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。     

核苷类抗生素代谢工程的研究现状及展望

核苷类抗生素是一类生物活性多样,主要来源于微生物的重要次级代谢产物,应用潜力巨大.一些核苷类抗生素生物合成基因簇的相继克隆及其基因功能的深入研究从分子水平揭示出此类抗生素独特的生物合成途径与调节机制,为今后利用代谢工程手段实现核苷类抗生素的定向改造提供了丰富的研究材料与理论基础.     

匹马菌素的生物合成研究进展

匹马菌素是纳塔链霉菌产生的一种安全、高效的多烯大环类真菌抑制剂,广泛应用于医药领域与食品工业.作为26元环的糖基化多烯大环聚酮,其生物合成基因簇长度约110kb,包含19个基因,编码5个聚酮合酶、10个修饰和转运蛋白及4个调控因子.本文分析了匹马菌素生物合成的基因基础、多烯聚酮合成过程、氧化和糖基化修饰与调控机理等最新研究进展,展望了利用组合生物合成进行基因簇改造的方案与应用前景.     

海洋放线菌Streptomyces costaricanus SCSIO ZS0073中fungichromin生物合成基因簇的分析鉴定

目的 对分离自我国广西红沙红树林湿地公园的放线菌菌株Streptomyces costaricanus SCSIO ZS0073进行基因组测序分析,并对其生产的制霉色基素(fungichromin)的生物合成基因簇进行分析鉴定。 方法 对S. costaricanus SCSIO ZS0073菌株进行基因组提取,利用Highseq4000和PacBio SMRT技术相结合的手段对该菌株进行了基因组完成图测序;利用生物信息学方法对基因组进行注释并寻找fungichromin的生物合成基因簇,对fungichromin生物合成骨架基因fgmJ1进行置换突变,确定fungichromin生物合成基因簇,结合fungichromin结构特征和其基因簇内遗传信息及聚酮化合物的生物合成原理,对其生物合成途径进行推测。 结果 全基因组测序发现S. costaricanus SCSIO ZS0073菌株基因组含有一条线性染色体和一个圆形质粒,基因组含有36个次级代谢产物生物合成基因簇。利用基因敲除手段对fungichromin的生物合成基因簇进行了确认并进行了生物合成途径推导。 结论 fungichromin生物合成基因簇的确定和生物合成途径的推导为该化合物的基因工程改造研究奠定了分子基础。     

罗米地辛类抗癌药物的生物合成

罗米地辛(又称FK228)是紫色色杆菌中非核糖体肽合酶(NRPS)和I型聚酮合酶(PKS)催化合成的一种缩酚酸肽类天然产物,具有组蛋白脱乙酰基酶抑制剂活性,现被用于皮肤T细胞淋巴瘤和外周T细胞淋巴瘤的临床治疗。我们对已发现的罗米地辛类天然产物的化学结构,生物合成基因簇和生物合成途径的研究现状进行了总结,在此基础上初步探讨了通过基因组挖掘和组合生物合成等生物学手段获取新的罗米地辛类天然产物的可能性。     

聚酮合成酶底物专一性的研究进展

聚酮合成酶能够合成包括许多大环内酯类抗生索在内的聚酮类天然产物，它的结构和功能的模块性为组合生物合成的产生和发展奠定了基础。聚酮合成酶的酰基转移酶功能域可以特异性地决定所选择酰基辅酶A的种类．决定产物的结构。近年来，针对许多来源不同、结构各异的聚酮合成酶的底物专一性已经从氨基酸序列、结构和功能等方面进行了大量的研究．为更有效地应用聚酮合成酶开发新型抗生索奠定了基础。     

一株海洋来源Streptomyces sp. SCSIO 1682中那西肽(nosiheptide)的分离鉴定及其生物合成基因簇分析

目的 对一株海洋来源的菌株SCSIO 1682进行16S rDNA分子生物学鉴定,对发酵产物中的抑菌活性化合物进行分离鉴定,并对抑菌活性化合物的生物合成基因簇进行分析。方法 通过构建基于16S rDNA序列的系统发育树来进行菌株鉴定;通过有机溶剂萃取、正相和反相硅胶层析等化学分离手段对菌株SCSIO 1682的次级代谢产物进行活性追踪分离纯化;运用HR-ESI-MS,₁H和₁₃C NMR等波谱手段对所得化合物进行结构解析;通过对菌株SCSIO 1682进行全基因组扫描测序鉴定所得化合物的生物合成基因簇。结果 该菌株被鉴定为Streptomyces sp. SCSIO 1682,所得抑菌活性化合物为那西肽,鉴定了那西肽的生物合成基因簇并进行生物信息学分析。结论 从海洋链霉菌Streptomyces sp. SCSIO 1682中分离鉴定了那西肽,那西肽生物合成基因簇的鉴定为利用组合生物合成和合成生物学技术对那西肽进行结构改造提供了新的基因资源。     

