海洋来源节菱孢菌ZSDS1-F3中PKS-NRPS类天然产物挖掘

【目的】以基因组信息为指导,定向激活海洋来源真菌Arthrinium arundinisZSDS1-F3中沉默的聚酮合成酶-非核糖体肽合成酶(PKS-NRPS)类生物合成基因簇,鉴定次级代谢产物结构。【方法】通过启动子工程和异源表达的策略激活实验室培养条件下沉默或低表达的生物合成基因簇,实现目标化合物的分离,通过HR-ESI-MS和NMR数据分析鉴定产物结构,结合基因重组和生物信息学分析结果推导化合物的生物合成途径。【结果】依据基因组生物信息学分析,从海洋来源真菌A. arundinis ZSDS1-F3中选取一个编码PKS-NRPS类次级代谢产物的生物合成基因簇开展研究,在宿主Aspergillus nidulansA1145中实现了基因簇的异源表达,从中分离到2个新化合物,并推导了其生物合成途径。【结论】基因组信息指导下的天然产物挖掘,可以目标明确地分离产物,加快真菌中新颖天然产物的发现步伐。     

枯草芽孢杆菌抗菌肽生物合成的研究进展

革兰氏阳性菌模式生物--枯草芽孢杆菌能分泌多种肽类及由肽类衍生的抗菌活性物质,按合成途径不同,可分为核糖体肽和非核糖体肽。其中,非核糖体肽分子量较小,一般为3000Da以下,其生物合成是通过多功能复合酶系--非核糖体肽链合成酶来完成的,多发生在菌体生长停止之后;而核糖体肽分子量较大,其合成多于菌体快速生长时期。非核糖体肽链合成酶和核糖体肽的合成及其调控均需基因参与,而这一系列基因就构成了各种抗菌肽生物合成的基因簇。对核糖体肽和非核糖体肽的生物合成及其相关调控机制进行了综述。     

海洋共附生微生物天然产物生物合成基因研究进展

对海洋无脊椎动物天然产物的研究表明,很多种活性物质的真正生产者是与其共生或附生的未培养微生物.克隆这些未培养微生物中特定活性物质的生物合成基因,不仅为活性物质的来源提供遗传学证据,也使通过异源表达相关生物合成基因来大量获取目标化合物成为可能.本文综述了来源于海绵、海鞘、苔藓虫、深海管状蠕虫和深海沉积物中共附生微生物天然产物生物合成基因簇的研究进展.     

非核糖体肽的生物合成研究进展

非核糖体肽(nonribosomal peptide, NRP)是由多种微生物通过非核糖体肽合成酶(nonribosomal peptide synthetase, NRPS)等催化合成的一类小分子多肽类次级代谢产物,具有抗菌、抗肿瘤、免疫抑制等多种生物活性,是一类重要的微生物药物,具有很高的临床应用价值。从目前已发现的小分子多肽类天然药物出发,综述了该类物质的生物功能、合成组装机制以及近年来在工程改造方面的进展,并提出了未来研究发展方向,对进一步通过组合生物合成等方式高效合成更多种类的小分子多肽类活性物质具有借鉴意义。     

非核糖体肽合成酶装配机制研究进展CSCD

非核糖体多肽(nonribosomal peptide,NRP)是天然生物活性产物一大类群,组成结构多样,具有多种重要的药用价值。在微生物中催化非核糖体多肽生物合成的是非核糖体肽合成酶(nonribosomal peptide synthetase,NRPS),NRPS是一类模块酶系,模块的组装在非核糖体多肽合成及其环化中起着关键作用。本文主要对非核糖体肽合成酶常规模块组装模式及3种非常规合成模式进行综述,为深入了解和应用非核糖体肽合成酶在抗生素类生物活性物质中的作用提供理论依据。     

林可链霉菌NRRL 2936中帕马霉素生物合成基因簇的异源 表达及调控基因功能研究

【背景】帕马霉素属于大环内酯类抗生素,具有较好的抗感染活性。该类化合物独特的化学结构和显著的生理活性受到了许多研究者的关注。同时,本实验室在林可链霉菌NRRL2936的全基因组序列中发现了帕马霉素的生物合成基因簇。【目的】尽管其生物合成途径已经得到了解析,但其生物合成基因簇中的2个调控基因功能尚不清楚。本研究从林可链霉菌NRRL2936的基因组文库中克隆了含有帕马霉素生物合成完整基因簇的质粒pJQK450,开展了质粒pJQK450在链霉菌中的异源表达,实现了帕马霉素的异源合成,并初步确定该生物合成基因簇中两个调控基因的功能。【方法】利用聚合酶链式反应递缩基因组文库筛选的方法,从林可链霉菌(Streptomyces lincolnensis)NRRL 2936基因组文库中筛选到了含有帕马霉素生物合成完整基因簇的Fosmid质粒pJQK450。然后,将该质粒转化到E.coli ET12567/pUZ8002中,利用大肠杆菌-链霉菌双亲接合转移的方法将pJQK450转入异源宿主中。对获得的异源表达菌株进行发酵产物的制备,采用耻垢分枝杆菌mc2155作为指示菌株进行帕马霉素生物活性测试,并结合LC-MS的分析确定帕马霉素的产生情况。最后,通过基因失活与回补的方法,考察帕马霉素生物合成基因簇中调控蛋白PamR1和PamR2对帕马霉素生物合成的影响。【结果】帕马霉素生物合成基因簇在天蓝色链霉菌M1154中实现了表达,证明PamR1和PamR2负调控了帕马霉素生物合成的过程。【结论】帕马霉素完整基因簇的成功异源表达,一方面便于其生物合成途径的遗传改造,为帕马霉素的生物合成及优产改造研究奠定了基础;另外,调控基因功能的研究为帕马霉素的产量优化提供了改造的目标。     

