抗癌药物榄香烯的生物合成及关键酶吉马烯A合酶的优化

榄香烯是我国拥有自主知识产权的抗肿瘤药物之一,因其抗肿瘤活性强、作用范围广、毒副作用轻微和不易产生耐药性等优点,被广泛应用于各类恶性肿瘤的临床治疗中。榄香烯的生产主要依靠药用植物温郁金的分离提取。但温郁金的榄香烯含量低、分离纯化难度大、得率低且成本高,严重阻碍了榄香烯的大规模生产与应用。随着合成生物学的发展,利用微生物构建细胞工厂用于生物合成天然药物成为研究热点,也为榄香烯的生产提供了新的思路。近年来,对榄香烯的生物合成研究在不断深入。研究者通过代谢工程、组合生物学和基因工程等手段,阐明榄香烯生物合成途径和关键酶,已经成功克隆了榄香烯生物合成途径上的一些关键酶基因,初步实现榄香烯的异源生物合成。本文以合成生物学研究思维概述榄香烯生物合成途径及其工程菌的优化,重点综述关键酶吉马烯A合酶(germacrane A synthase, GAS)。从限速酶基因的过表达和分流基因的敲除,融合表达酶工程策略、吉马烯A合酶的体外进化几方面,对其生物合成途径的改造策略进行阐述。同时,也分析提高异源生物合成榄香烯产量所面临的问题与挑战,为榄香烯的高效生物合成提供参考。     

人工促成檀香结香的研究

在自然情况下生长的檀香（ＳａｎｔａｌｕｍａｌｂｕｍＬ．）植株,约１０龄左右开始形成具芳香的心材（通称结香）,约３０—４０年方可砍伐利用．作者采用两年生的幼树,施用植物生长抑制剂ＰＧＩ１进行促成结香试验,结果证明采用１％３ｍｌ生长激素处理的植株,其檀香油和檀香醇的含量一般较对照和用水处理的植株高１—２倍．     

檀香奇木落户中华

提起檀香人们并不陌生,都知道它是一种珍贵的香料和重要的传统中药：檀香扇、檀香皂和植香型的香精、香水更是尽人皆知的高档商品。可是如果进一步问及檀香是怎么样的一种植物？利用的是哪一部分？真正了解的人就不太多了。虽然明代李时珍在其巨著（本草纲目）（1578）中对植香作过如下记载：“按大明一统志云：檀香出广东云南及占城真腊爪哇渤泥暹罗三佛齐回回等国,今的南造地亦皆有之。树叶皆似荔枝,皮青色而滑泽……”。因固于当时的条件,这些记载颇欠准确。可以判断,李时珍并未见过真正的檀香树。其实檀香（Santalumalbum）的天然…     

中国红豆杉紫杉烯合酶cDNA的分离、表达和鉴定

紫杉烯合酶是一种二萜环化酶,催化牛儿基牛儿基焦磷酸形成紫杉醇生物合成过程中的中间体紫杉烯.利用PCR扩增同源探针筛选cDNA文库,克隆了一个编码中国红豆杉(Taxus chinensis (Pilg.) Rehd.)紫杉烯合酶3′端的2 151 bp的cDNA片段,也通过PCR扩增得到了该基因5′端的611 bp的cDNA片段,将这两个cDNA片段拼接在一起,得到长2 712 bp的cDNA片段,具有一个2 586个碱基的开放阅读框架(ORF),编码包括质体转移肽在内的共862个氨基酸残基;该酶与太平洋红豆杉紫杉烯合酶有97%的同源性(identity),与其他植物萜类环化酶也有较高的同源性.利用融合表达载体pET-32a在大肠杆菌BL21trxB中表达,所表达的融合蛋白以包含体形式存在.包含体经过变性、复性和再折叠,利用His残基亲和凝胶柱层析得到融合的紫杉烯合酶.用毛细管气相色谱-质谱联用对酶促反应产物进行分析,结果表明,融合的紫杉烯合酶能催化产生4(5),11(12)-紫杉烯.     

