欧洲疮痂链霉菌F5的鉴定及其抗菌活性

【背景】木豆(Cajanus cajan)是一种具有多种药理活性的药用植物,目前对其根际功能放线菌的认识和研究有限,有必要对其应用开发潜力进行研究。【目的】从木豆根际土中筛选一株对植物病原菌和常见病原菌具有广谱拮抗活性的放线菌菌株,鉴定菌株的分类地位、相关代谢产物及可能的生物合成途径,为该菌株的开发应用提供数据支撑。【方法】以7种常见植物病原真菌及8种常见病原菌为指示菌,采用平板对峙法和滤纸片扩散法筛选具有广谱抗菌活性的放线菌菌株,基于形态观察与系统发育分析对该菌株进行分类鉴定,并通过高分辨质谱和超高效液相色谱-串联质谱(UPLC-MS/MS)对活性菌株的次生代谢产物进行鉴定与验证。采用PCR扩增菌株聚酮合成酶Ⅰ(polyketide synthase,PKS-Ⅰ)和非核糖体多肽合成酶(non-ribosomal peptide synthetase,NRPS)基因,明确其活性代谢产物可能的生物合成途径。【结果】通过抑菌试验筛选得到拮抗放线菌F5,确定其为欧洲疮痂链霉菌(Streptomyces europaeiscabiei),F5菌株基因组中含有编码PKS-Ⅰ和NRPS合成的相关基...     

阔苞菊内生放线菌的分离鉴定和抗菌活性研究

为了分离鉴定阔苞菊内生放线菌并研究其抑菌活性,通过与高氏一号培养基和1/10ATCC CL-173培养基的对比试验,选用改进的HV培养基从阔苞菊中分离纯化出48株内生放线菌;对其16S rDNA进行PCR扩增及基因测序,结果显示其中79.17%为链霉菌属菌株;对所提取放线菌基因组DNA进行BOX-PCR多样性指纹分析,结果与16Sr DNA序列分析的结果一致;利用滤纸片法测量其抑菌活性,其中菌株KBJ37、KBJ52、KBJ114对诺氏布丘氏菌、气单胞菌、乡间布丘氏菌、小牛葡萄球菌有抑制效果,仅有KBJ114对酿酒酵母有抑制效果;通过检测编码非核糖体多肽合成酶(NRPS)和聚酮化合物合酶基因Ⅰ、Ⅱ(PKS-Ⅰ和PKS-Ⅱ)的序列,来判断阔苞菊内生放线菌合成生物活性物质的潜力,结果显示97.92%的放线菌拥有合成生物活性物质的潜力。以上研究表明阔苞菊内生放线菌具有较为广谱的抑菌活性,且绝大多数都具有合成生物活性物质的潜力,为进一步探明阔苞菊内生放线菌资源多样性及药用价值提供了理论依据。     

河北九莲城淖尔可培养放线菌多样性及抗菌活性筛选

【目的】勘探干涸的九莲城淖尔土壤放线菌多样性并进行活性筛选,以期发现药用微生物资源,为新抗生素的发现奠定基础。【方法】采用15种分离培养基,以稀释涂布法分离放线菌;根据分离菌株的16S rRNA基因序列同源性分析放线菌多样性;发酵液经乙酸乙酯萃取,菌丝体经丙酮浸提,获得提取浓缩物样品;样品通过纸片扩散法进行抗菌活性初筛;抗菌阳性菌株采用PCR技术进行Ⅰ型聚酮合酶(PKS I)KS域、Ⅱ型聚酮合酶(PKS II)KS域和非核糖体多肽合成酶(NRPS)A结构域抗生素生物合成基因的检测。【结果】从11份盐湖土壤样品中分离纯化到251株放线菌,其分布于放线菌纲的10个目15个科31个属,其中优势菌属为链霉菌属和拟诺卡氏菌属;251株放线菌中包括57株耐(嗜)盐放线菌,其优势菌属为拟诺卡氏菌属(22株)和涅斯捷连科氏菌属(15株)。基于16S r RNA基因序列的系统发育分析显示,菌株J11Y309为糖霉菌科潜在新属,菌株J12GA03为分枝杆菌科潜在新种。96株放线菌活性检测结果显示,56株至少对1株检定菌具有抗菌活性,阳性率为58.3%;56株有活性的放线菌中,47株至少含有1种抗生素生物合成基因,其中17株同时具有3种抗生素生物合成基因。【结论】干涸的九莲城淖尔土壤中含有较为丰富的药用放线菌资源,具有从中发现放线菌新物种和新抗生素的潜力。     

