植物来源的二萜化合物的生物合成研究

二萜化合物广泛地存在于动物、植物和微生物中。由于其化学结构多样性和良好的生物活性,越来越多的二萜类化合物作为药物走进大众的视野,并受到越来越多的科学工作者的关注。绝大多数的二萜化合物来源于植物,但是随着植物药材的长期开发,野生植物药材资源日益减少,人工种植植物药材品质日渐退化,而揭示其中关键活性成分的生物合成机制,以合成生物学的策略改造的微生物作为"细胞工厂",是解决上述问题的重要手段。该文主要以二萜合酶为线索,概述了植物来源的二萜类化合物的生物合成研究进展,探究二萜化合物结构的多样性及其生物合成研究的前景和发展趋势。     

植物萜类合酶研究进展

随着植物中许多有价值的萜类化合物被发现和应用于人类生活 ,萜类生物合成途径的研究倍受重视。萜类合酶催化单萜、倍半萜和二萜生物合成 ,即分别催化GPP、FPP和GGPP形成单萜、倍半萜和二萜。本文叙述了近年来在植物萜类合酶催化机理、克隆策略和萜类生物工程的研究进展     

植物萜类合酶研究进展

随着植物中许多有价值的萜类化合物被发现和应用于人类生活,萜类生物合成途径的研究倍受重视。萜类合酶催化单萜、倍半萜和二萜生物合成,即分别催化GPP、FPP和GGPP形成单萜、倍半萜和二萜。本文叙述了近年来在植物萜类合酶催化机理、克隆策略和萜类生物工程的研究进展。     

过表达丹参GGPPS研究丹参酮类的生物合成途径

丹参酮是药用植物丹参(Salvia miltiorrhiza)中具有较强生物活性的脂溶性二萜醌类化合物,是目前国际上广泛认可的有效治疗心脑血管疾病的天然药物之一.本研究通过分析过表达萜类生物合成途径中的牻牛儿基牻牛儿基焦磷酸合酶(GGPPS)的转基因丹参,发现丹参酮类化合物与叶绿素具有共同的上游生物合成途径,而下游途径因组织的特异化而不同,从而生成不同的代谢产物.通过对丹参毛状根外施两个萜类生物合成途径的抑制剂Lovastatin和Fosmidomycin,在处理6周丹参酮积累达到稳定时测定丹参酮的含量,探明了丹参酮类的生物合成主要是通过质体中的MEP途径来完成,而非胞质中的MVA途径.本研究为丹参酮类的代谢生物学及合成生物学研究提供了证据和基础.     

细菌中倍半萜生物合成的研究进展

萜类化合物是一大类小分子天然产物,在生物体内扮演重要的角色。植物和真菌中萜类化合物的生物合成已被广泛研究,但是在真核生物中克隆或改造萜类化合物生物合成途径还有较大难度。许多细菌同样可以产生萜类化合物。在过去十多年间细菌萜类合酶的研究进展为我们对萜类化合物生物合成的理解做出了显著的贡献。这里我们主要关注细菌中合成的倍半萜化合物,概述其化学结构、倍半萜合酶对法尼基焦磷酸环化的机制、后修饰酶特别是氧化还原酶所参与的后修饰、代谢调控以及合成途径中尚未解决的问题等。     

异戊二烯类植物激素赤霉素和脱落酸的代谢调控

赤霉素和脱落酸在植物生理过程中具有重要的调控作用,其生物合成途径迄今已基本阐明。赤霉素与类胡萝卜素的生物合成途径具有共同前体牻牛儿基牻牛儿基二磷酸,而脱落酸则直接来自于类胡萝卜素。参与这两种植物激素和类胡萝卜素代谢过程的大多数酶基因已经从不同植物中获得克隆;各种调控方式也随着分子生物学的研究工作而得到鉴定。本文就近年来对赤霉素和脱落酸等代谢调控机制及其与植物类胡萝卜素代谢之间关系的研究工作做简要回顾。     

