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,是潜在的先导化合物,部分化合物已被开发成分子探针。本文综述了近年来微生物来源的戊二酰亚胺类天然产物及其生物合成研究,包括Iso-Migrastatin、Lactimidomyin、Cycloheximide、Streptimidone、Gladiostatin、Sesbanimide等,对戊二酰亚胺类天然产物的生物合成研究,将有效促进通过基因组挖掘策略寻找新型戊二酰亚胺类天然产物。     

多杀菌素生物合成的分子机理与定向遗传改造策略

多杀菌素是由土壤放线菌刺糖多孢菌产生的次生代谢产物．主要活性物质为spinosyn A和spinosyn D,是一种新型的高效广谱大环内酯类杀虫剂,因其独特的杀虫机理而兼具生物农药的安全性与化学农药的快速性．简述了多杀菌素生物合成的分子机理,包括聚酮链的生物合成与修饰、NDP糖的合成、修饰与连接转移以及多杀菌素生物合成基因表达调控．通过对多杀菌素生物合成的限速步骤及相关基因的分析．探讨了提高多杀菌素产量与合成其新衍生物的定向遗传改造策略,并展望了其应用发展前景．     

解淀粉芽孢杆菌SWB16菌株脂肽类代谢产物对球孢白僵菌的拮抗作用

【目的】筛选对球孢白僵菌(Beauveria bassiana)具有较强拮抗作用的细菌菌株及检测菌株脂肽类代谢产物的拮抗活性。【方法】通过形态学观察、生理生化实验、16S rRNA和gyrA基因序列分析鉴定目标菌株;用滤纸片扩撒法(K-B法)测定抑菌圈的直径;采用甲醇萃取菌株发酵液以提取脂肽类代谢产物,并显微观察提取物对白僵菌分生孢子及菌丝的拮抗作用;高效液相色谱-质谱联用方法及靶基因克隆技术检测菌株脂肽类代谢产物的主要成分和基因。【结果】从植物盾叶薯蓣(Dioscorea zingiberensis C.H.Wright)组织内分离得到了一株对球孢白僵菌具有较强拮抗活性的菌株SWB16,该菌株属于解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其脂肽类提取物对球孢白僵菌分生孢子的发芽和菌丝生长均具有明显的抑制作用,质谱检测表明提取物的主要成分是芬枯草菌素和伊枯草菌素,从菌株基因组中克隆到编码芬枯草菌素和伊枯草菌素的fenB基因和ituA基因。【结论】解淀粉芽孢杆菌(B.amyloliquefaciens)SWB16菌株能产生脂肽类抗生素并对球孢白僵菌具有拮抗作用,拮抗活性显示该菌株对防治家蚕等经济昆虫的白僵病具有潜在的应用价值。     

专性海洋放线菌盐孢菌的研究进展

盐孢菌属(Salinispora)作为首个被报道的专性海洋放线菌,主要分布于热带和亚热带海洋沉积环境中,在海绵、海鞘中也有发现。与其他大多数放线菌一样,盐孢菌属的菌株可以产生大量具有抗细菌、抗病毒、抗肿瘤细胞活性、结构新颖的次级代谢产物且表现出物种特异性。全基因组序列分析显示,盐孢菌属菌株基因组中超过10%的基因序列与次级代谢产物合成相关,但绝大多数生物合成基因簇编码的产物未被发现,表明盐孢菌属还存在巨大的生物合成潜能,有待深入发掘。目前新的培养方法、测序技术及生物信息学、基因组发掘技术、合成生物学技术的发展对提升盐孢菌属菌株新型药物的生产潜力发挥重要作用。本文对盐孢菌属的物种多样性、系统分类与化合物发现等方面的研究进行了系统综述。     

隐性次级代谢产物生物合成基因簇的激活及天然产物定向发现

传统的"活性-化合物"天然药物发现方法导致大量已知化合物被重复分离,大大加剧了新药发现的难度。规模化基因组测序揭示了微生物基因组中存在大量的隐性(cryptic)次级代谢产物生物合成基因簇,如何激活这些隐性基因簇成为当今世界天然产物研究领域的难点与热点。本文从途径特异性和多效性两个角度综述了隐性生物合成基因簇激活策略;同时,对基因组信息指导下结构导向(structure-guided)的化合物定向分离技术进行了归纳。隐性基因簇的激活为定向发掘具有优良活性的新型天然产物提供了新的契机。     