Streptomyces coelicolor A3(2)中新颖Ⅰ型羊毛硫肽的挖掘及异源表达

【目的】Ⅰ型羊毛硫肽通常具有广泛的生物活性,且抑菌机制独特,较少产生耐药性,因而在临床上具有很好的应用前景。本文对Streptomyces coelicolor A3(2)基因组上2个新颖的Ⅰ型羊毛硫肽生物合成基因簇进行研究,以实现目标羊毛硫肽的表达。【方法】首先,通过antiSMASH分析S. coelicolor A3(2)基因组序列,挖掘羊毛硫肽生物合成基因簇,使用BLAST进行基因功能注释,选择可能参与生物合成过程的基因;然后利用基因组装技术构建异源表达质粒,通过接合转移在链霉菌底盘细胞中进行异源表达;最后对发酵产物进行高效液相色谱、质谱及生物活性检测。【结果】通过添加启动子元件重构S. coelicolor A3(2)上基因簇3 (8.9 kb)和基因簇24 (9.0 kb),得到pYES-ColE1-SCO-cluster3和pYES-ColE1-SCO-cluster24。pYES-ColE1-SCO-cluster3在底盘细胞Streptomyces coelicolor M1152和Streptomycessp. A14中成功表达,得到潜在目标化合物coelin 3;pYES-ColE1-SCO-cluster24在底盘细胞Streptomyces sp. ZM13中成功表达,得到潜在目标化合物coelin 24。其中coelin 3对Bacillus subtilis 168和Escherichia coli ATCC 25922具有抑制作用,并且抑菌圈均达到28 mm。【结论】本研究成功使用启动子激活和异源表达策略实现了coelin 3和coelin 24的表达和活性测试,为后续新颖的羊毛硫肽结构解析和作用机制研究奠定了基础。     

基于CRISPR/Cas系统的天然产物生物合成基因簇克隆和编辑技术

合成生物学和基因组测序技术的快速发展使挖掘和高效合成天然产物进入了一个全新的时代。由于多数原始菌株生长缓慢、难以培养及遗传改造困难等问题,导致天然产物生物合成基因簇的激活和高效表达受到严重制约。基于此,将原始菌株来源的基因簇转移到操作简便、遗传背景清晰的模式宿主中进行异源表达成为天然产物发现和产量提高的一种有效手段。其中,基因簇的克隆与编辑是实现天然产物异源表达的一个主要限速步骤。CRISPR/Cas技术的应用极大地提高了大型基因簇克隆和编辑的效率,有效促进了微生物来源新药的发现。本文针对基于CRISPR/Cas开发的基因簇克隆和编辑技术进行了系统梳理和全面总结,探讨相关技术在天然产物挖掘和高效合成中的应用及其重要意义。     

非核糖体肽合成酶工程改造研究进展

非核糖体肽合成酶合成的非核糖体肽类天然产物具有丰富的结构和多样的功能,在医药、农业、工业等领域具有广泛的应用潜力。利用合成生物技术工程改造非核糖体肽合成酶,在微生物细胞工厂中组合生物合成新型非核糖体肽分子顺应绿色化学的发展理念,是国内外学者关注的热点。文中归纳了3种不同的非核糖体肽合成酶工程改造策略,并对近年来相关领域的研究进展进行综述。     

提高放线菌次级代谢产物产量方法的研究进展

放线菌的次级代谢产物一直是抗生素等药物开发的重要来源。通过基因技术来提高放线菌次级代谢产物产量的方法已被广泛接受。结合近年的研究成果,概述了利用基因技术过表达或者敲除代谢途经关键基因及加倍基因簇,诱变核糖体相关蛋白的基因,以及异源表达外源基因簇等提高次级代谢产物产量的方法。旨在为寻找新药及改造高产菌株提供相应的技术参考。     

The tallysomycin biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insights into the biosynthesis of the bleomycin family of antitumor antibiotics

The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.     