武陵山放线菌多样性

[目的]为了探究武陵山放线菌多样性,以便从新放线菌菌株中发现新的潜在药物先导化合物.[方法]从武陵山采集280份土样,采用纯培养的方法,用4种培养基分离到1134株放线菌.选择其中30株代表菌进行了初步分类鉴定;以3株细菌和7株农作物致病真菌作为指示菌,检测其抑菌活性;利用特异性引物扩增的方法,检测是否具有聚酮合酶(PKS Ⅰ、PKSⅡ)基因、非核糖体多肽合成酶(NRPS)基因和多烯类化合物合成酶(CYP)基因.[结果]分离到的武陵山放线菌中,链霉菌占70%以上,还有小单孢菌等8个科13个属,其中有5个菌株是潜在的新种.选取的30株实验菌对细菌、真菌有不同程度的抗菌活性;其中含有4类化合物合成基因的菌株占23%～60%.[结论]武陵山原始森林土壤中,放线菌多样性很丰富,且存在很多未开发的稀有类群.有抑菌活性的菌株,可用于进一步的药物开发利用.     

大香格里拉土壤放线菌组成分析及生物活性测定

【目的】探究大香格里拉地区的土壤放线菌组成及其抑菌和酶活性,为放线菌新药物先导化合物和高活性酶的筛选提供资源。【方法】从大香格里拉地区不同海拔高度采集220份土样,用4种培养基,分别进行了常温放线菌和低温放线菌的分离。从常温放线菌中选择25株代表菌株进行了初步分类鉴定。采用琼脂扩散法,检测了其对4株细菌和7株农作物致病真菌的抑菌活性;利用特异性引物扩增法,筛选了其聚酮合酶(PKSⅠ、PKSⅡ)基因、非核糖体多肽合成酶(NRPS)基因和多烯类化合物合成酶(CYP)基因。同时,检测了低温菌的多种酶活性。【结果】25株常温代表菌的系统发育分析显示它们分属于放线菌目的6个亚目、12个科、15个属。其中14株菌的NRPS及11株的CYP基因筛选呈阳性。低温条件下分离到的111株放线菌,88%属于耐冷菌,12%是嗜冷菌,它们中的大部分能利用明胶、纤维素,甲壳素等。【结论】研究结果显示,大香格里拉森林土壤蕴涵着种类和活性丰富的放线菌,为放线菌资源的开发利用和保护提供了新依据。     

西藏仲巴五彩沙漠放线菌资源勘探及生物活性筛选

【背景】细菌耐药性问题日益严峻,新抗生素的研发速度远远落后于临床需要,从特殊生境中挖掘微生物药物资源有望解决以上问题。【目的】勘探西藏仲巴五彩沙漠土壤放线菌多样性并进行生物活性筛选,为发现药用放线菌资源、开发新型抗生素奠定基础。【方法】采用8种分离培养基,通过平板稀释涂布法分离放线菌;根据分离菌株的16S r RNA基因序列同源性分析放线菌多样性;采用PCR技术对分离的放线菌菌株进行II型聚酮合酶(PKS-II)酮缩酶结构域KS、非核糖体多肽合成酶(NRPS)腺苷酸化结构域A、安莎类抗生素生物合成前体3-氨基-5-羟基-苯甲酸合酶(AHBA)保守区、黄素腺嘌呤二核苷酸卤化酶(Halo)保守区抗生素生物合成基因检测;对生物合成基因检测阳性的菌株进行液体发酵,发酵液经乙酸乙酯萃取、菌体经丙酮浸提,获得提取浓缩物样品进行抑菌活性和抗氧化活性筛选。【结果】从4份土样中分离纯化到231株放线菌,分布于7个属,其中链霉菌为优势菌属。68株放线菌的生物合成基因分析显示至少具有1种生物合成基因簇,其中6株同时具有4种生物合成基因簇;进一步的抑菌活性检测显示所有检测的菌株至少表现为对1株检定菌具有抑菌活性,其中8株具有广谱抗菌活性;抗氧化活性筛选结果为13株显示总抗氧化能力阳性,10株具有较好的羟自由基清除能力,3株显示较强的氧自由基清除能力。【结论】西藏仲巴五彩沙漠土壤中含有较丰富的放线菌药用资源,具有从中发现放线菌新菌种和开发新抗生素的潜力。     

