New aminocoumarin antibiotics from genetically engineered Streptomyces strains

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A(1) are produced by different Streptomyces strains and are potent inhibitors of DNA gyrase. The biosynthetic gene clusters of all three antibiotics have been cloned and sequenced, and the function of most genes contained therein has been elucidated. In the last years, a number of "unnatural" aminocoumarins could be generated using the genetic information for the biosynthesis of these antibiotics. The investigated enzymes of aminocoumarin biosynthesis have less-than-perfect substrate specificity, facilitating the production of new antibiotics by various methods. Several new aminocoumarins could be produced by targeted genetic manipulation in the natural producers, but also in heterologous host Streptomyces coelicolor after expression of the respective gene cluster. Mutasynthesis experiments, i. e. generation of a cloQ-defective mutant of the clorobiocin producer and feeding of 13 different structural analogs of 3-dimethylallyl-4-hydroxybenzoic acid to this mutant, allowed the isolation of 32 new aminocoumarins. These compounds contained, instead of the genuine 3-dimethylallyl-4-hydroxybenzoyl moiety, the externally added analogs as the acyl components in their structures. Production of new aminocoumarins was also achieved by chemoenzymatic synthesis in vitro. Several biosynthetic enzymes have been heterologously expressed, purified und used for chemoenzymatic synthesis. The structures of the new aminocoumarins were elucidated by NMR and mass spectroscopy. Their inhibitory activity on gyrase in vitro as well as their antibacterial activity was determined. These results give further insight into the structure-activity relationships of aminocoumarins.     

Combinatorial biosynthesis, metabolic engineering and mutasynthesis for the generation of new aminocoumarin antibiotics

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1 are produced by different Streptomyces strains. They are potent inhibitors of bacterial gyrase and topoisomerase IV, and novobiocin has been licensed as antibiotic for clinical use (Albamycin). They also have potential applications in oncology. The biosynthetic gene clusters of all three antibiotics have been cloned and sequenced, and the function of nearly all genes contained therein has been elucidated. Rapid and versatile methods have been developed for the heterologous expression of these biosynthetic gene clusters, and in Streptomyces coelicolor M512 as heterologous host these antibiotics were produced in yields comparable to those in the natural producer strains. lambda RED-mediated homologous recombination was used for genetic modification of the gene clusters in Escherichia coli. The phage PhiC31 attachment site and integrase functions were introduced into the cosmid backbones and employed for stable integration of the clusters into the genome of the heterologous hosts. Modification of the clusters by single or multiple gene replacements or gene deletions resulted in the formation of numerous new aminocoumarin derivatives, providing an efficient tool for the rational generation of antibiotics with modified structure. Additionally, many new antibiotics were generated by mutasynthesis experiments, i.e. the targeted deletion of genes required for the biosynthesis of a certain structural moiety of the antibiotic, and the replacement of this moiety by structural analogs which were added to the culture broth. The diversity of new structures obtained by this approach could be expanded by further genetic modifications of the gene deletion mutants, especially by expression of heterologous biosynthetic enzymes with appropriate substrate specificity.     

宁夏枸杞根际土壤链霉菌V-1-3次级代谢产物分析

摘要：目的 获得链霉菌V-1-3的基因组序列信息,分析其次级代谢产物生物合成基因簇并预测其代谢产物,为发现潜在新抗生素奠定基础。方法 基于16S rRNA基因序列进行菌株属水平鉴定,利用Illumina HiSeq+PacBio测序技术对菌株V-1-3进行基因组测序,采用antiSMASH(v6.0.1)在线工具分析次级代谢产物生物合成基因簇,液-质联用技术检测产生的次级代谢产物。结果 链霉菌V-1-3基因组序列全长8 243 417 bp,平均(G+C)含量为72.14 %,共编码7578个基因,预测到33个生物合成基因簇,利用液-质联用检测到4个代谢物：oxalomycin B、geosmin、coelichelin和ishigamide。结论 来自盐碱地的链霉菌V-1-3具有丰富的次级代谢产物生物合成基因簇,能产生多种次级代谢产物,具有进一步发掘新抗生素的价值。     

链霉菌抗生素生物合成的基因调控

链霉菌中抗生素生物合成受到许多调控基因的调控,它们通过途径特异性调控方式、全局性调控以及双组分调控方式对基因表达进行调控.概述这些调控基因将有利于改良抗生素生产菌株及新型抗生素的研究,并为改造链霉菌抗生素生物合成相关基因、提高产量提供理论依据.本文归纳了国内外链霉菌抗生素生物合成基因簇各调控基因及其研究进展.     