高等植物牻牛儿基牻牛儿基焦磷酸合成酶基因的研究进展

牻牛儿基牻牛儿基焦磷酸合成酶(GGPPS)在植物体内催化牻牛儿基牻牛儿基焦磷酸(GGPP)的合成,GGPP是萜类物质、类胡萝卜素、叶绿素及几个重要植物激素合成的前体物,是联系植物体内多条重要次生代谢通路的节点物质。本文综述了植物GGPPS基因近年来的生物学功能研究进展和该基因家族的遗传分类情况,以及GGPPS小亚基基因的重要调控作用,拟为深入研究植物GGPPS基因的生物学功能和萜类含量调控的遗传工程提供新认识和新思路。     

竹叶花椒ZaGGPPS基因克隆与表达分析

为了揭示竹叶花椒萜类代谢的分子机理及嫁接对其风味的影响,该文依据转录组数据设计特异性引物,采用RT-PCR方法从竹叶花椒(Zanthoxylum armatum)中克隆得到一个全新的牻牛儿基牻牛儿基焦磷酸合成酶(GGPPS)基因的全长cDNA序列,命名为ZaGGPPS,并利用NCBI、ProParam、SignalP 4.1 server、DNAMAN和MEGA 7.0软件对ZaGGPPS基因进行生物信息学分析,并比较其在嫁接树和实生树中的表达量。结果表明:ZaGGPPS包含完整的cDNA开放阅读框(OFR),由1 086 bp组成,编码361个氨基酸。其蛋白的相对分子量为39 079.14 Da,理论等电点pI为6.38。Blast比对结果显示该蛋白质属于GGPPS家族蛋白,含有2个GGPPS蛋白特有的天冬氨酸富集基序,分别是"DDXXXXD"和"DDXXD",以及5个特征性功能结构域。系统进化树结果显示竹叶花椒与芸香科植物甜橙(Citrus sinensis)、克里曼丁桔(C. clementina)、柚子(C. maxima)等亲缘关系较近。荧光定量PCR检测显示,ZaGGPPS基因在竹叶花椒中的表达量从高到低分别为实生树的叶、嫁接树的叶、实生树的茎、嫁接树的茎。牻牛儿基牻牛儿基焦磷酸合成酶是竹叶花椒萜类化合物生物合成途径中的关键酶,通过嫁接可影响ZaGGPPS基因在叶和茎中的表达量。该文对竹叶花椒ZaGGPPS基因进行了克隆与分析,为后续深入研究竹叶花椒香气形成的分子机理及利用分子生物学手段选育优良品种提供理论依据。     

两种短命植物春萌秋萌个体生态生物学特征比较

短命植物是新疆北部荒漠生态系统中重要组成部分,对稳定该区沙漠生态系统、丰富物种多样性具有重要的意义。以往对短命植物的研究多集中在春季萌发的短命植物,秋季萌发的短命植物研究很少,对秋季萌发短命植物和春季萌发短命植物的生态生物学特征的异同以及它们对稳定荒漠生态系统的意义缺乏了解。选择小车前(Plantago minuta)和尖喙牻牛儿苗(Erodium oxyrrhynchum)为研究对象,采用野外定点标记观测和室内分析结合的方法,研究了两种短命植物春季萌发和秋季萌发两种表现型在冠幅、生物量、物候期、菌根侵染率等生态生物学特征方面的差异性。结果表明:秋季萌发的小车前和尖喙牻牛儿苗能够在覆雪下越冬,其单株的冠幅、叶片数量、干重等均远远大于同一时期生长的春季萌发的小车前和尖喙牻牛儿苗,尤其是单株结种数量更是比春季萌发小车前和尖喙牻牛儿苗的高13.0和4.4倍;秋季萌发的小车前和尖喙牻牛儿苗的花期分别比春季萌发小车前和尖喙牻牛儿苗早14和7 d,植株黄枯期和成熟期分别提早3和4 d;此外,秋季萌发小车前和尖喙牻牛儿苗各时期的菌根侵染率也显著高于同期春季萌发小车前和尖喙牻牛儿苗。本研究得到以下结论:秋季萌发的小车前和尖喙牻牛儿苗在稳定和扩大其后代种群繁衍能力、提高防风固沙能力、稳定荒漠生态系统等方面具有重要意义。     