中国红豆杉紫杉烯合酶cDNA的分离、表达和鉴定

紫杉烯合酶是一种二萜环化酶 ,催化牛儿基牛儿基焦磷酸形成紫杉醇生物合成过程中的中间体紫杉烯。利用PCR扩增同源探针筛选cDNA文库 ,克隆了一个编码中国红豆杉 (Taxuschinensis (Pilg .)Rehd .)紫杉烯合酶 3′端的 2 15 1bp的cDNA片段 ,也通过PCR扩增得到了该基因 5′端的 6 11bp的cDNA片段 ,将这两个cDNA片段拼接在一起 ,得到长 2 712bp的cDNA片段 ,具有一个 2 5 86个碱基的开放阅读框架 (ORF) ,编码包括质体转移肽在内的共 86 2个氨基酸残基 ;该酶与太平洋红豆杉紫杉烯合酶有 97%的同源性 (identity) ,与其他植物萜类环化酶也有较高的同源性。利用融合表达载体pET_32a在大肠杆菌BL2 1trxB中表达 ,所表达的融合蛋白以包含体形式存在。包含体经过变性、复性和再折叠 ,利用His残基亲和凝胶柱层析得到融合的紫杉烯合酶。用毛细管气相色谱 质谱联用对酶促反应产物进行分析 ,结果表明 ,融合的紫杉烯合酶能催化产生 4(5 ) ,11(12 )_紫杉烯     

利用Bio-BglBrick方法表达紫杉醇生物合成酶

目的:进行紫杉醇药物生物合成前五步催化酶二磷酸盐合酶(GGPPS)、紫杉二烯合酶(TS)、紫杉二烯5α羟化酶(THY5α)、紫杉二烯5α-O-乙酰转移酶(TAT)和紫杉烷10β羟化酶(TDH)基因在大肠杆菌异源生物合成途径的组建及串联,实现单个表达及串联表达,并试图通过连续生物催化获得紫杉醇中间体紫衫二烯。方法:依据合成生物学中Brick基因组装方法,通过对载体pET30a的酶切位点进行定向改造,设计独特的BglⅡ/BamHⅠ和XbaⅠ/SpeⅠ串联表达盒,在大肠杆菌BM Rosetta(DE3)中表达产物。结果:设计多基因表达盒,实现紫杉醇药物生物合成前五步催化酶的单个表达,GGPPS和TS以及THY5α、TAT和TDH的串联表达。结论:利用BglBrick/BioBrick基因组装方法,可以实现紫杉醇生物合成催化酶的快速组装及后续表达。     

代谢工程酵母菌合成紫杉烯的研究

紫杉烯是紫杉醇生物合成的重要中间体,为在酿酒酵母(Saccharomyces cerevisiae)中建立一个生物合成紫杉烯的代谢途径,克隆了酵母的羟甲基戊二酰CoA(3-hydroxy-3-methylglutarylcoenzyme A,HMG-CoA)还原酶基因和=牛儿基=牛儿基二磷酸(geranylgeranyl diphosphate,GGDP)合酶基因,并构建了其融合表达载体pGBT9/HG;同时构建了包含紫杉烯合酶基因的表达载体pADH/TS;将这两个表达载体共转化酵母细胞,通过GC-MS分析检测工程酵母的代谢产物,结果表明获得的工程酵母能够合成紫杉烯,即在酵母细胞中建立了一个合成紫杉烯的代谢途径。     

药用植物裂环烯醚萜苷类化合物生物合成途径关键酶基因研究进展

裂环烯醚萜苷类化合物是植物体内产生的具有保肝、消炎、降血糖血脂等多重生物活性的次级代谢产物,在临床上应用广泛。该文依据近年来国内外有关裂环烯醚萜苷类化合物生物合成途径及关键酶基因的挖掘与调控机理研究进展,主要对药用植物裂环烯醚萜苷类化合物生物合成途径、关键酶(GPPS、GES、G10H、8HGO、IS、IO、7DLGT、DL7H、LAMT、SLS)与编码基因的研究进展进行综述,为进一步阐明其生物合成途径机制与关键酶调控作用、提高有效活性成分积累、减缓药用植物野生资源紧张等问题提供参考。     

粤北阴香不同器官中精油成分研究

采用水蒸汽同步蒸馏法分别对采自粤北的阴香的果、叶、枝条提取精油,并用气质联用仪对所提取的精油进行了成分分析和鉴定。共鉴定了41种成分,主要成分为龙脑（68．5％～73．8％）、芳香醇、丁子香烯、橙花叔醇、榄香烯、柠檬醛、莰烯、葑醇、愈创烯、香叶烯、枞萜、萜品醇、蒎烯等。阴香不同器官（果、叶、枝）中的精油成分及所占比例均有差异。     