Partial activation of a silent angucycline-type gene cluster from a rubromycin beta producing Streptomyces sp. PGA64

In the course of DNA-fingerprinting our strain collection for antibiotic biosynthesis genes, two different type II polyketide synthase (PKS) gene clusters were observed from Streptomyces sp. PGA64. Phylogenetic analysis placed these together with known rubromycin and angucycline biosynthetic gene clusters. The host strain itself has a very clean production profile of secondary metabolites, which composes mainly of rubromycin beta under typical fermentation conditions. Sequencing of a 16.5 kb fragment from the putative angucycline cluster revealed eight genes that were homologous to typical type II PKS genes responsible for synthesizing aromatic polyketides. These genes were especially similar to genes from known angucycline biosynthetic gene clusters and also synteny to these clusters was observed. In addition, three genes were recognized that are needed for priming the minimal PKS complex before polyketide synthesis can initiate, but which are not normally found to cluster with antibiotic biosynthesis genes. A putative repressor gene that was dissimilar to repressor genes found from well-characterized antibiotic biosynthesis gene clusters was also discovered. Gene disruption of the repressor resulted in partial activation of the cluster and production of two angucycline metabolites, UWM6 and rabelomycin. The results confirm that the DNA-fingerprinting method we have developed can be used to correctly detect compounds that are not visible in chemical screens.     

Combinatorial natural products: from cloning to analysis

Medicinal compounds from plants represent one of the largest and most diverse groups of plant secondary metabolites. The advent of advanced bioinformatics tools and modern genetic technology allowed for manipulation of biosynthetic pathways with the potential of generating novel chemical entities. First, public databases of secondary metabolite related enzymes were interrogated to identify relevant plant genes from vinca rosea (Catharanthus roseus) and other species. Genes of interest were tested after cloning by transfection into tobacco cell cultures using DNA viral vectors. The biosynthetic enzymes coded by these genes were over-expressed in the host. Automated solvent extraction procedure was employed to extract secondary metabolites from plant leaf tissues and transfected tobacco cell culture samples. The composition of the extracts was analyzed by state of the art bioanalytical methods such as high performance liquid chromatography and capillary electrophoresis to monitor changes in secondary metabolite patterns.     

雷帕链霉菌次级代谢产物及其生物合成的研究进展

摘要：雷帕链霉菌(Streptomyces rapamycinicus)是一种重要的工业菌株,主要用于生产新型大环内酯类抗生素——雷帕霉 素。该抗生素具有抗真菌、抗肿瘤、免疫抑制和抗衰老等众多生物活性,临床上主要用作器官移植的免疫抑制剂以及抗肿瘤药 物。全基因组测序表明,雷帕链霉菌野生型菌株NRRL5491基因组全长12.7Mb,编码多达48个次级代谢产物生物合成基因簇(共 长达3Mb),证明其具备强大的次级代谢潜力。除雷帕霉素以外,至今已有多种活性天然产物被鉴定,包括放线菌酸、尼日利亚 菌素、洋橄榄叶素、安莎类抗生素和六烯类抗生素等,相关合成基因簇及其生物合成途径已被解析。本文将就雷帕链霉菌中各 种次级代谢产物的生物学功能、生物合成基因簇及其生物合成过程等研究进展进行总结梳理,并就如何更好挖掘雷帕链霉菌中 的活性天然产物进行简单展望与讨论。     

微生物非核糖体多肽的合成基因簇挖掘及其合成研究进展

摘要：非核糖体多肽因其独特的生物活性受到关注,它结构复杂、种类繁多。许多微生物能利用非核糖体肽合成酶(non-ribosomal peptide synthase,NRPSs)合成这些非核糖体多肽。NRPSs是一类分子巨大的蛋白复合物,由一系列催化结构域组成,能识别、激活、转运氨基酸底物并按特定顺序合成非核糖体肽(non-ribosomal peptide,NRPs)。其模块化结构涉及的基因在基因组上成簇排列。基于基因组挖掘技术可以预测基因组上的次级代谢产物合成基因簇,并发现新的次级代谢产物。针对非核糖体多肽的生物合成基因簇的分布、挖掘、模块化结构以及合成等方面的研究进行分析,有望为理解非核糖体肽合成酶系统合成生物活性物质的机制和挖掘新型生物活性物质提供依据。     

刺糖多胞菌合成多杀菌素的基因研究

多杀菌素是由刺糖多孢菌产生的一种新型大环内酯类混合次级代谢产物,为新型、高效、安全的生物杀虫剂。该类化合物由一个12元大环内酯以及一个中性糖和一个胺糖构成。多杀菌素生物合成基因大部分聚集在刺糖多孢菌基因组上约80kb大小的区域中。该区域DNA已被完全测序,并通过破坏目的基因的方法,对这一区域DNA的特性和功能进行了深入研究。多杀菌素生物合成基因簇包括5个编码Ⅰ型聚酮合成酶的基因和14个与大环内酯结构修饰有关的基因,另外还有4个编码鼠李糖的基因未包含在上述基因簇中。     

Mutational biosynthesis of a new antibiotic, streptomutin A, by an idiotroph of Streptomyces griseus.