罗布泊盐湖可培养放线菌多样性及PKS Ⅱ功能基因筛选

目的:挖掘新疆罗布泊盐湖可培养放线菌的资源和探索该环境放线菌产生Ⅱ型聚酮合成酶(PKS)基因的潜能。方法:从新疆罗布泊盐湖采集到10份样品,采用不同盐浓度的9种选择性培养基分离放线菌,并通过PCR技术扩增其16S r DNA序列、聚酮合成酶(PKS)基因分子筛选,测序后进行系统发育分析和BLAST比较研究。结果:试验结果共得42株放线菌,包括6个属,链霉菌属(Streptomyces)和拟诺卡氏菌属(Nocardiopsis)的类群最多,糖霉菌属(Glycomyces)、放线多孢菌属(Actinopolyspora)、微球菌(Micrococcus)和栖白蚁菌属(Isoptericola)的菌株相对较少。研究获得新的放线菌分类单元,其16S r DNA基因序列与已知菌株的序列相似性低于97%。42株放线菌中共有29株产生PKSⅡ基因,主要是链霉菌属和拟诺卡氏菌属。结论:研究结果表明罗布泊盐湖放线菌具有丰富的资源多样性和较强合成抗生素的潜在能力。     

一株抗万古霉素耐药菌的抗生素产生菌AR1148的鉴定

从土壤中分离得到一株放线菌AR1148,其代谢产物对万古霉素耐药肠球菌有较明显的抑菌活性。该菌株的基内菌丝无横隔,气生菌丝丰茂,分枝较好,孢子椭圆形,表面光滑。菌丝细胞壁工型,含有L-2,6-二氨基庚二酸（LL-DAP）和甘氨酸。菌株AR1148归属于链霉菌属金色类群。根据16SrDNA序列分析,该菌株与现有种差异明显,聚类在不同的分支。定名为Streptomyces sp．AR1148。     

一株抗MRSA的土壤放线菌的分离与鉴定

目的：从土壤中分离筛得一株对MRSA有明显抑菌活性的菌株。方法：分离得到一株放线菌NB0701,通过其生理生化反应,细胞壁化学组成、菌株细胞壁、糖型以及rRNA基因的序列进行了分析。结果：菌株细胞壁Ⅰ型,糖型C型,rRNA基因的序列分析其与Streptomyces parvus在16SrDNA中Blast比对相似率达99.9%。结论：定名为Streptomyces parvus subsp NB。     

北极海洋链霉菌604F的卤化酶基因克隆及特征

【目的】本研究旨在克隆来自北极海洋、具有合成卤化物潜力的链霉菌(Streptomyces sp.)604F中的一个卤化酶基因,为后续克隆卤化物合成基因簇、分离鉴定卤化物提供指导。【方法】利用琼脂块法初步测试抗菌活性,借助液相色谱-飞行时间串联质谱技术(LC-Tof MS)初步寻找Streptomyces sp.604F发酵粗提液中的卤化物,并以简并PCR扩增合成次级代谢产物的指示基因(I型、II型聚酮合酶及非核糖体多肽合成酶编码基因);根据依赖黄素腺嘌呤二核苷酸的卤化酶基因保守区设计的简并引物,扩增基因片段并测序分析;采用高效热不对称交错PCR(hiTAIL-PCR)技术扩增卤化酶基因全长。【结果】Streptomyces sp.604F具有较强的抗白色念珠菌活性,其基因组同时含有编码I型聚酮合酶、II型聚酮合酶和非核糖体多肽合成酶基因,以及卤化酶基因;通过染色体步移克隆该卤化酶基因全长,共1443 bp,编码一个新的非色氨酸卤化酶,在合成已知卤化物的卤化酶数据库中,与其同源关系最近的为一类参与合成糖肽类次级代谢产物的卤化酶。【结论】Streptomyces sp.604F具有新的非色氨酸卤化酶,且推测参与糖肽类化合物的卤化修饰,为后续寻找目的卤化物提供了指导,也为研究该合成基因簇奠定基础。     

Engineered biosynthesis of a novel amidated polyketide, using the malonamyl-specific initiation module from the oxytetracycline polyketide synthase

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.     

Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.     

A novel quinone-forming monooxygenase family involved in modification of aromatic polyketides