真菌天然产物异源生产研究进展

真菌天然产物是天然药物的重要来源之一,大规模真菌基因组序列测序的完成表明真菌具有产生丰富的次级代谢产物的潜能。然而,许多真菌或生长缓慢,或不适宜在实验室条件下培养,或难以进行遗传操作,或化合物产量极低等,这些因素导致大量有价值的真菌天然产物无法获得。利用异源表达系统对真菌天然产物进行生产是发现新天然产物及解析其生物合成途径的有效手段,并为定向的以合成生物学的手段去合成重要活性分子奠定基础。本文对目前用于真菌天然产物生产的各种异源表达系统进行了综述,并结合最新的DNA组装技术展望了异源表达系统在真菌天然产物研究中的应用价值和前景。     

基于基因组挖掘的农抗120活性成分制霉菌素和丰加霉素的发现和鉴定

【目的】分析刺孢吸水链霉菌北京变种(农抗120产生菌)基因组和次级代谢产物组分,研究并鉴定农抗120产生菌中未被发现的活性组分。【方法】利用antiSMASH在线分析农抗120产生菌Streptomyces hygrospinosusvar.beijingensis基因组信息,锁定可能的制霉菌素和丰加霉素生物合成基因簇。利用HPLC和LC-MS等分析方法对农抗120产生菌发酵产物进行分析,同时利用制霉菌素和丰加霉素标准品作为对照,以鉴定该菌株代谢组分中的次级代谢产物。此外,通过构建目标基因簇大片段缺失突变株,并对所得突变株发酵产物进行检测,以确定生物合成基因簇与目的代谢产物的对应关系。【结果】本研究综合利用基因组序列分析、基因缺失突变株构建以及代谢产物检测方法,鉴定了农抗120产生菌中制霉菌素和丰加霉素两种活性成分,并确定了负责这些化合物合成的基因簇。【结论】本研究所构建的多重基因簇失活突变株为挖掘刺孢吸水链霉菌北京变种更多的天然次级代谢产物奠定了基础。     

红树林放线菌及其天然产物研究进展北大核心CSCD

红树林是热带亚热带潮间带的木本植物群落。为从海洋环境寻找新的天然产物,红树林已经成为放线菌资源收集、天然产物分离鉴定及其生物合成机制研究的热点。从巴哈马红树林分离的盐孢菌所产生的盐孢菌素(Salinosporamide A)已经进入临床研究。从红树林分离的放线菌类群已经达到8个亚目11科24属,并发现新属3个,新种31个。从红树林放线菌分离的新天然产物包括生物碱、喹啉等芳香类、阿扎霉素等大环类脂及吲哚衍生物等,大多数天然产物来源于红树林链霉菌。新型吲哚咔唑、吲哚倍半萜及厦霉素等以新颖的结构备受关注,相关生物合成途径已被揭示。     

基于生物合成机制的天然产物结构多样性研究

天然产物尤其是次级代谢产物在药物化学和化学生物学中扮演重要角色.基于天然产物获得结构多样性的类似物对于新药的筛选和医学研究具有重要意义.天然产物均由生物体代谢产生,在了解其生物合成机制的基础上,对生物合成过程进行合理化改造,可以极大地丰富天然产物的结构多样性,获得许多具有重要生理活性和有机化学不易合成的天然产物类似物.本文以硫肽类抗生素中的硫链丝菌素和聚酮聚肽类化合物为例,对生物合成方法在天然产物结构多样性中的应用进行总结和展望.     

链霉菌底盘细胞的开发现状及其应用

天然产物及其衍生物在现代医疗中扮演着举足轻重的角色,其生物活性多样性以及化学结构的丰富性是新药研发的源泉和动力。利用纯化学方法合成天然产物在技术和成本上有很大的困难,加上许多天然产物的原始产生菌具有培养条件苛刻、产量低下等缺点,而且大量基因簇在原始菌株中是沉默的,这使得利用合成生物学思想来指导天然产物生物合成基因簇的异源表达具有重大意义。作为抗生素、抗肿瘤活性物质、免疫抑制剂等次级代谢产物主要来源的放线菌一直是研究者们关注的焦点,特别是随着基因测序技术的飞速发展,人们发现链霉菌基因组中包含着极为丰富的天然产物生物合成基因簇资源。这意味着开发链霉菌底盘细胞作为异源表达宿主有其得天独厚的优势。本综述从底盘细胞开发的意义入手,重点阐述链霉菌底盘细胞构建的策略及现状,随后通过实例阐述了各种底盘链霉菌的实际应用。     