三烯生育酚生物学功能及合成调控研究进展

介绍了三烯生育酚的生物合成途径,重点综述了三烯生育酚在神经保护、抗癌、降低胆固醇以及抗氧化等方面的优越生物学功能,以及利用关键酶的高效表达和前体物质水平的提高等植物代谢工程手段提高植物体内三烯生育酚生物合成水平的研究进展。     

Molecular regulation of santalol biosynthesis in Santalum album L.

Santalum album L. commonly known as East-Indian sandal or chandan is a hemiparasitic tree of family santalaceae. Santalol is a bioprospecting molecule present in sandalwood and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. Santalol is a sesquiterpene synthesized through mevalonate or non-mevalonate pathways. First step of santalol biosynthesis involves head to tail condensation of isopentenyl pyrophosphate (IPP) with its allylic co-substrate dimethyl allyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP; C10 — a monoterpene). GPP upon one additional condensation with IPP produces farnesyl pyrophosphate (FPP; C15 — an open chain sesquiterpene). Both the reactions are catalyzed by farnesyl diphosphate synthase (FDS). Santalene synthase (SS), a terpene cyclase catalyzes cyclization of open ring FPP into a mixture of cyclic sesquiterpenes such as α-santalene, epi-β-santalene, β-santalene and exo bergamotene, the main constituents of sandal oil. The objective of the present work was to generate a comprehensive knowledge on the genes involved in santalol production and study their molecular regulation. To achieve this, sequences encoding farnesyl diphosphate synthase and santalene synthase were isolated from sandalwood using suppression subtraction hybridization and 2D gel electrophoresis technology. Functional characterization of both the genes was done through enzyme assays and tissue-specific expression of both the genes was studied. To our knowledge, this is the first report on studies on molecular regulation, and tissue-specific expression of the genes involved in santalol biosynthesis.     

Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus.     

Geographic and Phenotypic Variation in Heartwood and Essential‐Oil Characters in Natural Populations of Santalum austrocaledonicum in Vanuatu

Phenotypic variation in heartwood and essential‐oil characters of Santalum austrocaledonicum was assessed across eleven populations on seven islands of Vanuatu. Trees differed significantly in their percentage heartwood cross‐sectional area and this varied independently of stem diameter. The concentrations of the four major essential‐oil constituents (α‐santalol, β‐santalol, (Z)‐β‐curcumen‐12‐ol, and cis‐nuciferol) of alcohol‐extracted heartwood exhibited at least tenfold and continuous tree‐to‐tree variation. Commercially important components α‐ and β‐santalol found in individual trees ranged from 0.8–47% and 0–24.1%, respectively, across all populations, and significant (P<0.05) differences for each were found between individual populations. The Erromango population was unique in that the mean concentrations of its monocyclic ((Z)‐β‐curcumen‐12‐ol and cis‐nuciferol) sesquiterpenes exceeded those of its bi‐ and tricyclic (α‐ and β‐santalol) sesquiterpenes. Heartwood colour varied between trees and spanned 65 colour categories, but no identifiable relationships were found between heartwood colour and α‐ and β‐santalol, although a weak relationship was evident between colour saturation and total oil concentration. These results indicate that the heartwood colour is not a reliable predictive trait for oil quality. The results of this study highlight the knowledge gaps in fundamental understanding of heartwood biology in Santalum genus. The intraspecific variation in heartwood cross‐sectional area, oil concentration, and oil quality traits is of considerable importance to the domestication of sandalwood and present opportunities for the development of highly superior S. austrocaledonicum cultivars that conform to the industry's International Standards used for S. album.     