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。     

2-脱氧链霉胺类抗生素生物合成基因的研究进展

含2-脱氧链霉胺类（2-DOS）氨基糖苷抗生素是临床常用抗生素。近几年对2-脱氧链霉胺类抗生素的分子水平研究取得重大进展，得到了2-DOS合成中的关键酶基因，以及丁酰苷菌素、妥布霉素、卡那霉素、庆大霉素等部分生物合成基因簇克隆。本文对2-脱氧链霉胺类抗生素生物合成基因研究进展进行了综述。     

产黄青霉青霉素生物合成途径的亚细胞分区

产黄青霉(P.chrysogenum)的青霉素生物合成途径分三步(图1),至20世纪九十年代各步酶的编码基因皆已克隆,所构成的基因簇,亦克隆至不产青霉素的异种,并获表达.青霉素生物合成途径的亚细胞分区研究,则建立于这种基础上,还借助于高产的工业菌株.     

The biosynthetic gene clusters of aminocoumarin antibiotics

Plants and microorganisms are the most important sources of secondary metabolites in nature. For research in the functional genomics of secondary metabolism, and for the biotechnological application of such research by genetic engineering and combinatorial biosynthesis, most microorganisms offer a unique advantage to the researcher: the biosynthetic genes for a specific secondary metabolite are not scattered over the genome, but rather are clustered in a well-defined, contiguous region - the biosynthetic gene cluster of that metabolite. This is exemplified in this review for the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A (1), which are potent inhibitors of DNA gyrase. Cloning, sequencing and analysis of the biosynthetic gene clusters of these three antibiotics revealed that the structural differences and similarities of the compounds are perfectly reflected by the genetic organisation of the biosynthetic gene clusters. The function of most biosynthetic genes could be identified by gene inactivation experiments as well as by heterologous expression and biochemical investigation. The prenylated benzoic acid moiety of novobiocin and clorobiocin, involved in the interaction with gyrase, is structurally similar to metabolites found in plants. However, detailed investigations of the biosynthesis revealed that the biosynthetic pathway and the enzymes involved are totally different from those identified in plants.     

Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis

Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis is playing an increasingly important role in natural product-based drug discovery and development programmes. This review highlights the requirements and challenges associated with this conceptually simple strategy of using surrogate hosts for the production of natural products in good yields and for the generation of novel analogues by combinatorial biosynthesis methods, taking advantage of the recombinant DNA technologies and tools available in the model hosts. Specific topics addressed include: i) the mobilisation of biosynthetic gene clusters using different vector systems; ii) the selection of suitable model heterologous hosts; iii) the requirement of post-translational protein modifications and precursor supply within the model hosts; iv) the influence of promoters and pathway regulators; and v) the choice of suitable fermentation conditions. Lastly, the use of heterologous expression in combinatorial biosynthesis is addressed. Future directions for model heterologous host engineering and the optimisation of natural product biosynthetic gene cluster expression in heterologous hosts are also discussed.     

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.     

Plasticity of the streptomyces genome-evolution and engineering of new antibiotics

Streptomyces is a genus of soil dwelling bacteria with the ability to produce natural products that have found widespread use in medicine. Annotation of Streptomyces genome sequences has revealed far more biosynthetic gene clusters than previously imagined, offering exciting possibilities for future combinatorial biosynthesis. Experiments to manipulate modular biosynthetic clusters to create novel chemistries often result in no detectable product or product yield is extremely low. Understanding the coupling between components in these hybrid enzymes will be crucial for efficient synthesis of new compounds. We are using new algebraic approaches to predict protein properties, and homologous recombination to exploit natural evolutionary constraints to generate novel functional enzymes. The methods and techniques developed could easily be adapted to study modular, multi-interacting complex systems where appreciable biochemical and comparative sequence data are available, for example, clinically significant non-ribosomally synthesised peptides and polyketides.     

