin biosynthetic gene cluster, gouC and gouD were disrupted by PCR-targeting method to generate pGOUe-ΔC and pGOUe-ΔD. Both pGOUe-ΔC and pGOUe-ΔD were introduced into Streptomyces coelicolor M1146 by intergeneric conjugation, thus gouC and gouD disrpution mutants (Ml146-GOUe-AC and M1146-GOUe-ΔD) were obtained. The gougerotin production of M1146-GOUe-ΔC and M1146-GOUe-ΔD were assayed by HPLC analysis. The intermediates accumulated in these mutants were purified and subjected to MS and NMR analyses for structure determinations. Bioassay of these intermediates against tumor cell line were also carried out. RESULTS: Disruption mutants of gouC and gouD failed to produce gougerotin and the mutants accumulated different gougerotin intermediates, which lost their ability to inhibit cancer cell proliferation. CONCLUSION: gouC and gouD are key structual genes in the biosynthesis of gougerotin peptidyl moieties. This study will pave the way for the elucidation of gougerotin biosynthetic pathway.


Assuntos
Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Estrutura Molecular , Família Multigênica , Nucleosídeos de Pirimidina/biossíntese , Nucleosídeos de Pirimidina/química , Streptomyces/química , Streptomyces/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
15.
FEMS Microbiol Rev ; 40(4): 554-73, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27288284

RESUMO

Streptomyces bacteria are the major source of antibiotics and other secondary metabolites. Various environmental and physiological conditions affect the onset and level of production of each antibiotic by influencing concentrations of the ligands for conserved global regulatory proteins. In addition, as reviewed here, well-known autoregulators such as γ-butyrolactones, themselves products of secondary metabolism, accumulate late in growth to concentrations allowing their effective interaction with cognate binding proteins, in a necessary prelude to antibiotic biosynthesis. Most autoregulator binding proteins target the conserved global regulatory gene adpA, and/or regulatory genes for 'cluster-situated regulators' (CSRs) linked to antibiotic biosynthetic gene clusters. It now appears that some CSRs bind intermediates and end products of antibiotic biosynthesis, with regulatory effects interwoven with those of autoregulators. These ligands can exert cross-pathway effects within producers of more than one antibiotic, and when excreted into the extracellular environment may have population-wide effects on production, and mediate interactions with neighbouring microorganisms in natural communities, influencing speciation. Greater understanding of these autoregulatory and cross-regulatory activities may aid the discovery of new signalling molecules and their use in activating cryptic antibiotic biosynthetic pathways.


Assuntos
Antibacterianos/biossíntese , Regulação Bacteriana da Expressão Gênica , Interações Microbianas/fisiologia , Streptomyces/fisiologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Ligantes , Ligação Proteica , Transdução de Sinais , Streptomyces/genética
16.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1017-1018: 10-17, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26937589

RESUMO

Phenolic compounds are major components of tea flavour, in which catechins and flavonol glycosides play important roles in the astringent taste of tea infusion. However, the flavonol glycosides are difficult to quantify because of the large variety, as well as the inefficient seperation on chromatography. In this paper, a total of 15 flavonol glycosides in the tea plant (Camellia sinensis) were identified by the high performance liquid chromatography (HPLC) coupled to a time-of-flight mass spectrometer (TOF-MS), and a quantitative method was established based on multiple reaction monitoring (MRM) mode of ultra-high performance liquid chromatography (UPLC) coupled to a triple quadrupole mass spectrometer (QQQ-MS/MS). It provided the limit of detection and quantification to the order of picogram, which was more sensitive than the HPLC detection of the order of nanogram. The relative standard deviations of the intra- and inter-day variations in retention time and signal intensity (peak area) of six analytes were less than 0.26% and 4%, respectively. The flavonol glycosides of four tea cultivars were relatively quantified using the signal intensity (peak area) of product ion, in which six flavonol glycosides were quantified by the authentic standards. The results showed that the flavonol mono-, di- and tri-glycoside mostly accumulated in young leaves of the four tea cultivars. Notably, the myricetin 3-O-galactoside was the major component among the six flavonol glycosides detected.


Assuntos
Camellia sinensis/química , Cromatografia Líquida de Alta Pressão/métodos , Flavonóis/análise , Glicosídeos/análise , Espectrometria de Massas em Tandem/métodos , Limite de Detecção
17.
J Biol Chem ; 291(10): 5406-17, 2016 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-26750095

RESUMO

Chlorothricin, isolated from Streptomyces antibioticus, is a parent member of spirotetronate family of antibiotics that have long been appreciated for their remarkable biological activities. ChlF1 plays bifunctional roles in chlorothricin biosynthesis by binding to its target genes (chlJ, chlF1, chlG, and chlK). The dissociation constants of ChlF1 to these genes are ∼ 102-140 nm. A consensus sequence, 5'-GTAANNATTTAC-3', was found in these binding sites. ChlF1 represses the transcription of chlF1, chlG, and chlK but activates chlJ, which encodes a key enzyme acyl-CoA carboxyl transferase involved in the chlorothricin biosynthesis. We demonstrate that the end product chlorothricin and likewise its biosynthetic intermediates (demethylsalicycloyl chlorothricin and deschloro-chlorothricin) can act as signaling molecules to modulate the binding of ChlF1 to its target genes. Intriguingly, a correlation between the antibacterial activity and binding ability of signaling molecules to the regulator ChlF1 is clearly observed. These features of the signaling molecules are associated with the glycosylation of spirotetronate macrolide aglycone. The findings provide new insights into the TetR family regulators responding to special structure of signaling molecules, and we reveal the regulatory mini-network mediated by ChlF1 in chlorothricin biosynthesis for the first time.