RppA is a type III polyketide synthase (PKS) that catalyzes condensation of five molecules of malonyl-CoA to form 1,3,6,8-tetrahydroxynaphthalene (THN). In Streptomyces antibioticus IFO13271 and several other Streptomyces species, an open reading frame, named momA, is present as a neighbor of rppA. MomA belonged to the "cupin" superfamily because it contained a set of two motifs that is responsible for binding one equivalent of metal ions. MomA catalyzed monooxygenation of the THN produced from malonyl-CoA by the action of RppA to form flaviolin. In addition, it used several polyketides as substrates and formed the corresponding quinones. MomA required redox-active transition metal ions (Ni(2+), Cu(2+), Fe(3+), Fe(2+), Mn(2+), and Co(2+)) for its activity, whereas it was inhibited by a redox-inert transition metal ion (Zn(2+)). MomA neither possessed any flavin prosthetic group nor required nicotinamide cofactors for monooxygenation, which shows that MomA as a member of the cupin superfamily is a novel monooxygenase. Consistent with the catalytic property of MomA, WhiE-ORFII showing similarity in amino acid sequence to MomA and containing a cupin domain also catalyzed monooxygenation of THN. whiE-ORFII is located immediately upstream of the "minimal PKS" gene within the whiE type II PKS gene cluster for biosynthesis of a gray spore pigment in Streptomyces coelicolor A3(2), and a number of whiE-ORFII homologues are present in the biosynthetic gene cluster for polyketides of type II in various Streptomyces species. These findings show that a novel class of quinone-forming monooxygenases is involved in modification of aromatic polyketides synthesized by PKSs of types II and III.     

The lipopeptide antibiotic A54145 biosynthetic gene cluster from Streptomyces fradiae

Ca(2+)-dependent cyclic lipodepsipeptides are an emerging class of antibiotics for the treatment of infections caused by Gram-positive pathogens. These compounds are synthesized by nonribosomal peptide synthetase (NRPS) complexes encoded by large gene clusters. The gene cluster encoding biosynthetic pathway enzymes for the Streptomyces fradiae A54145 NRP was cloned from a cosmid library and characterized. Four NRPS-encoding genes, responsible for subunits of the synthetase, as well as genes for accessory functions such as acylation, methylation and hydroxylation, were identified by sequence analysis in a 127 kb region of DNA that appears to be located subterminally in the bacterial chromosome. Deduced epimerase domain-encoding sequences within the NRPS genes indicated a D: -stereochemistry for Glu, Lys and Asn residues, as observed for positionally analogous residues in two related compounds, daptomycin, and the calcium-dependent antibiotic (CDA) produced by Streptomyces roseosporus and Streptomyces coelicolor, respectively. A comparison of the structure and the biosynthetic gene cluster of A54145 with those of the related peptides showed many similarities. This information may contribute to the design of experiments to address both fundamental and applied questions in lipopeptide biosynthesis, engineering and drug development.     

The Streptomyces peucetius dpsC Gene Determines the Choice of Starter Unit in Biosynthesis of the Daunorubicin Polyketide

The starter unit used in the biosynthesis of daunorubicin is propionyl coenzyme A (CoA) rather than acetyl-CoA, which is used in the production of most of the bacterial aromatic polyketides studied to date. In the daunorubicin biosynthesis gene cluster of Streptomyces peucetius, directly downstream of the genes encoding the beta-ketoacyl:acyl carrier protein synthase subunits, are two genes, dpsC and dpsD, encoding proteins that are believed to function as the starter unit-specifying enzymes. Recombinant strains containing plasmids carrying dpsC and dpsD, in addition to other daunorubicin polyketide synthase (PKS) genes, incorporate the correct starter unit into polyketides made by these genes, suggesting that, contrary to earlier reports, the enzymes encoded by dpsC and dpsD play a crucial role in starter unit specification. Additionally, the results of a cell-free synthesis of 21-carbon polyketides from propionyl-CoA and malonyl-CoA that used the protein extracts of recombinant strains carrying other daunorubicin PKS genes to which purified DpsC was added suggest that this enzyme has the primary role in starter unit discrimination for daunorubicin biosynthesis.     

Organization of the biosynthetic gene cluster for the polyketide antitumor macrolide, pladienolide, in Streptomyces platensis Mer-11107

Pladienolides are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107. They show strong antitumor activity and are a potential lead in the search for novel antitumor agents. We sequenced the 65-kb region covering the biosynthetic gene cluster, and found four polyketide synthase genes (pldAI-pldAIV) composed of 11 modules, three genes involved in post-modifications (pldB-D), and a luxR-family regulatory gene (pldR). The thioesterase domain of pldAIV was more dissimilar to that of polyketide synthase systems synthesizing 12/14-membered macrolide polyketides than to that of systems synthesizing other cyclic polyketides. The pldB gene was identified as a 6-hydroxylase belonging to a cytochrome P450 of the CYP107 family. This was clarified by a disruption experiment on pldB, in which the disruptant produced 6-dehydroxy pladienolide B. Two genes located downstream of pldB, designated pldC and pldD, are thought to be a probable genes for 7-O-acetylase and 18, 19-epoxydase respectively.     