Microorganisms producing antibiotics have been genetically converted by earlier workers to mutants which cannot produce antibiotic without supplementation with a moiety of the antibiotic. These antibiotics include neomycin, kanamycin, paromomycin, butirosin, sisomicin, ribostamycin and novobiocin. Success has not been reported for organisms producing guanidinocyclitol antibiotics such as streptomycin. We mutagenized conidia of the streptomycin-producing Streptomyces griseus strain 7-455F3 with nitrosoguanidine at pH 7.0. Non-producers of streptomycin were visually selected by the agar-plug technique using Bacillus subtilis. We successfully isolated mutant MIT-A5 which produces no streptomycin unless streptidine is added to the agar medium. The streptidine-dependent phenotype was confirmed in submerged culture in flasks. Attempts to produce new antibiotics by feeding aminocyclitols to mutant MIT-A5 failed. However a new antibiotic (streptomutin A) was produced by supplementation with the guanidinocyclitol, 2-deoxystreptidine. We propose the term "mutational biosynthesis" for the production of new metabolites by the use of mutants blocked in the biosynthetic pathway to the secondary metabolite. We further propose the term "idiotroph" to properly describe such mutants.     

Genetics of Dothistromin Biosynthesis in the Peanut Pathogen Passalora arachidicola

The peanut leaf spot pathogen Passalora arachidicola (Mycosphaerella arachidis) is known to produce dothistromin, a mycotoxin related to aflatoxin. This is a feature shared with the pine needle pathogen Dothistroma septosporum (Mycosphaerella pini). Dothistromin biosynthesis in D. septosporum commences at an unusually early stage of growth in culture compared to most other fungal secondary metabolites, and the biosynthetic genes are arranged in fragmented groups, in contrast to aflatoxin gene clusters. Dothistromin biosynthetic genes were identified and studied in P. arachidicola to determine if the attributes described in D. septosporum are shared by another dothistromin-producing species within the Class Dothideomycetes. It was shown that dothistromin biosynthesis is very similar in the two species with regard to gene sequence and gene synteny. Functional complementation of D. septosporum mutants with P. arachidicola dothistromin genes was also possible. These similarities support a vertical mode of dothistromin gene transmission. P. arachidicola also produced dothistromin at an early growth stage in culture, suggesting that this type of regulation pattern may be relevant to the biological role of dothistromin.     

表观遗传修饰应用于真菌次级代谢调控研究的最新进展

真菌次级代谢产物的生物合成与其基因簇所在染色体的表观遗传状态密切相关,通过表观遗传修饰能够调控真菌的次级代谢过程.分子表观遗传修饰方法主要通过敲除或过表达表观遗传相关酶类的编码基因,而化学表观遗传修饰方法则是外源加入化学表观遗传修饰酶抑制剂.二者都能促进基因的转录,进而激活沉默的生物合成基因簇,提高真菌次级代谢产物的化...     

微生物次级代谢产物生物合成基因簇与药物创新

微生物产生众多结构和生物活性多样的次级代谢产物，其生物合成基因簇的克隆是药物创新和产量提高的必要前提。迄今为止已有超过150种生物合成基因簇通过各种方式被克隆，并被用于组合生物合成、体外糖类随机化、代谢工程的定向改造。我们研究室已经克隆并测定了氨基糖苷类井冈霉索／有效霉索、多烯类抗生素FR-008／克念菌索、聚醚类南昌霉索、聚酮类梅岭霉索、杂合聚酮一多肽类略唑霉索等生物合成基因簇。深入的基因功能分析揭示了他们独特的生物合成途径和调节机理，为正在进行的组合生物合成结构改造和代谢工程产量提高奠定了基础。     

洛伐他汀生物合成及其相关基因研究进展

洛伐他汀是一种有效的降胆固醇药物，这种真菌次生代谢产物及其衍生物是胆固醇生物合成限速酶——HMG-CoA还原酶的竞争性抑制剂。文章综述了用同位素标记前体研究洛伐他汀生物合成和洛伐他汀生物合成相关基因的克隆、功能分析等方面的研究进展。     