Molecular breeding of carotenoid biosynthetic pathways

The burgeoning demand for complex, biologically active molecules for medicine, materials science, consumer products, and agrochemicals is driving efforts to engineer new biosynthetic pathways into microorganisms and plants. We have applied principles of breeding, including mixing genes and modifying catalytic functions by in vitro evolution, to create new metabolic pathways for biosynthesis of natural products in Escherichia coli. We expressed shuffled phytoene desaturases in the context of a carotenoid biosynthetic pathway assembled from different bacterial species and screened the resulting library for novel carotenoids. One desaturase chimera efficiently introduced six rather than four double bonds into phytoene, to favor production of the fully conjugated carotenoid, 3, 4,3',4'-tetradehydrolycopene. This new pathway was extended with a second library of shuffled lycopene cyclases to produce a variety of colored products. One of the new pathways generates the cyclic carotenoid torulene, for the first time, in E. coli. This combined approach of rational pathway assembly and molecular breeding may allow the discovery and production, in simple laboratory organisms, of new compounds that are essentially inaccessible from natural sources or by synthetic chemistry.     

Elucidation of the complete biosynthetic pathway of the main triterpene glycosylation products of Panax notoginseng using a synthetic biology platform

UDP-glycosyltransferase (UGT)-mediated glycosylation is a widespread modification of plant natural products (PNPs), which exhibit a wide range of bioactivities, and are of great pharmaceutical, ecological and agricultural significance. However, functional annotation is available for less than 2% of the family 1 UGTs, which currently has 20,000 members that are known to glycosylate several classes of PNPs. This low percentage illustrates the difficulty of experimental study and accurate prediction of their function. Here, a synthetic biology platform for elucidating the UGT-mediated glycosylation process of PNPs was established, including glycosyltransferases dependent on UDP-glucose and UDP-xylose. This platform is based on reconstructing the specific PNPs biosynthetic pathways in dedicated microbial yeast chassis by the simple method of plug-and-play. Five UGT enzymes were identified as responsible for the biosynthesis of the main glycosylation products of triterpenes in Panax notoginseng, including a novel UDP-xylose dependent glycosyltransferase enzyme for notoginsenoside R1 biosynthesis. Additionally, we constructed a yeast cell factory that yields >1 g/L of ginsenoside compound K. This platform for functional gene identification and strain engineering can serve as the basis for creating alternative sources of important natural products and thereby protecting natural plant resources.     

Saponins in cereals

Saponins are a diverse family of secondary metabolites that are produced by many plant species, particularly dicots. These molecules commonly have potent antifungal activity and their natural role in plants is likely to be in protection against attack by pathogenic microbes. They also have a variety of commercial applications including use as drugs and medicines. The enzymes, genes and biochemical pathways involved in the synthesis of these complex molecules are largely uncharacterized for any plant species. Cereals and grasses appear to be generally deficient in saponins with the exception of oats, which produce both steroidal and triterpenoid saponins. The isolation of genes for saponin biosynthesis from oats is now providing tools for the analysis of the evolution and regulation of saponin biosynthesis in monocots. These genes may also have potential for the development of improved disease resistance in cultivated cereals.     

Significant natural product biosynthetic potential of actinorhizal symbionts of the genus frankia, as revealed by comparative genomic and proteomic analyses

Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus.     

Biological activities associated to the chemodiversity of the brown algae belonging to genus <Emphasis Type="Italic">Lobophora</Emphasis> (Dictyotales,Phaeophyceae)

Although Lobophora belongs to a marine algal family (Dictyotaceae) that produces a large array of secondary metabolites, it has received little attention compared to other genera, such as Dictyota, in terms of natural compounds isolation and characterization. However, metabolites produced by Lobophora species have been found to exhibit a wide array of bioactivities including pharmacological (e.g. antibacterial, antiviral, antioxidant, antitumoral), pesticidal, and ecological. This review aims to report the state-of-the-art of the natural products isolated from Lobophora species (Dictyotales, Phaeophyceae) and their associated bioactivities. All bioactivities documented in the literature are reported, therefore including studies for which pure active substances were described, as well as studies limited to extracts or fractions. From the early 1980s until today, 49 scientific works have been published on Lobophora chemistry and bioactivity, among which 40 have reported bioactivities. Only six studies, however, have identified, characterized and tested no less than 23 bioactive pure compounds (three C₂₁ polyunsaturated alcohols, three fatty-acids, a macrolactone, 11 polyketides, a few sulfated polysaccharides, three sulfolipids, a tocopherol derivative). The present review intends to raise awareness of chemists and biologists given the recent significant taxonomic progress of this brown algal genus, which holds a promising plethora of natural products yet to be discovered with ecological and pharmacological properties.     