植物萜类生物合成中的后修饰酶

萜类化合物由于其结构类型丰富多样而被称为"terpenome".除了参与植物生长发育、环境应答等生理过程,萜类化合物还应用于医药、有机化工等领域.萜类的生物合成大致可分为前体形成、骨架构建以及后修饰三部分,基本骨架通常由萜类合酶催化形成,进一步在后修饰酶的作用下产生数以万计的萜类化合物.结合我们对香茶菜二萜生物合成的初步研究结果,本文主要针对近年来植物萜类生物合成中的一些有代表性的后修饰酶包括P450单氧酶、双键还原酶、酰基转移酶和糖基转移酶,进行研究现状分析与展望.     

Biochemical and molecular analyses of gibberellin biosynthesis in fungi

The plant hormone, gibberellin (GA), regulates plant growth and development. It was first isolated as a superelongation-promoting diterpenoid from the fungus, Gibberella fujikuroi. G. fujikuroi uses different GA biosynthetic intermediates from those in plants to produce GA3. Another class of GA-producing fungus, Phaeosphaeria sp. L487, synthesizes GA1 by using the same intermediates as those in plants. A molecular analysis of GA biosynthesis in Phaeosphaeria sp. has revealed that diterpene cyclase and cytochrome P450 monooxygenases were involved in the plant-like biosynthesis of GA1. Fungal ent-kaurene synthase is a bifunctional cyclase. Subsequent oxidation steps are catalyzed by P450s, leading to biologically active GA1. GA biosynthesis in plants is divided into three steps involving soluble enzymes and membrane-bound cytochrome P450. The activation of plant GAs is catalyzed by soluble 2-oxoglutarate-dependent dioxygenases, which is in contrast to the catalysis of fungal GA biosynthesis. This difference suggests that the origin of fungal GA biosynthesis is evolutionally independent of that in plants.     

An unexpected diterpene cyclase from rice: functional identification of a stemodene synthase

We have cloned a novel diterpene synthase (OsKSL11) from rice that produces stemod-13(17)-ene from syn-copalyl diphosphate. Notably, this gene sequence was not predicted from the extensive sequence information available for rice, nor, despite extensive phytochemical investigations, has this diterpene or any derived natural product previously been reported in rice plants. OsKSL11 represents the first identified stemodene synthase, which catalyzes the committed step in biosynthesis of the stemodane family of diterpenoid natural products, some of which possess antiviral activity. In addition, OsKSL11 is highly homologous to the mechanistically similar stemarene synthase recently identified from rice, making this pair of diterpene cyclases an excellent model system for investigating the enzymatic determinants for differential product outcome. The unexpected nature of this cyclase and its product parallels recent observations of previously unrecognized natural products metabolism in Arabidopsis thaliana, suggesting that many, if not all, plant species will prove to have extensive biosynthetic capacity.     

Bacterial diterpene synthases: new opportunities for mechanistic enzymology and engineered biosynthesis

Diterpenoid biosynthesis has been extensively studied in plants and fungi, yet cloning and engineering diterpenoid pathways in these organisms remain challenging. Bacteria are emerging as prolific producers of diterpenoid natural products, and bacterial diterpene synthases are poised to make significant contributions to our understanding of terpenoid biosynthesis. Here we will first survey diterpenoid natural products of bacterial origin and briefly review their biosynthesis with emphasis on diterpene synthases (DTSs) that channel geranylgeranyl diphosphate to various diterpenoid scaffolds. We will then highlight differences of DTSs of bacterial and higher organism origins and discuss the challenges in discovering novel bacterial DTSs. We will conclude by discussing new opportunities for DTS mechanistic enzymology and applications of bacterial DTS in biocatalysis and metabolic pathway engineering.     