Isolation of cDNAs and functional characterisation of two multi-product terpene synthase enzymes from sandalwood, Santalum album L

Sandalwood, Santalum album (Santalaceae) is a small hemi-parasitic tropical tree of great economic value. Sandalwood timber contains resins and essential oils, particularly the santalols, santalenes and dozens of other minor sesquiterpenoids. These sesquiterpenoids provide the unique sandalwood fragrance. The research described in this paper set out to identify genes involved in essential oil biosynthesis, particularly terpene synthases (TPS) in S. album, with the long-term aim of better understanding heartwood oil production. Degenerate TPS primers amplified two genomic TPS fragments from S. album, one of which enabled the isolation of two TPS cDNAs, SamonoTPS1 (1731 bp) and SasesquiTPS1 (1680 bp). Both translated protein sequences shared highest similarity with known TPS from grapevine (Vitis vinifera). Heterologous expression in Escherichia coli produced catalytically active proteins. SamonoTPS1 was identified as a monoterpene synthase which produced a mixture of (+)-α-terpineol and (−)-limonene, along with small quantities of linalool, myrcene, (−)-α-pinene, (+)-sabinene and geraniol when assayed with geranyl diphosphate. Sesquiterpene synthase SasesquiTPS1 produced the monocyclic sesquiterpene alcohol germacrene D-4-ol and helminthogermacrene, when incubated with farnesyl diphosphate. Also present were α-bulnesene, γ-muurolene, α- and β-selinenes, as well as several other minor bicyclic compounds. Although these sesquiterpenes are present in only minute quantities in the distilled sandalwood oil, the genes and their encoded enzymes described here represent the first TPS isolated and characterised from a member of the Santalaceae plant family and they may enable the future discovery of additional TPS genes in sandalwood.     

Quantitative co-occurrence of sesquiterpenes; a tool for elucidating their biosynthesis in Indian sandalwood, Santalum album

A chemotaxonomic approach was used to investigate biosynthetic relationships between heartwood sesquiterpenes in Indian sandalwood, Santalum album L. Strong, linear relationships exist between four structural classes of sesquiterpenes; alpha- and beta-santalenes and bergamotene; gamma- and beta-curcumene; beta-bisabolene and alpha-bisabolol and four unidentified sesquiterpenes. All samples within the heartwood yielded the same co-occurrence patterns, however wood from young trees tended to be more variable. It is proposed that the biosynthesis of each structural class of sesquiterpene in sandalwood oil is linked through common carbocation intermediates. Lack of co-occurrence between each structural class suggests that four separate cyclase enzymes may be operative. The biosynthesis of sandalwood oil sesquiterpenes is discussed with respect to these co-occurrence patterns. Extractable oil yield was correlated to heartwood content of each wood core and the oil composition did not vary significantly throughout the tree.     

Identification of Trans-β-santalol and epi-cis-β-santalol in east indian sandalwood oil

An analysis of East Indian sandalwood oil (Santalum album) has resulted in the isolation and identification of trans-β-santalol and epi-cis-β- santalol.     

Repellent effect of santalol from sandalwood oil against Tetranychus urticae (Acari: Tetranychidae)

Thirty-four essential oils were screened for their repellent activities against the twospotted spider mite, Tetranychus urticae Koch (Acarina: Tetranychidae), at 0.1% concentration level using choice and no-choice laboratory bioassays. Of these, 20 essential oils showed significant repellencies against T. urticae in the choice tests. In subsequent no-choice tests using these 20 essential oils, only sandalwood oil showed significant repellency against T. urticae. Total number of eggs oviposited by T. urticae was significantly lower than controls in the choice tests when the kidney bean leaves were treated with 1 of 14 essential oils. The significant repellency of sandalwood oil against T. urticae lasted at least for 5 h at the 0.1% concentration level. Our GC-MS analysis indicated that the major components of the sandalwood oil were alpha-santalol (45.8%), beta-santalol (20.6%), beta-sinensal (9.4%), and epi-beta-santalol (3.3%). Santanol, a mixture of the two main components in the sandalwood oil, appears to be responsible for the repellency of sandalwood oil against T. urticae.     

StructureOdor Relationships of Semisynthetic β‐Santalol Analogs

A series of eleven β‐santalol analogs, including nine new derivatives, was prepared by semisynthesis from natural (?)‐(Z)‐β‐santalol and studied by gas chromatography‐olfactometry (GC‐O) to characterize their olfactory properties and potencies. These compounds and 45 others selected in the literature were used to build three olfactophores by molecular modelling. Three models were obtained that gather structural and physicochemical constraints that will be useful for further design of new sandalwood odorants.