An atypical orthologue of 6-pyruvoyltetrahydropterin synthase can provide the missing link in the folate biosynthesis pathway of malaria parasites

Folate metabolism in malaria parasites is a long-standing, clinical target for chemotherapy and prophylaxis. However, despite determination of the complete genome sequence of the lethal species Plasmodium falciparum, the pathway of de novo folate biosynthesis remains incomplete, as no candidate gene for dihydroneopterin aldolase (DHNA) could be identified. This enzyme catalyses the third step in the well-characterized pathway of plants, bacteria, and those eukaryotic microorganisms capable of synthesizing their own folate. Utilizing bioinformatics searches based on both primary and higher protein structures, together with biochemical assays, we demonstrate that P. falciparum cell extracts lack detectable DHNA activity, but that the parasite possesses an unusual orthologue of 6-pyruvoyltetrahydropterin synthase (PTPS), which simultaneously gives rise to two products in comparable amounts, the predominant of which is 6-hydroxymethyl-7,8-dihydropterin, the substrate for the fourth step in folate biosynthesis (catalysed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; PPPK). This can provide a bypass for the missing DHNA activity and thus a means of completing the biosynthetic pathway from GTP to dihydrofolate. Supported by site-directed mutagenesis experiments, we ascribe the novel catalytic activity of the malarial PTPS to a Cys to Glu change at its active site relative to all previously characterized PTPS molecules, including that of the human host.     

TNT biotransformation: when chemistry confronts mineralization

The recent increase and availability of whole genome sequences have revised our view of the metabolic capabilities of microorganisms. From these data, a large number of orphan biosynthesis pathways have been identified by bio-informatics. Orphan biosynthetic pathways are gene clusters for which the encoded natural product is unknown. It is worthy to note that the number of orphan pathways coding for putative natural products outnumbers by far the number of currently known metabolites for a given organism. Whilst Streptomyces coelicolor was known to produce only 4 secondary metabolites, the genome analysis revealed 18 additional orphan biosynthetic pathways. It is intriguing to note that this is not a particular case because analysis of other microbial genomes originating from myxobacteria, cyanobacteria and filamentous fungi showed the presence of a comparable or even larger number of orphan pathways. The discovery of these numerous pathways represents a treasure trove, which is likely to grow exponentially in the future, uncovering many novel and possibly bio-active compounds. The few natural products that have been correlated with their orphan pathway are merely the tip of the iceberg, whilst plenty of metabolites await discovery. The recent strategies and methods to access these promising hidden natural products are discussed in this review.     

Genome-enabled determination of amino acid biosynthesis in Xanthomonas campestris pv. campestris and identification of biosynthetic pathways for alanine, glycine, and isoleucine by 13C-isotopologue profiling

To elucidate the biosynthetic pathways for all proteinogenic amino acids in Xanthomonas campestris pv. campestris, this study combines results obtained by in silico genome analysis and by (13)C-NMR-based isotopologue profiling to provide a panoramic view on a substantial section of bacterial metabolism. Initially, biosynthesis pathways were reconstructed from an improved annotation of the complete genome of X. campestris pv. campestris B100. This metabolic reconstruction resulted in the unequivocal identification of biosynthesis routes for 17 amino acids in total: arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Ambiguous pathways were reconstructed from the genome data for alanine, glycine, and isoleucine biosynthesis. (13)C-NMR analyses supported the identification of the metabolically active pathways. The biosynthetic routes for these amino acids were derived from the precursor molecules pyruvate, serine, and pyruvate, respectively. By combining genome analysis and isotopologue profiling, a comprehensive set of biosynthetic pathways covering all proteinogenic amino acids was unraveled for this plant pathogenic bacterium, which plays an important role in biotechnology as a producer of the exopolysaccharide xanthan. The data obtained lay ground for subsequent functional analyses in post-genomics and biotechnology, while the innovative combination of in silico and wet lab technology described here is promising as a general approach to elucidate metabolic pathways.     

Engineering natural products using combinatorial biosynthesis and biocatalysis

Many biologically active natural products are produced by the host organisms using dedicated biosynthetic pathways. The programming rules of these pathways may be rationally manipulated through combinatorial biosynthesis to produce natural products that contain structural variations or enhanced pharmacological properties. Furthermore, these pathways contain enzymes that can be harvested as powerful biocatalysts for the synthesis of both new drugs and existing blockbuster therapeutics. This review will highlight recent advances in exploring natural product biosynthetic pathways for new compounds, novel enzymes and useful biocatalysts.