基于非核糖体肽的文献计量分析

许多临床上的重要抗生素来源于微生物生产的非核糖体肽类天然产物或者聚酮-非核糖体肽杂合体类天然产物,本文选取了近5年Web of Science上关于非核糖体肽的国际期刊文献,采用文献计量、统计分析等方法展示非核糖体肽研究领域的热点方向,探究了该领域的发展趋势,以期为进一步研究提供参考。     

Identification of a biosynthetic gene cluster and the six associated lipopeptides involved in swarming motility of Pseudomonas syringae pv. tomato DC3000

Pseudomonas species are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to "mine" the genome of Pseudomonas syringae pv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility of P. syringae pv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites.     

非核糖体多肽合成酶研究进展

细菌和真菌采用非核糖体系统合成一些重要的多肽类物质.近年来的研究表明,在该系统中发挥关键作用的是一类分子巨大的非核糖体多肽合成酶.它们由顺序排列的组件构成,酶分子结构本身即蕴涵着多肽合成的信息.对非核糖体多肽合成酶结构和功能的了解,使人们期望可以通过对这类酶的修饰和重组来合成一些新的多肽类物质.     

Where chemistry meets biology: the chemoenzymatic synthesis of nonribosomal peptides and polyketides

Intensive studies on modular biosynthetic assembly line machinery have provided researchers with a profound knowledge of how nonribosomal peptides and polyketides used in different therapeutic areas are produced in nature. This has opened the door for projects aiming to manipulate the assembly of these small molecules by directed and combinatorial approaches to produce novel compounds both in vivo and in vitro. Here, we highlight a set of recent chemoenzymatic attempts towards the synthesis of nonribosomal peptides and polyketides that aim to generate these structurally demanding compounds through the combined utilization of synthetic chemical tools and recombinant natural product metabolic enzymes.     

非核糖体肽合成酶(NRPSs)作用机理与应用的研究进展

许多微生物能利用非核糖体肽合成酶(NRPSs)合成结构复杂、种类繁多的的生物活性肽。非核糖体肽因其独特的理化特性和药理学特性已被广泛关注,极具商业开发潜力。NRPSs由多个模块组成,模块的不同空间排列顺序决定其多肽产物的氨基酸序列特异性。NRPSs以多载体巯基化模板机理进行多肽合成,其底物特异性由腺苷酰化结构域和缩合结构域共同实现。目前,人们已经利用天然的NRPSs、某些特定结构域、将已知NRPSs的模块或特定结构域进行组合甚至杂合组合而构建成的新的NRPSs来合成目的多肽。     

Cloning and characterization of a new hetero-gene cluster of nonribosomal peptide synthetase and polyketide synthase from the cyanobacterium Microcystis aeruginosa K-139

Two nonribosomal peptide synthetase genes responsible for the biosynthesis of microcystin and micropeptin in Microcystis aeruginosa K-139 have been identified. A new nonribosomal peptide synthetase gene, psm3, was identified in M. aeruginosa K-139. The gene is a cluster extending 30 kb and comprising 13 bidirectionally transcribed open reading frames arranged in two putative operons. psm3 encodes four adenylation proteins, one polyketide synthase, and several unique proteins, especially Psm3L consisting of halogenase, acyl-CoA binding protein-like protein, and acyl carrier protein. Alignment of the binding pocket of the adenylation domain and an ATP-PPi exchange analysis using a recombinant protein with the adenylation domain of Psm3B showed that Psm3G and Psm3B activate aspartic acid and tyrosine, respectively. Although disruption of psm3 did not reveal the product produced by Psm3, we identified microviridin B and aeruginosin K139 in the cells of M. aeruginosa K-139. The above-mentioned results indicated that M. aeruginosa possesses at least five nonribosomal peptide synthetase gene clusters.     