海洋微生物次级代谢产物生物合成的研究进展

海洋微生物次级代谢产物往往具有新颖的化学结构,蕴含着独特的生物合成途径、酶学机理和不同于陆生放线菌次级代谢产物的生物合成机制。自从2000年第一例海洋微生物天然产物enterocin的生物合成基因簇被阐明以来,迄今已克隆和鉴定了27种海洋微生物次级代谢产物的完整生物合成基因簇。这些次级代谢产物的生物合成主要源于四种途径,包括聚酮合酶途径,非核糖体肽合成酶途径,聚酮-非核糖体肽合成酶杂合途径,以及其他途径。本文综述了近年来一些重要海洋微生物活性次级代谢产物的生物合成途径,以及组合生物合成技术在海洋微生物次级代谢产物结构多样化方面的应用。     

Comparison of Expression of Secondary Metabolite Biosynthesis Cluster Genes in Aspergillus flavus,A. parasiticus,and A. oryzae

Fifty six secondary metabolite biosynthesis gene clusters are predicted to be in the Aspergillus flavus genome. In spite of this, the biosyntheses of only seven metabolites, including the aflatoxins, kojic acid, cyclopiazonic acid and aflatrem, have been assigned to a particular gene cluster. We used RNA-seq to compare expression of secondary metabolite genes in gene clusters for the closely related fungi A. parasiticus, A. oryzae, and A. flavus S and L sclerotial morphotypes. The data help to refine the identification of probable functional gene clusters within these species. Our results suggest that A. flavus, a prevalent contaminant of maize, cottonseed, peanuts and tree nuts, is capable of producing metabolites which, besides aflatoxin, could be an underappreciated contributor to its toxicity.     

Draft genome sequence of potato crop bacterial isolates and nanoparticles-intervention for the induction of secondary metabolites biosynthesis

IntroductionInsights about the effects of gold nanoparticles (AuNPs) on the biosynthetic manipulation of unknown microbe secondary metabolites could be a promising technique for prospective research on nano-biotechnology.AimIn this research, we aimed to isolate a fresh, non-domesticated unknown bacterium strain from a common scab of potato crop located in Saudi Arabia and study the metabolic profile.MethodologyThis was achieved through genomic DNA (gDNA) sequencing using Oxford Nanopore Technology. The genomic data were subjected to several bioinformatics tools, including canu-1.9 software, Prokka, DFAST, Geneious Prime, and AntiSMASH. We exposed the culture of the bacterial isolate with different concentrations of AuNPs and investigated the effects of AuNPs on secondary metabolites biosynthesis using several analytical techniques. Furthermore, Tandem-mass spectrometric (MS/MS) technique was optimized for the characterization of several significant sub-classes.ResultsThe genomic draft sequence assembly, alignment, and annotation have verified the bacterial isolate as Priestia megaterium. This bacterium has secondary metabolites related to different biosynthetic gene clusters. AuNPs intervention showed an increase in the production of compounds with the molecular weights of 254 and 270 Da in a direct-dependent manner with the increase of the AuNPs concentrations.ConclusionThe increase in the yields of compound 1 and 2 concomitantly with the increase in the concentration of the added AuNPs provide evidences about the effects of nanoparticles on the biosynthesis of the secondary metabolites. It contributes to the discovery of genes involved in different biosynthetic gene clusters (BGCs) and prediction of the structures of the natural products.     

链霉菌沉默生物合成基因簇调控的研究进展

本文以天蓝色链霉菌(Streptomyces coelicolor)中黄色色素coelimycin生物合成调控及开发策略的研究进展为代表,介绍了链霉菌中沉默生物合成基因簇调控激活的新进展,为链霉菌天然产物基因簇的挖掘和新次级代谢产物的发现提供研究思路。     

New aminocoumarin antibiotics derived from 4-hydroxycinnamic acid are formed after heterologous expression of a modified clorobiocin biosynthetic gene cluster

Three new aminocoumarin antibiotics, termed ferulobiocin, 3-chlorocoumarobiocin and 8'-dechloro-3-chlorocoumarobiocin, were isolated from the culture broth of a Streptomyces coelicolor M512 strain expressing a modified clorobiocin biosynthetic gene cluster. Structural analysis showed that these new aminocoumarins were very similar to clorobiocin, with a substituted 4-hydroxycinnamoyl moieties instead of the prenylated 4-hydroxybenzoyl moiety of clorobiocin. The possible biosynthetic origin of these moieties is discussed.