4-Nitrotryptophan is a substrate for the non-ribosomal peptide synthetase TxtB in the thaxtomin A biosynthetic pathway

Thaxtomin A, a cyclic dipeptide with a nitrated tryptophan moiety, is a phytotoxic pathogenicity determinant in scab-causing Streptomyces species that inhibits cellulose synthesis by an unknown mechanism. Thaxtomin A is produced by the action of two non-ribosomal peptide synthetase modules (TxtA and TxtB) and a complement of modifying enzymes, although the order of biosynthesis has not yet been determined. Analysis of a thaxtomin dual module knockout mutant and single module knockout mutants revealed that 4-nitrotryptophan is an intermediate in thaxtomin A biosynthesis prior to backbone assembly. The 4-nitrotryptophan represents a novel substrate for non-ribosomal peptide synthetases. Through identification of N -methyl-4-nitrotryptophan in a single module knockout and the use of adenylation domain specificity prediction software, TxtB was identified as the non-ribosomal peptide synthetase module specific for 4-nitrotryptophan.     

Molecular detection of streptomycin-producing streptomycetes in Brazilian soils.

Actinomycetes were isolated from soybean rhizosphere soil collected as two field sites in Brazil. All the isolates were identified as Streptomyces species and were screened for streptomycin production and the presence of two genes, strA and strB1, known to be involved in streptomycin biosynthesis in Streptomyces griseus. Antibiotic resistance profiles were determined for 53 isolates from cultivated and uncultivated sites, and approximately half the strains were streptomycin resistance. Clustering by the unweighted pair group method with averages indicated the presence of two major clusters, with the majority of resistant strains from cultivated sites being placed in cluster 1. Only representatives from this cluster contained strA. Streptomycetes containing strA and strB1 were phenotypically diverse, and only half could be assigned to known species. Sequence comparison of 16S rRNA and trpBA (tryptophan synthetase) genes revealed that streptomycin- producing streptomycetes were phylogenetically diverse. It appeared that a population of streptomycetes had colonized the rhizosphere and that a proportion of these were capable of streptomycin production.     

Identification of a Gene Involved in the Synthesis of a Dipeptidyl Peptidase IV Inhibitor in Aspergillus oryzae

WYK-1 is a dipeptidyl peptidase IV inhibitor produced by Aspergillus oryzae strain AO-1. Because WYK-1 is an isoquinoline derivative consisting of three l-amino acids, we hypothesized that a nonribosomal peptide synthetase was involved in its biosynthesis. We identified 28 nonribosomal peptide synthetase genes in the sequenced genome of A. oryzae RIB40. These genes were also identified in AO-1. Among them, AO090001000009 (wykN) was specifically expressed under WYK-1-producing conditions in AO-1. Therefore, we constructed wykN gene disruptants of AO-1 after nonhomologous recombination was suppressed by RNA interference to promote homologous recombination. Our results demonstrated that the disruptants did not produce WYK-1. Furthermore, the expression patterns of 10 genes downstream of wykN were similar to the expression pattern of wykN under several conditions. Additionally, homology searches revealed that some of these genes were predicted to be involved in WYK-1 biosynthesis. Therefore, we propose that wykN and the 10 genes identified in this study constitute the WYK-1 biosynthetic gene cluster.     

A glutamic acid 3-methyltransferase encoded by an accessory gene locus important for daptomycin biosynthesis in Streptomyces roseosporus

In many peptide antibiotics, modified amino acids are important for biological activity. The amino acid 3-methyl-glutamic acid (3mGlu) has been found only in three cyclic lipopeptide antibiotics: daptomycin and the A21978C family produced by Streptomyces roseosporus, calcium-dependent antibiotic produced by Streptomyces coelicolor and A54145 produced by Streptomyces fradiae. We studied the non-ribosomal peptide synthetase genes involved in A21978C biosynthesis and the downstream genes, dptG, dptH, dptI and dptJ predicted to encode a conserved protein of unknown function, a thioesterase, a methyltransferase (MTase) and a tryptophan 2,3-dioxygenase respectively. Deletion of dptGHIJ reduced overall lipopeptide yield and led to production of a series of novel A21978C analogues containing Glu12 instead of 3mGlu12. Complementation by only dptI, or its S. coelicolor homologue, glmT, restored the biosynthesis of the 3mGlu-containing compounds in the mutant. Compared with A21978C, the Glu12-containing derivatives were less active against Staphylococcus aureus. Further genetic analyses showed that members of the dptGHIJ locus cooperatively contributed to optimal A21978C production; deletion of dptH, dptI or dptJ genes reduced the yield significantly, while expression of dptIJ or dptGHIJ from the strong ermEp* promoter substantially increased lipopeptide production. The results indicate that these genes play important roles in the biosynthesis of daptomycin, and that dptI encodes a Glu MTase.