Recent advances regarding diterpene cyclase genes in higher plants and fungi

Cyclic diterpenoids are commonly biosynthesized from geranylgeranyl diphosphate (GGDP) through the formation of carbon skeletons by specific cyclases and subsequent chemical modifications, such as oxidation, reduction, methylation, and glucosidation. A variety of diterpenoids are produced in higher plants and fungi. Rice produces four classes of diterpene phytoalexins, phytocassanes A to E, oryzalexins A to F, oryzalexin S, and momilactones A and B. The six diterpene cyclase genes involved in the biosynthesis of these phytoalexins were identified and characterized. Fusicoccin A was produced by the phytopathogenic Phomopsis amygdali and served as a plant H(+)-ATPase activator. A PaFS, encoding a fungal diterpene synthase responsible for fusicoccin biosynthesis, was isolated. The PaFS is an unusual chimeric diterpene synthase that possesses not only terpene cyclase activity (the formation of fusicoccadiene, a biosynthetic precursor of fusicoccin A), but also prenyltransferase activity (the formation of GGDP). Thus, we identified a unique multifunctional diterpene synthase family in fungi.     

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.     

Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin

A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627-1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.     

Stemar-13-ene synthase, a diterpene cyclase involved in the biosynthesis of the phytoalexin oryzalexin S in rice

In suspension-cultured rice cells, diterpenoid phytoalexins are produced in response to exogenously applied elicitors. We isolated a cDNA encoding a diterpene cyclase, OsDTC2, from suspension-cultured rice cells treated with a chitin elicitor. The OsDTC2 cDNA was overexpressed in Escherichia coli as a fusion protein with glutathione S-transferase, and the recombinant OsDTC2 was indicated to function as stemar-13-ene synthase that converted syn-copalyl diphosphate to stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S. The level of OsDTC2 mRNA in suspension-cultured rice cells began to increase 3 h after addition of the elicitor and reached the maximum after 8 h. The expression of OsDTC2 was also induced in UV-irradiated rice leaves. In addition, we indicated that stemar-13-ene accumulated in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves.     

Identification of diterpene biosynthetic gene clusters and functional analysis of labdane-related diterpene cyclases in Phomopsis amygdali

Two diterpene biosynthesis gene clusters in the fusicoccin-producing fungus, Phomopsis amygdali, were identified by genome walking from PaGGS1 and PaGGS4 which encode the geranylgeranyl diphosphate (GGDP) synthases. The diterpene cyclase-like genes, PaDC1 and PaDC2, were respectively located proximal to PaGGS1 and PaGGS4. The amino acid sequences of these two enzymes were similar to those of fungal labdane-related diterpene cyclases. Recombinant PaDC1 converted GGDP mainly into phyllocladan-16 alpha-ol via (+)-copalyl diphosphate (CDP) and trace amounts of several labdane-related hydrocarbons which had been identified from the P. amygdali F6 mycelia. Since phyllocladan-16 alpha-ol had not been identified in P. amygdali F6 mycelia, we isolated phyllocladan-16 alpha-ol from the mycelia. Recombinant PaDC2 converted GGDP into (+)-CDP. Furthermore, we isolated the novel diterpenoid, phyllocladan-11 alpha,16 alpha,18-triol, which is a possible metabolite of phyllocladan-16 alpha-ol in the mycelia. We propose that genome walking offers a useful strategy for the discovery of novel natural products in fungi.     

Gibberellin biosynthesis in bacteria: Separate ent-copalyl diphosphate and ent-kaurene synthases in Bradyrhizobium japonicum

Gibberellins are ent-kaurene-derived diterpenoid phytohormones produced by plants, fungi, and bacteria. The distinct gibberellin biosynthetic pathways in plants and fungi are known, but not that in bacteria. Plants typically use two diterpene synthases to form ent-kaurene, while fungi use only a single bifunctional diterpene synthase. We demonstrate here that Bradyrhizobium japonicum encodes separate ent-copalyl diphosphate and ent-kaurene synthases. These are found in an operon whose enzymatic composition indicates that gibberellin biosynthesis in bacteria represents a third independently assembled pathway relative to plants and fungi. Nevertheless, sequence comparisons also suggest potential homology between diterpene synthases from bacteria, plants, and fungi.