非核糖体肽合成酶的末端硫酯酶与多肽环化

非核糖体肽合成酶（nonribosomal peptide synthetases,NRPSs）能以多载体巯基化模板机制合成各种结构复杂、种类繁多的次生代谢非核糖体环肽.根据环肽末端环化的方式,可分为两大类：大环内酯型和内酰胺型.负责非核糖体环肽最终环化的硫酯酶（thioesterase,TE）属于α/β水解酶超家族.该家族包括：脂酶、蛋白酶、酯酶等,其共有特征是含有保守的催化三元件（Ser-His-Asp）,起到终止反应和释放产物的功能. TE具有区域定向性（regiospecific）、化学定向性（chemospecific）及立体定向性（stereospecific）的特点,在非核糖体肽（nonribosomal peptide,NRP）的合成反应中具有决定性作用,直接影响到最终环肽的生成. 同时,TE由于其特有的环化和水解的双重活性,在体外的线性多肽环化中越来越受到众多学者的关注. 综合国内外相关文献,本文着重从TE介导下的产物释放机制和影响因素两个方面综述非核糖体末端硫酯酶的研究进展及其应用.     

Nonribosomal peptide synthesis and toxigenicity of cyanobacteria.

Nonribosomal peptide synthesis is achieved in prokaryotes and lower eukaryotes by the thiotemplate function of large, modular enzyme complexes known collectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural-product or combinatorial biosynthesis (R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, and C. Khosla, Science 262:1546-1557, 1993; T. Stachelhaus, A. Schneider, and M. A. Marahiel, Science 269:69-72, 1995). Most nonribosomal peptides from microorganisms are classified as secondary metabolites; that is, they rarely have a role in primary metabolism, growth, or reproduction but have evolved to somehow benefit the producing organisms. Cyanobacteria produce a myriad array of secondary metabolites, including alkaloids, polyketides, and nonribosomal peptides, some of which are potent toxins. This paper addresses the molecular genetic basis of nonribosomal peptide synthesis in diverse species of cyanobacteria. Amplification of peptide synthetase genes was achieved by use of degenerate primers directed to conserved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacterial toxin microcystin was shown for both cultured and uncultured samples. Blot hybridizations, DNA amplifications, sequencing, and evolutionary analysis revealed a broad distribution of peptide synthetase gene orthologues in cyanobacteria. The results demonstrate a molecular approach to assessing preexpression microbial functional diversity in uncultured cyanobacteria. The nonribosomal peptide biosynthetic pathways detected may lead to the discovery and engineering of novel antibiotics, immunosuppressants, or antiviral agents.     

<Emphasis Type="Italic">In silico</Emphasis> analysis of methyltransferase domains involved in biosynthesis of secondary metabolites

Background  

Secondary metabolites biosynthesized by polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) family of enzymes constitute several classes of therapeutically important natural products like erythromycin, rapamycin, cyclosporine etc. In view of their relevance for natural product based drug discovery, identification of novel secondary metabolite natural products by genome mining has been an area of active research. A number of different tailoring enzymes catalyze a variety of chemical modifications to the polyketide or nonribosomal peptide backbone of these secondary metabolites to enhance their structural diversity. Therefore, development of powerful bioinformatics methods for identification of these tailoring enzymes and assignment of their substrate specificity is crucial for deciphering novel secondary metabolites by genome mining.     

Structural change of the enterobactin synthetase in crowded solution and its relation to crowding-enhanced product specificity in nonribosomal enterobactin biosynthesis

Significant conformational change is detected by circular dichroism and fluorimetry for the major component of the enterobactin synthetase in crowded solutions mimicking the intracellular environment. The structural change correlates well with the extent of the crowding-induced side product suppression in nonribosomal enterobactin synthesis. In contrast, protein-stabilizing solvophobic agents such as glycerol have no effect on the formation of side products, excluding crowding-induced protein stability as a cause for the observed enhancement of the product specificity of the synthetase. These results strongly support that macromolecular crowding is an indispensable physiological factor for normal functioning of the nonribosomal enterobactin synthetase by altering the active sites to increase its product specificity.     

Nonribosomal Nature of Novel, Stable Ribonucleic Acid Species Accumulated by Blue-Green Bacteria

The facultatively chemoheterotrophic blue-green bacterium Aphanocapsa 6714 accumulates two novel, stable ribonucleic acid species when deprived of sources of carbon and energy. At least one of these species is nonribosomal.     

Exploring the biosynthesis of natural products and their inherent suitability for the rational design of desirable compounds through genetic engineering

Much effort has been invested in studying how natural products are biosynthesized, and great advances have been made in understanding how these compounds acquire their structural complexity and biological activities. In recent years, significant progress has been made due to the devoted efforts of scientists in this field and to technological advancements. Numerous details, applications, and innovative findings have been elucidated by scientists using biochemical, genetic, and molecular biological approaches. Here I present a comprehensive overview of highly valued biosynthetic proteins, polyketide synthase and nonribosomal peptide synthetase. I begin with "Diels-Alderase" a captivating enzyme that has the ability to catalyze a Diels-Alder reaction valued by chemists for its usefulness in chemical synthesis. A handful of these enzymes have been characterized and chemically authenticated. The most well understood enzyme of this category is macrophomate synthase. Secondly, I focus on the polyketide and nonribosomal peptide biosynthetic pathways and the enzyme assembly for producing its metabolite. Many important natural products are produced by this biosynthetic pathway as secondary metabolites, such as erythromycin, rifamycin, and FK520, as antibiotics and an immunosuppressive, respectively. I conclude with a discussion of nonribosomal peptides and their mechanistic pathways. Special attention will be devoted to de novo production of echinomycin in a heterologous manner, the earliest example of totally engineered biosynthesis of the biologically active form of a nonribosomal peptide host in Escherichia coli.     

Improved heterologous production of the nonribosomal peptide‐polyketide siderophore yersiniabactin through metabolic engineering and induction optimization

Biosynthesis of complex natural products like polyketides and nonribosomal peptides using Escherichia coli as a heterologous host provides an opportunity to access these molecules. The value in doing so stems from the fact that many compounds hold some therapeutic or other beneficial property and their original production hosts are intractable for a variety of reasons. In this work, metabolic engineering and induction variable optimization were used to increase production of the polyketide‐nonribosomal peptide compound yersiniabactin, a siderophore that has been utilized to selectively remove metals from various solid and aqueous samples. Specifically, several precursor substrate support pathways were altered through gene expression and exogenous supplementation in order to boost production of the final compound. The gene expression induction process was also analyzed to identify the temperatures and inducer concentrations resulting in highest final production levels. When combined, yersiniabactin production was extended to ～175 mg L^?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1412–1417, 2016     

Amino acid selectivity in the aminoacylation of coenzyme A and RNA minihelices by aminoacyl-tRNA synthetases

Coenzyme A (CoA-SH), a cofactor in carboxyl group activation reactions, carries out a function in nonribosomal peptide synthesis that is analogous to the function of tRNA in ribosomal protein synthesis. The amino acid selectivity in the synthesis of aminoacyl-thioesters by nonribosomal peptide synthetases is relaxed, whereas the amino acid selectivity in the synthesis of aminoacyl-tRNA by aminoacyl-tRNA synthetases is restricted. Here I show that isoleucyl-tRNA synthetase aminoacylates CoA-SH with valine, leucine, threonine, alanine, and serine in addition to isoleucine. Valyl-tRNA synthetase catalyzes aminoacylations of CoA-SH with valine, threonine, alanine, serine, and isoleucine. Lysyl-tRNA synthetase aminoacylates CoA-SH with lysine, leucine, threonine, alanine, valine, and isoleucine. Thus, isoleucyl-, valyl-, and lysyl-tRNA synthetases behave as aminoacyl-S-CoA synthetases with relaxed amino acid selectivity. In contrast, RNA minihelices comprised of the acceptor-TpsiC helix of tRNA(Ile) or tRNA(Val) were aminoacylated by cognate synthetases selectively with isoleucine or valine, respectively. These and other data support a hypothesis that the present day aminoacyl-tRNA synthetases originated from ancestral forms that were involved in noncoded thioester-dependent peptide synthesis, functionally similar to the present day nonribosomal peptide synthetases.     

Draft Genome Sequence of Paenibacillus sp. Strain OSY-SE,a Bacterium Producing the Novel Broad-Spectrum Lipopeptide Antibiotic Paenibacterin

A strain of Paenibacillus sp., OSY-SE, was isolated from soil and found to produce a novel lipopeptide antibiotic. The antibiotic, paenibacterin, is active against Gram-negative and Gram-positive bacterial pathogens. Paenibacterin is biosynthesized by a nonribosomal peptide synthetase pathway. Here we report the draft genome sequence of Paenibacillus sp. OSY-SE.     

Characterization of a gene cluster responsible for the biosynthesis of anticancer agent FK228 in Chromobacterium violaceum No. 968

A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned, and partially characterized in Chromobacterium violaceum no. 968. First, a genome-scanning approach was applied to identify three distinctive C. violaceum no. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate the genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative flavin adenine dinucleotide-dependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system should enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum no. 968 for the generation of structural analogs as anticancer drug candidates.     

An adherent mucus layer attenuates the genotoxic effect of colibactin

The human gastrointestinal tract is a complex ecosystem in which epithelial cells and microorganisms of the intestinal microbiota live in symbiosis. Certain members of the microbiota, in particular Escherichia coli strains of the B2 phylotype, carry the polyketide synthase‐island encoding the genotoxin colibactin. Colibactin is a nonribosomal peptide or polyketide‐nonribosomal peptide hybrid of still unsolved structure, which induces DNA double strand breaks (DSBs) in eukaryotic cells. However, direct contact between live bacteria and host cell is required in order to elicit these genotoxic effects. In this study, we used a variety of cell culture models, among them, a 3D cell culture approach based on decellularised small intestinal submucosa, to investigate whether the intestinal mucus layer has the potential to interfere with colibactin activity. We demonstrate that the expression of mucins and the formation of an adherent mucus layer significantly increased with increasing complexity of cell culture. Moreover, we show that the presence of an adherent mucus layer on epithelial cells attenuates the genotoxic activity of colibactin, by preventing the induction of DNA‐DSBs. Removal of the adherent mucus layer restored the occurrence of DNA‐DSBs.     

Nostophycin biosynthesis is directed by a hybrid polyketide synthase-nonribosomal peptide synthetase in the toxic cyanobacterium Nostoc sp. strain 152

Cyanobacteria are a rich source of natural products with interesting pharmaceutical properties. Here, we report the identification, sequencing, annotation, and biochemical analysis of the nostophycin (npn) biosynthetic gene cluster. The npn gene cluster spans 45.1 kb and consists of three open reading frames encoding a polyketide synthase, a mixed polyketide nonribosomal peptide synthetase, and a nonribosomal peptide synthetase. The genetic architecture and catalytic domain organization of the proteins are colinear in arrangement, with the putative order of the biosynthetic assembly of the cyclic heptapeptide. NpnB contains an embedded monooxygenase domain linking nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) catalytic domains and predicted here to hydroxylate the nostophycin during assembly. Expression of the adenylation domains and subsequent substrate specificity assays support the involvement of this cluster in nostophycin biosynthesis. Biochemical analyses suggest that the loading substrate of NpnA is likely to be a phenylpropanoic acid necessitating deletion of a carbon atom to explain the biosynthesis of nostophycin. Biosyntheses of nostophycin and microcystin resemble each other, but the phylogenetic analyses suggest that they are distantly related to one another.     

The small MbtH-like protein encoded by an internal gene of the balhimycin biosynthetic gene cluster is not required for glycopeptide production

The balhimycin biosynthetic gene cluster of the glycopeptide producer Amycolatopsis balhimycina includes a gene (orf1) with unknown function. orf1 shows high similarity to the mbtH gene from Mycobacterium tuberculosis. In almost all nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters, we could identify a small mbtH-like gene whose function in peptide biosynthesis is not known. The mbtH-like gene is always colocalized with the NRPS genes; however, it does not have a specific position in the gene cluster. In all glycopeptide biosynthetic gene clusters the orf1-like gene is always located downstream of the gene encoding the last module of the NRPS. We inactivated the orf1 gene in A. balhimycina by generating a deletion mutant. The balhimycin production is not affected in the orf1-deletion mutant and is indistinguishable from that of the wild type. For the first time, we show that the inactivation of an mbtH-like gene does not impair the biosynthesis of a nonribosomal peptide.