Cloning and identification of saprolmycin biosynthetic gene cluster from Streptomyces sp. TK08046

Saprolmycins A–E are anti-Saprolegnia parasitica antibiotics. To identify the gene cluster for saprolmycin biosynthesis in Streptomyces sp. TK08046, polymerase chain reaction using aromatase and cyclase gene-specific primers was performed; the spr gene cluster, which codes for angucycline biosynthesis, was obtained from the strain. The cluster consists of 36 open reading frames, including minimal polyketide synthase, ketoreductase, aromatase, cyclase, oxygenase, and deoxy sugar biosynthetic genes, as defined by homology to the corresponding genes of the urdamycin, Sch-47554, and grincamycin biosynthetic gene clusters in Streptomyces fradiae, Streptomyces sp. SCC-2136, and Streptomyces lusitanus, respectively. To establish the function of the gene cluster, an expression cosmid vector containing all 36 open reading frames was introduced into Streptomyces lividans TK23. The transformant was confirmed to express the biosynthetic genes and produce saprolmycins by liquid chromatography–mass spectrometry analysis of the extract.     

Characterization of the locus of genes encoding enzymes producing heptadepsipeptide micropeptin in the unicellular cyanobacterium Microcystis

The gene cluster involved in producing the cyclic heptadepsipeptide micropeptin was cloned from the genome of the unicellular cyanobacterium Microcystis aeruginosa K-139. Sequencing revealed four genes encoding non-ribosomal peptide synthetases (NRPSs) that are highly similar to the gene cluster involved in cyanopeptolins biosynthesis. According to predictions based on the non-ribosomal consensus code, the order of the mcnABCE NPRS modules was well consistent with that of the biosynthetic assembly of cyclic peptides. The biochemical analysis of a McnB(K-139) adenylation domain and the knock-out of mcnC in a micropeptin-producing strain, M. viridis S-70, revealed that the mcn gene clusters were responsible for the production of heptadepsipeptide micropeptins. A detailed comparison of nucleotide sequences also showed that the regions between the mcnC and mcnE genes of M. aeruginosa K-139 retained short stretches of DNA homologous to halogenase genes involved in the synthesis of halogenated cyclic peptides of the cyanopeptolin class including anabaenopeptilides. This suggests that the mcn clusters of M. aeruginosa K-139 have lost the halogenase genes during evolution. Finally, a comparative bioinformatics analysis of the congenial gene cluster for depsipetide biosynthesis suggested the diversification and propagation of the NRPS genes in cyanobacteria.     

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.     

Biosynthesis of a natural polyketide-isoprenoid hybrid compound, furaquinocin A: identification and heterologous expression of the gene cluster

Furaquinocin (FQ) A, produced by Streptomyces sp. strain KO-3988, is a natural polyketide-isoprenoid hybrid compound that exhibits a potent antitumor activity. As a first step toward understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we have cloned an FQ A biosynthetic gene cluster by taking advantage of the fact that an isoprenoid biosynthetic gene cluster generally exists in flanking regions of the mevalonate (MV) pathway gene cluster in actinomycetes. Interestingly, Streptomyces sp. strain KO-3988 was the first example of a microorganism equipped with two distinct mevalonate pathway gene clusters. We were able to localize a 25-kb DNA region that harbored FQ A biosynthetic genes (fur genes) in both the upstream and downstream regions of one of the MV pathway gene clusters (MV2) by using heterologous expression in Streptomyces lividans TK23. This was the first example of a gene cluster responsible for the biosynthesis of a polyketide-isoprenoid hybrid compound. We have also confirmed that four genes responsible for viguiepinol [3-hydroxypimara-9(11),15-diene] biosynthesis exist in the upstream region of the other MV pathway gene cluster (MV1), which had previously been cloned from strain KO-3988. This was the first example of prokaryotic enzymes with these biosynthetic functions. By phylogenetic analysis, these two MV pathway clusters were identified as probably being independently distributed in strain KO-3988 (orthologs), rather than one cluster being generated by the duplication of the other cluster (paralogs).     

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.     

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。     

Lantibiotic engineering: molecular characterization and exploitation of lantibiotic-synthesizing enzymes for peptide engineering

Lanthionine-containing peptide antibiotics called lantibiotics are produced by a large number of Gram-positive bacteria. Nukacin ISK-1 produced by Staphylococcus warneri ISK-1 is type-A(II) lantibiotic. Ribosomally synthesized nukacin ISK-1 prepeptide (NukA) consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events including unusual amino acid formation by the modification enzyme NukM, cleavage of leader peptide and export by the dual functional ABC transporter NukT, finally yielding a biologically active peptide. Unusual amino acids in lantibiotics contribute to biological activity and also structural stability against proteases. Thus, lantibiotic-synthesizing enzymes have a high potentiality for peptide engineering by introduction of unusual amino acids into desired peptides with altering biological and physicochemical properties, e.g., activity and stability, termed lantibiotic engineering. We report the establishment of a heterologous expression of nukacin ISK-1 biosynthetic gene cluster by the nisin-controlled expression system and discuss our recent progress in understanding of the biosynthetic enzymes for nukacin ISK-1 such as localization, molecular interaction in biophysical and biochemical aspects. Substrate specificity of the lantibiotic-synthesizing enzymes was evaluated by complementation of the biosynthetic enzymes (LctM and LctT) of closely related lantibiotic lacticin 481 for nukacin ISK-1 biosynthesis. We further explored a rapid and powerful tool for introduction of unusual amino acids by co-expression of hexa-histidine-tagged NukA and NukM in Escherichia coli.