Assuntos
Aminoglicosídeos/biossíntese , Antibacterianos/biossíntese , Produtos Finais de Glicação Avançada/metabolismo , Streptomyces antibioticus/metabolismo , Aminoglicosídeos/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transdução de Sinais , Streptomyces antibioticus/genética
18.
Wei Sheng Wu Xue Bao ; 56(9): 1371-84, 2016 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-29738206

RESUMO

Salinomycin is a monocarboxylic acid polyether antibiotics produced by Streptomyces albus. It has strong inhibiting and killing activity against most gram-positive bacteria and various coccidiums with low adverse impact on environment. In addition, salinomycin can specifically inhibit the growth of a variety of cancer cells and cancer stem cells via targeting to multiple sites, and is a promising anti-tumor drug candidate. To obtain high yield salinomycinproducing strain, conventional mutation techniques and modern molecular genetic methods have been used. Meanwhile, bioactivity and selectivity of salinomycin could be improved by modifying the chemical structure and changing drug delivery methods. Here, we summarize the key strategies for enhancing salinomycin production and review the progresses in optimizing its drug activity and targeting properties. The future research focus is also addressed.


Assuntos
Antibacterianos/biossíntese , Antineoplásicos/metabolismo , Piranos/metabolismo , Streptomyces/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Piranos/química , Piranos/farmacologia , Streptomyces/genética
19.
Wei Sheng Wu Xue Bao ; 55(6): 707-18, 2015 Jun 04.
Artigo em Chinês | MEDLINE | ID: mdl-26562995

RESUMO

OBJECTIVE: We expressed a nikkomycin biosynthetic gene cluster in the well-characterized surrogate Streptomyces coelicolor M1146. METHODS: By using PCR-targeting method, we replaced the promoters of sanG and sanF in pNIK, which contains nikkomycin biosynthetic gene cluster, with the hrdB promoter to generate pNIKm. We transferred pNIK and pNIKm into S. coelicolor M1146 by intergeneric conjugation and obtained M1146-NIK and M1146-NIKm, respectively. We then evaluated expression of the gene cluster in the heterologous host by RT-PCR. Furthermore, we also compared the antifugal activity and nikkomycin production of M1146-NIK and M1146-NIKm by bioassay against Alternaria longipes and HPLC analysis. RESULTS: M1146-NIK and M1146-NIKm exhibited antifungal activity, and they can produce a trace amount of nikkomycin X, nikkomycin Z and pseudo-Z. There was a substantial accumulation of uridine in M1146-NIK, whereas substantial accumulations of uridine, ribofuranosyl-4-formyl-4-imidazolone and pyridylhomothreonine were observed in M1146-NIKm. CONCLUSION: We successfully expressed the nikkomycin biosynthetic gene cluster in the heterologous host and identified nikkomycins and some of its key biosynthetic intermediates. This study will provide the basis for enzymatic reaction of the condensation between the two nikkomycin moieties and for the generation of hybrid antibiotics by combinatorial biosynthesis.


Assuntos
Aminoglicosídeos/biossíntese , Antifúngicos/metabolismo , Proteínas de Bactérias/genética , Streptomyces/genética , Streptomyces/metabolismo , Aminoglicosídeos/química , Antifúngicos/química , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Engenharia Metabólica , Família Multigênica , Regiões Promotoras Genéticas
20.
Microb Cell Fact ; 14: 173, 2015 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-26525981

RESUMO

BACKGROUND: Streptomyces, as the main source of antibiotics, has been intensively exploited for discovering new drug candidates to combat the evolving pathogens. Disruption of wblA, an actinobacteria-specific gene controlling major developmental transition, can cause the alteration of phenotype and morphology in many species of Streptomyces. One wblA homologue was found in Streptomyces ansochromogenes 7100 by using the Basic Local Alignment Search Tool. It is interesting to identify whether novel secondary metabolites could be produced by the wblA disruption mutant as evidenced in other Streptomyces. RESULTS: The wblA disruption mutant of S. ansochromogenes 7100 (ΔwblA) was constructed by homologous recombination. ΔwblA failed to produce spores and nikkomycin, the major product of S. ansochromogenes 7100 (wild-type strain) during fermentation. Antibacterial activity against Staphylococcus aureus and Bacillus cereus was observed with fermentation broth of ΔwblA but not with that of the wild-type strain. To identify the antibacterial compounds, the two compounds (compound 1 and compound 2) produced by ΔwblA were characterized as 16-membered macrolides by mass spectrometry and nuclear magnetic resonance spectroscopy. The chemical structure of these compounds shows similarity with tylosin, and the bioassays indicated that the two compounds inhibited the growth of a number of gram-positive bacteria. It is intriguing that they displayed much higher activity than tylosin against Streptococcus pneumoniae. CONCLUSIONS: Two novel tylosin analogues (compound 1 and 2) were generated by ΔwblA. Bioassays showed that compound 1 and 2 displayed much higher activity than tylosin against Streptococcus pneumoniae, implying that these two compounds might be used to widen the application of tylosin.


Assuntos
Antibacterianos/química , Proteínas de Bactérias/genética , Glicosídeos/química , Streptomyces/genética , Tilosina/análogos & derivados , Aminoglicosídeos/farmacologia , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Conformação Molecular , Mutação , Fenótipo , Streptococcus pneumoniae/efeitos dos fármacos , Streptomyces/química , Streptomyces/metabolismo , Tilosina/química , Tilosina/isolamento & purificação , Tilosina/metabolismo , Tilosina/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA