抗菌和细胞毒活性海洋细菌的筛选及其次生代谢基因证据

从不同海域的海水、海泥和海洋生物中分离海洋细菌,利用琼脂扩散法和MTT法对细菌培养液的乙酸乙酯提取物进行了抗菌和细胞毒活性筛选,并对具有细胞毒活性的细菌菌株进行了16SrRNA系统发生学分析和多聚酮合酶(PKSⅠ型)、非核糖体肽合成酶(NRPS)的筛选。结果显示,在分离到的346株海洋细菌中,42株细菌具有抗菌活性,12株具有细胞毒活性。对12株具有细胞毒活性的细菌菌株进行了16SrRNA系统发生学分析,它们分别属于Agrobacterium,Pseudoalteromons,Bacillus,Paracoccus,Rheinheimera,Aerococcus,Exiguobacterium和Alteromonas8个属。对这12株具有细胞毒活性的细菌菌株进行进一步的PKS和NRPS筛选,得到了4株含有PKSⅠ型的KS结构域或NPRS的A结构域的海洋细菌,为从聚酮和非核糖体肽等生物合成途径去深入研究其次生代谢产物提供了基因学的证据。     

芍药内生真菌的鉴定及产生活性次生代谢产物的评估

【目的】研究药用植物芍药(Paeonia lactiflora Pall.)内生真菌的种群多样性,同时对其可能存在的聚酮合酶(Polyketide synthase,PKS)和非核糖体多肽合成酶(Non-ribosomal peptide synthetase,NRPS)基因多样性进行评估,预测芍药内生真菌产生活性次生代谢产物的潜力。【方法】采用组织分离法获得芍药根部内生真菌菌株,结合形态学特征和ITS序列分析,进行鉴定;利用兼并性引物对内生真菌中存在的聚酮合酶(PKS)基因和非核糖体多肽合成酶(NRPS)基因进行PCR扩增及序列测定分析,构建系统发育树,明确芍药内真菌PKS基因序列和NRPS基因序列的系统进化地位。【结果】从芍药组织块中共分离得到105株内生分离物,去重复后获得52株内生真菌,菌株ITS基因序列信息显示,52株芍药内生真菌隶属于7目、13科、15属,其中小球腔菌属(Leptosphaeria)、土赤壳属(Ilyonectria)和镰孢属(Fusarium)为优势种群;从52株内生真菌中筛选获得13株含PKS基因片段的菌株,8株含NRPS基因片段的菌株,部分菌株功能基因的氨基酸序列与Gen Bank中已知化合物的合成序列具有一定的同源性,预示芍药根部内生真菌具有合成丰富多样的次生代谢产物的潜力。【结论】药用植物芍药根部具有丰富的内生真菌资源,且具有产生活性次生代谢产物的潜力,值得进一步开发研究和应用。     

多聚酮的微生物合成及其多样性研究进展

多聚酮多是由微生物产生的一大类天然产物,在结构和功能上具有多样性。很多聚酮具有抗细菌、抗真菌、抗寄生虫、抗肿瘤等生物活性,有很大的临床应用价值。随着研究的不断深入,一方面更多的天然产物聚酮被发现。另一方面对它们合成相关酶的作用机制研究也更加深入。主要对多聚酮的合成机制以及多聚酮合成类型的多样性展开论述。     

埃博霉素（Epothilones）的PKS/NRPS杂合基因簇

埃博霉素是由粘细菌纤维堆囊菌产生的一类具有促微管聚合活性的大环内酯类化合物。埃博霉素生物合成的多酶复合体是一个由多个功能模块组成,同时含有多聚酮合酶（PKS）和非核糖体肽合成酶（NRPS）的大操纵子。根据同位素标记试验结果和合成酶全基因簇功能的推测,埃博霉素的生物合成包括聚酮链的引发、链合成的起始和噻唑环的形成、链的延伸和转移、链合成的终止释放和环化、及产物的后修饰5个阶段。埃博霉素的PKS/NRPS杂合基因簇是开展组合生物合成研究的良好材料。     