Biosynthesis of hybrid peptide-polyketide natural products

The structural and catalytic similarities between non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) support the idea of combining individual NRPS and PKS modules for combinatorial biosynthesis. Recent advances in cloning and characterization of biosynthetic gene clusters for naturally occurring hybrid polyketide-peptide metabolites have provided direct evidence for the existence of hybrid NRPS-PKS systems, thus setting the stage to investigate the molecular basis for intermodular communication between NRPS and PKS modules. Reviewed in this article are biosynthetic data pertinent to hybrid peptide-polyketide biosynthesis published up to late 2000. Hybrid peptide-polyketide natural products can be divided into two classes: (i) those whose biosyntheses do not involve functional interaction between NRPS and PKS modules; and (ii) those whose biosyntheses are catalyzed by hybrid NRPS-PKS systems involving direct interactions between NRPS and PKS modules. It is the latter systems that are most likely amenable to combinatorial biosynthesis. The same catalytic sites appear to be conserved in both hybrid NRPS-PKS and normal NRPS or PKS systems, with the exception of the ketoacyl synthase domains in hybrid NRPS-PKS systems which are unique. Specific linkers may play a critical role in communication, facilitating the transfer of the growing intermediates between the interacting NRPS and/or PKS modules. In addition, phosphopantetheinyl transferases with broad carrier protein specificity are essential for the production of functional hybrid NRPS-PKS megasynthetases. These findings should now be taken into consideration in engineered biosynthesis of hybrid peptide-polyketide natural products for drug discovery and development.     

Enediyne natural products: biosynthesis and prospect towards engineering novel antitumor agents

This review gives a brief account on the current status of enediyne biosynthesis and the prospective of applying combinatorial biosynthesis methods to the enediyne system for novel analog production. Methods for cloning enediyne biosynthetic gene clusters are first reviewed. A unified paradigm for enediyne biosynthesis, characterized with (a) the enediyne PKS, (b) the enediyne PKS accessory enzymes, and (c) tailoring enzymes, is then presented. Strategies and tools for novel enediyne analog production by combinatorial biosynthesis are finally discussed. The results set the stage to decipher the molecular mechanism for enediyne biosynthesis and lay the foundation to engineer novel enediynes by combinatorial biosynthesis for future endeavors.     

非核糖体肽类化合物的组合生物合成策略研究进展

很多微生物能够利用非核糖体肽合酶合成结构复杂、种类繁多、生物活性多样的小分子肽类化合物.组合生物合成是对控制抗生素生物合成的基因簇进行阻断、置换、重组或异源表达等遗传操作,从而达到利用生物技术和环境友好的手段构建化合物衍生物库的目的.组合生物合成在增加天然活性化合物的数量,改良天然化合物的生物学活性,提高天然化合物的产量,开发创新药物和酶制剂等领域都具有重要应用价值.近年来,非核糖体肽的组合生物合成研究取得了重要进展.本文就非核糖体肽合酶的组合生物合成研究策略,从模块定点突变、替换、插入、删除、模块“洗牌”与异源表达等角度进行了综述.     

阿维菌素产生菌的生物技术改造研究进展

目的综述近年来运用基因工程技术对阿维菌素产生菌改良的研究进展。方法在查阅国内外文献近100篇的基础上,介绍了阿维菌素的生物合成途径,产生多组分的3个关键酶及阿维菌素产生菌改良的研究进展,包括选择性生产有效组分,产生新抗生素、杂合抗生素,改进生产工艺以及提高菌种产抗生素量。结果目前国内外均已经构建了只产生B组分及寡霉素基因缺失或失活的工程菌。分别运用突变结合理性化筛选和特定基因重组提高活性高的组分的产量,并且通过基因改造产生多种阿维菌素的衍生物;将透明颤菌的血红蛋白基因引入阿维菌素产生菌,改进氧的供应,阿维菌素的产量不断提高。阿维菌素生物合成调控机制和组合生物学改造聚酮合成酶等方面仍需深入研究。结论利用生物技术改造阿维菌素产生菌在组分改造、结构修饰、产品收率、生产工艺改进等方面已取得显著进展。对阿维菌素和其他聚酮体药物产生菌的生物合成、基因改造起着重要作用,使生产简化,成本降低,药物应用更广泛。     

恩拉霉素与雷莫拉宁生物合成研究进展

目前耐药革兰阳性菌在医院感染中的比例不断上升,具有新型革兰阳性病原菌抑制机制的恩拉霉素和雷莫拉宁成为目前研究的热点.含有17个氨基酸的肽类抗生素,恩拉霉素和雷莫拉宁的生物合成基因簇已分别从链霉菌Streptomyces fungicidicus ATCC21013和游动放线菌Actinoplanes sp.ATCC33076中克隆到.本文综述了恩拉霉素与雷莫拉宁生物合成途径研究的最新进展,为进一步利用基因工程手段对其进行结构改造提供依据.