Ⅱ型硫脂酶与次生代谢产物合成北大核心CSCD

Ⅱ型硫脂酶(type Ⅱ thioesterases,TEⅡ)属于α/β水解酶,含有保守的催化三元件(Ser-His-Asp),广泛存在于非核糖体肽合成酶(nonribosomal peptide synthetases,NRPS)和聚酮合成酶(polyketide synthases,PKS)系中。与Ⅰ型硫脂酶(type Ⅰ thioesterases,TEⅠ)不同,TEⅡ作为一个独立的蛋白质行使硫脂键水解功能。以往的数据研究发现,合成途径中TEⅡ基因缺失导致聚酮(polyketides,PK)或非核糖体肽(nonribosomal peptide,NRP)的产量显著降低。本文通过对硫酯酶(thioesterases,TE)的聚类分析,显示序列同源性方面TE聚类成两个不同的进化枝,一个含有所有TEⅠ,第二个包含所有TEⅡ。对TEⅡ的多序列比对及结构分析,结果显示TEⅡ序列中的标记序列"GHSMG"为保守序列,结构的差异性导致TEⅡ功能的差异性,综合前期相关数据研究,对TEⅡ在生物合成中的功能途径进行了概括性论述,并对其今后在次生代谢物合成研究中的应用进行了展望,以期加深对TEⅡ的认识和理解。     

戊二酰亚胺类天然产物生物合成研究进展

微生物天然产物是天然药物的重要组成部分,而天然产物的良好生物活性很大程度上取决于发挥药效的结构基团.这些特殊药效基团的生物合成,通常是利用小分子羧酸、氨基酸等结构简单的初级代谢产物,经过复杂的生物化学过程,最终合成结构复杂活性多样的天然产物.戊二酰亚胺类天然产物是一类重要的细菌来源天然产物,它们具有良好的生物活性,是潜...     

紫菜外生细菌抑菌活性及其多聚酮合酶(PKS Ⅰ)基因筛选

[目的]基于紫菜外生细菌抑菌活性的研究,本文对具有广谱抑菌活性的菌株进行了多聚酮合酶(Polyketide synthase Ⅰ,PKS Ⅰ)基因的筛选,以期获得PKS Ⅰ阳性菌株及探讨紫菜藻际微生物区系细菌的拮抗机制与PKS Ⅰ途径的关系.[方法]利用琼脂柱法筛选出具广谱抑菌活性的菌株31株,以其基因组DNA为模板,设计引物扩增酮基合成酶(Ketosynthase, KS)片段基因并将其克隆到pMD19-T Vector,筛选出PKS Ⅰ阳性菌,进行16S rDNA测序分析.[结果]紫菜外生细菌表现出广谱抑菌性.从温州病烂紫菜外生菌中筛选出3株具强抑菌活性的PKS Ⅰ阳性菌,BLAST比对结果显示:菌株WPhG3、WPySw1和WPySw2扩增得到PKS Ⅰ的KS结构域核苷酸序列所对应的氨基酸序列与Bacillus subtilis subsp. subtilis str. 168(NP. 389602)、Bacillussubtilis (ABR19776)和Aspergillus carbonarius(AAZ99721)的PKS Ⅰ的KS结构域的同源性分别达到98%、99%和98%;16S rDNA系统发生学分析显示它们均与Bacillus的同源性最高.[结论]紫菜藻际微生物群落组成复杂,通过多条途径调节藻际微生物区系的平衡.PKS Ⅰ途径可能是温州病烂紫菜外生菌Bacillus表现抑菌活性的一种方式.     

非核糖体肽合成酶催化的非常规装配模式

非核糖体肽合成酶(NRPSs)催化形成复杂肽类天然产物, 其中很多显示了很好的生物学活性和医疗价值。常规NRPSs具有模块化和线性催化的特点, 然而在生物合成研究过程中也发现了很多具有非常规装配模式的NRPSs。本文针对其中4种非常规装配模式: 重复使用、非线性、模块跳跃和非核糖体前肽模式, 结合一些代表性例子做一小型综述。     

生物表面活性剂Surfactin生产菌株的定向改造策略

表面活性素(Surfactin)是芽胞杆菌属(Bacillussp.)代谢产生的脂肽类生物表面活性剂,是由非核糖体肽合成酶(NRPS)催化而得的一种次级代谢产物。由于surfactin具有稳定性好、可被降解、表面活性好等理化性质以及抑菌、抗肿瘤等生物活性,在医药、农业、食品、化妆品、石油开采等方面都具有很大的应用潜力。但是,天然菌株产率低、生产成本高等特点限制了surfactin的规模化应用。本文对surfactin的合成机理进行了简要阐述,并针对目前提升surfactin产量和改变结构组分的4种定向改造策略(启动子工程、强化外排分泌、改造NRPS结构域和脂肪酸链合成酶系)进行了综述,最后对surfactin的研究方向进行了展望。     

放线菌环二肽类活性天然产物生物合成研究进展

环二肽(cyclodipeptide,CDP)是一类由2个α-氨基酸缩合而成的最小环肽分子,也可称为二酮哌嗪类化合物(diketopiperazines,DKPs)。CDP具有稳定的DKP环状骨架结构,活性广泛而显著,药用前景良好,发掘意义重大。放线菌是重要的CDP生产菌,同时具有非核糖体肽合成酶(nonribosomal peptide synthetase, NRPS)与环二肽合酶(cyclodipeptide synthase, CDPS)两种DKP骨架合成催化酶,并从中发现多种骨架结构修饰酶,研究开发价值巨大。本文系统介绍了放线菌CDP类活性化合物的DKP骨架合成途径及其结构修饰机制两方面的研究工作,以期为新型CDP类天然产物的发掘、新颖CDP分子生物合成机制的阐明及合成生物学设计与应用等领域的研究与实践提供参考。     

Novel insights into evolution of protistan polyketide synthases through phylogenomic analysis

Polyketide synthase (PKS) enzymes are large multi-domain complexes that structurally and functionally resemble the fatty acid synthases involved in lipid metabolism. Polyketide biosynthesis of secondary metabolites and hence functional PKS genes are widespread among bacteria, fungi and streptophytes, but the Type I was formerly known only from bacteria and fungi. Recently Type I PKS genes were also uncovered in the genomes of some alveolate protists. Here we show that the newly sequenced genomes of representatives of other protist groups, specifically the chlorophytes Ostreococcus tauri, O. lucimarinus, and Chlamydomonas reinhardtii, and the haptophyte Emiliania huxleyi also contain putative modular Type I PKS genes. Based on the patchy phylogenetic distribution of this gene type among eukaryotic microorganisms, the question arises whether they originate from recent lateral gene transfer from bacteria. Our phylogenetic analyses do not indicate such an evolutionary history. Whether Type I PKS genes originated several times independently during eukaryotic evolution or were rather lost in many extant lineages cannot yet be answered. In any case, we show that environmental genome sequencing projects are likely to be a valuable resource when mining for genes resembling protistan PKS I genes.     

Phylogenetic affiliation and determination of bioactive compounds of bacterial population associated with organs of mud crab,Scylla olivacea

Mud crab belongs to the genus Scylla is an economically valuable and preferred species for costal aquaculture in Asian countries, including India. In recent years, there has been a tremendous expansion of Scylla farming, which has led to increasing research on its habit and habitats. However, there has been no study undertaken to understand the role of the bacterial population associated with the different organs of the mud crab, Scylla olivacea. In total, 43 isolates were recovered from four selected parts of the crab (carapace, n?=?18; abdomen n?=?11; leg, n?=?8; and hand, n?=?6), and the 16S rRNA gene was used to identify the bacterial isolates. The antimicrobial potential along with the detection of modular polyketide synthase (PKSI), cytochrome P450 hydroxylase (CYP) and non-ribosomal peptide synthetase (NRPS) gene clusters were investigated to show a relationship among the biosynthetic genes with their useful aspects. Additionally, the potential three strains (BPS_CRB12, BPS_CRB14 and BPS_CRB41), which showed significant antimicrobial activities, also showed the presence of twenty volatile compounds (VOCs) using GC–MS analysis. We conclude that the strain Aneurinibacillus aneurinilyticus BPS_CRB41 could be source for the production of bioactive compounds.     

Analysis of the enzymatic domains in the modular portion of the coronafacic acid polyketide synthase

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant pathogen Pseudomonas syringae. The CFA polyketide synthase (PKS) consists of two open reading frames (ORFs) that encode type I multifunctional proteins and several ORFs that encode monofunctional proteins. Sequence comparisons of the modular portions of the CFA PKS with other prokaryotic, modular PKSs elucidated the boundaries of the domains that are involved in the individual stages of polyketide assembly. The two β-ketoacyl:acyl carrier protein synthase (KS) domains in the modular portion of the CFA PKS exhibit a high degree of similarity to each other (53%), but are even more similar to the KS domains of DEBS, RAPS, and RIF. Cfa6 possesses two acyltransferases- AT0, which is associated with a loading domain, and AT1, which uses ethylmalonyl-CoA (eMCoA) as a substrate for chain extension. Cfa7 contains an AT that uses malonyl-CoA as a substrate for chain extension. The Cfa6 AT0 shows 35 and 32% similarity to the DEBS1 and NidA1 AT0s, respectively, and 32 and 36% similarity to the Cfa6 and Cfa7 AT1s. Sequence motifs have previously been identified that correlate with AT substrates. The motifs in Cfa6 AT1 were found to correlate reasonably well with those predicted for methylmalonyl-CoA (mMCoA) ATs. The motifs in the AT of Cfa7 correlated more poorly with those predicted for MCoA ATs. Three ACP domains occur in the modular proteins of the COR PKS. The loading domain-associated ACP0 showed 38% similarity to the loading domain ACP0s of DEBS1 and NidA1 and 32–36% similarity to the two module-associated ACPs of the COR PKS. It exhibited a higher degree of similarity to the module-associated ACPs of RAPS. The two module-associated ACPs show 39% similarity to each other, but appear more closely related to module-associated ACP domains in RAPS and RIFS. Furthermore, the DH and KR domains of Cfa6 and Cfa7 show greater similarity to DH and KR domains in RAPS and RIFS than to each other. The CFA PKS includes a thioesterase domain (TE I) that resides at the C-terminus of Cfa7 and a second thioesterase, which exists as a separate ORF (Cfa9, a TE II). Analysis of a Cfa7 thioesterase mutant demonstrated that the TE domain is required for the production of CFA. The co-existence of TE domains within modular PKSs along with physically separated, monofunctional TEs (TE IIs) has been reported for a number of modular polyketide and non-ribosomal peptide synthases (NRPS). An analysis of the two types of thioesterases using Clustal X yielded a dendrogram showing that TE IIs from PKSs and NRPSs are more closely related to each other than to domain TEs from either PKSs or NRPSs. Furthermore, the dendrogram indicates that both types of TE IIs are more closely related to TE domains associated with PKSs than to TE domains in NRPSs. Finally, the overall % G+C content and the % G+C content at the third codon for all of the PKS genes in the COR cluster suggest that these genes may have been recruited from a gram-positive bacterium.     

The Screening of Antimicrobial Bacteria with Diverse Novel Nonribosomal Peptide Synthetase (NRPS) Genes from South China Sea Sponges

Nonribosomal peptide synthetase (NRPS) adenylation (A) domain genes were investigated by polymerase chain reaction for 109 bacteria isolated from four South China Sea sponges, Stelletta tenuis, Halichondria rugosa, Dysidea avara, and Craniella australiensis. Meanwhile, the antimicrobial bioassay of bacteria with NRPS genes were carried out to confirm the screening of NRPS genes. Fifteen bacteria were found to contain NRPS genes and grouped into two phyla Firmicutes (13 of 15) and Proteobacteria (two of 15) according to 16S rDNA sequences. Based on the phylogenetic analysis of the conserved A domain amino acid sequences, most of the NRPS fragments (11 of 15) showed below 70% similarity to their closest relatives suggesting the novelty of these NRPS genes. All of the 15 bacteria with NRPS genes have antimicrobial activities, with most of them exhibiting activity against multiple indicators including fungi and gram-positive and gram-negative bacteria. The different antimicrobial spectra indicate the chemical diversity of biologically active metabolites of sponge-associated bacteria and the possible role of bacterial symbionts in the host’s antimicrobial chemical defense. Phylogenetic analysis based on the representative NRPS genes shows high diversity of marine NRPS genes. The combined molecular technique and bioassay strategy will be useful to obtain sponge-associated bacteria with the potential to synthesize bioactive compounds. An erratum to this article can be found at     

4-香豆酰-CoA连接酶(4CL)和聚酮合酶(PKS1)的融合蛋白在莱茵衣藻中表达促进树莓酮积累

树莓酮具有重要的医药价值,如抗流感、预防糖尿病等.为了在莱茵衣藻Chlamydomonas reinhardtii中获得树莓酮,本研究将树莓酮合成的最后两个步骤中的酶,即4-香豆酰-CoA连接酶(4-coumaryl-CoA ligase,4CL)和聚酮合酶(Polyketide synthase,PKS1)通过甘氨酸...     

Effect of pH on growth of acid stream algae

Five species of algae (Chlamydomonas applanata var. acidophila, Euglena mutabilis, Gloeochrysis turfosa, Hormidium rivulare, Stichococcus bacillaris) were isolated from a stream at pH 2·6–3·1, and their laboratory growth studied. Growth of all species could occur at pH values lower than those from which they were isolated, the lowermost limits being quite similar to those recorded for the particular species growing anywhere in England. Morphological differences were apparent with all five species at the lowermost pH values. These took place with Stichococcus bacillaris at pH values at which there was little reduction in growth rate, but with the other species obvious differences in morphology were correlated with a marked reduction in growth rate below the optimum rate. At the uppermost pH value tested, however, no obvious morphological differences were apparent. The effect of including 10% stream water in the medium was rather similar for all five species. No influence on growth rate was detectable at the lower pH values, but higher pH values led to a decrease in growth rate as compared with that found in medium lacking stream water.     

Biosynthetic Pathway of Cephabacins in Lysobacter lactamgenus: Molecular and Biochemical Characterization of the Upstream Region of the Gene Clusters for Engineering of Novel Antibiotics

The cephabacins, one of the beta-lactam antibiotics, are produced by Lysobacter lactamgenus. The previous studies the cephabacin biosynthesis were limited to a gene cluster that encodes the gene products responsible for the biosynthesis of the cephem nucleus. The long-term goal of this research is to elucidate the metabolic diversity and biosynthetic pathway of cephabacins and to design and/or discover new pharmacologically active compounds by engineering the cephabacin biosynthetic pathway in L. lactamgenus. In this study, we have cloned and sequenced a 24-kb fragment of a DNA locus upstream of the previously reported but incomplete putative ORF9 of L. lactamgenus. This contains three putative ORFs (the complete ORF9, ORF10, and ORF11) transcribed in the same direction and one putative ORF (ORF12) in the opposite direction. The isolated DNA locus extends the previously cloned part of the DNA locus containing the genes responsible for biosynthesis of the cephem nucleus up to 45 kb. The 42-kb fragment of the 45-kb gene cluster is located between a potential TATA box just upstream of the ORF11 and a termination loop just downstream of the previously reported bla gene. The complete ORF9 contains three nonribosomal peptide synthetase (NRPS) modules and one polyketide synthase (PKS) module and the ORF11 contains one NRPS module. The complete ORF9 also contains a putative thioesterase domain at the C-terminal end. We predicted the amino acid specificity of the four NRPSs by generating specificity binding pockets and expressed one of the NRPSs to confirm the amino acid specificity. The adenylation domain of the NRPS1, which is the last module of the NRPSs, showed significant amino acid specificity for L-arginine. These findings are in perfect agreement with the composition that was expected for the structure of cephabacins which contain an acetate residue, an L-arginine, and one to three L-alanines at the C-3' position of the cephem nucleus of cephabacins. The ORF10, encoding a putative ABC transporter which might be involved in conferring resistance against cephabacins, was identified between the complete ORF9 and the ORF11. Therefore, the complete ORF9, ORF10, ORF11 reported here and the other genes previously reported constitute an operon for the biosynthesis of cephabacins in L. lactamgenus. Based on our results, the biosynthetic pathways of acetate and elongated peptide moieties and a mechanism by which cephabacins are assembled by connecting the peptide moiety synthesized by the gene products of the complete ORF9 and the ORF11 to the C-3' position of the cephem nucleus synthesized by the gene products of pcbAB, pcbC, cefE, cefF, and cefD have been elucidated.     

Comprehensive analysis of distinctive polyketide and nonribosomal peptide structural motifs encoded in microbial genomes

We developed a highly accurate method to predict polyketide (PK) and nonribosomal peptide (NRP) structures encoded in microbial genomes. PKs/NRPs are polymers of carbonyl/peptidyl chains synthesized by polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We analyzed domain sequences corresponding to specific substrates and physical interactions between PKSs/NRPSs in order to predict which substrates (carbonyl/peptidyl units) are selected and assembled into highly ordered chemical structures. The predicted PKs/NRPs were represented as the sequences of carbonyl/peptidyl units to extract the structural motifs efficiently. We applied our method to 4529 PKSs/NRPSs and found 619 PKs/NRPs. We also collected 1449 PKs/NRPs whose chemical structures have been determined experimentally. The structural sequences were compared using the Smith-Waterman algorithm, and clustered into 271 clusters. From the compound clusters, we extracted 33 structural motifs that are significantly related with their bioactivities. We used the structural motifs to infer functions of 13 novel PKs/NRPs clusters produced by Pseudomonas spp. and Burkholderia spp. and found a putative virulence factor. The integrative analysis of genomic and chemical information given here will provide a strategy to predict the chemical structures, the biosynthetic pathways, and the biological activities of PKs/NRPs, which is useful for the rational design of novel PKs/NRPs.