Taxol biosynthesis and molecular genetics-紫杉醇生物合成途径

1.1细胞色素P450研究进展1.1.1细胞色素P450细胞色素P450(cytochrome P450或CYP,简称P450）是一个古老的以血红素为辅基的B族细胞色素蛋白酶基因超家族，广泛存在于细菌、真菌、植物以及动物等各种生物体内[1]，通常与质体、线粒体、内质网、高尔基体等细胞器膜结合。

还原态P450与CO结合后在450nm处能检测到最大吸收峰，故命名为P450。

因其能使疏水性分子插入一个氧原子而变得更具有亲水性或者活性，因此又称之为单加氧酶(mixed-function oxidase,简称MFO)[2]。

P450酶系作为自然界中生物催化剂，它所催化的反应类型多样，最典型的反应是把分子氧还原为水的同时，将其中一个氧原子转移至底物形成产物，催化反应为[3]:RH+O2+NADPH+H+ROH+H2O+NADP+1958年，在大鼠肝微粒体中第一次发现P450。

D.S Frear于1969年首次在棉花(Gossypium hirsutum L.)中发现了它的存在[4]。

此后，大量的研究表明在拟南芥(Arabidopsis thaliana L.)[5]、小麦(Triticum aestivum L.)[6]、苜蓿(Medicago sativa L.)[7]、蓖麻(Ricinus communis L.)[8]等许多植物中也均有P450存在。

P450酶系在植物中参与多种代谢反应，发挥重要的催化作用。

[1]Omura T(1999).Forty years of cytochrome P450.Biochem BiophysRes Commun,266(3):690~698.[2]Nelson D R,Kaymans L,Kamataki T,et al.P450superfamily:updateon new sequence,gene mapping,accession numbers andnomenclature[J].Pharmacogenetics,1996,6:1-42.[3]Ortiz de Montellano PR.Cytochrome P450:structure,mechanism,and biochemistry[M],3rd ed.Kluwer Academic/Plenum Press,New York,2005,183-245.[4]Frear DS,Swanson HR,Tanaka FS.N-Demethylation of substituted3-(phenyl)-1-methylureas:isolation and characterization of a microsomal mixed function oxidase from cotton.Phytochemistry, 1969,8(11):2157–2169.[5]Paquette SM,Bak S,Feyereisen R.Intron-exon organization andphylogeny in a large superfamily,the paralogous cytochrome P450 genes of Arabidopsis thaliana.DNA Cell Biol,2000,19(5): 307–317.[6]Murphy PJ,West CA.The role of mixed function oxidases in kaurenemetabolism in Echinocystis macrocarpa Greene endosperm.Arch Biochem Biophys,1969,133(2):395–407.[7]Li LY,Cheng H,Gai JY,Yu DY.Genome-wide identifycation andcharacterization of putative cytochrome P450genes in the model legume Medicago truncatula.Planta,2007,226(1):109–123. [8]Lew FL,West CA.(-)-kaur-16-en-7β-ol-19-oic acid,an intermediatein gibberellin biosynthesis.Phytochemistry,1971,10(9): 2065–2076.1.1.2细胞色素P450结构特征在细胞色素P450超基因家族中，不同成员之间在氨基酸序列上具有高度的变异性，但其空间结构上却保持较高的相似性，P450蛋白三级结构主要由C端的α-螺旋结构和N端的β-折叠结构组成[1,2]。

化学发光法生物素标记核酸检测试剂盒产品编号 产品名称包装 D3308化学发光法生物素标记核酸检测试剂盒1000cm 2产品简介：化学发光法生物素检测试剂盒(Chemiluminescent Biotin-labeled Nucleic Acid Detection Kit)是一种通过Streptavidin-HRP 及后续的BeyoECL Moon 试剂来实现化学发光检测Biotin 标记核酸的检测试剂盒。

适用于Southern blot 、Northern blot 、ribonuclease protection assay (RPA)或EMSA 等实验中，采用生物素标记的DNA 或RNA 探针时的检测。

本试剂盒不适用于生物素标记蛋白的检测。

本试剂盒同时还提供了封闭液、洗涤液等检测时所需的配套试剂。

本试剂盒采用了高质量的Streptavidin-HRP Conjugate ，HRP 和Streptavidin 共价交联的比例大于3，这样比采用Streptavidin 和Biotin-HRP conjugate 两种试剂进行检测要更方便，并且灵敏度更高。

采用了非特异性结合比avidin 更低的strepatavidin ，使检测结果背景更低灵敏度更高。

本试剂盒没有提供生物素探针标记相关的试剂，生物素标记的DNA 探针或EMSA 探针的制备可以相应地使用碧云天生产的生物素3'末端DNA 标记试剂盒(D3106)或EMSA 探针生物素标记试剂盒(GS008)。

本试剂盒可以用于检测至少10块10×10cm 有生物素标记EMSA 探针的膜，即共1000cm 2。

包装清单：产品编号 产品名称包装 D3308-1 BeyoECL Moon A 液 55ml D3308-2 BeyoECL Moon B 液 55ml D3308-3 Streptavidin-HRP Conjugate100µl D3308-4 封闭液 380ml D3308-5 洗涤液(5X)250ml D3308-6检测平衡液 250ml —说明书1份保存条件：D3308-3 Streptavidin-HRP Conjugate 在-20ºC 保存，其余可4ºC 保存。

Hereditas (Beijing) 2021年5月, 43(5): 459―472 收稿日期: 2020-10-10; 修回日期: 2021-03-04基金项目:国家自然科学基金项目(编号：U1903201, 31670298, 31771413, 21702100, 21907051)和教育部创新团队项目(编号：IRT_14R27)资助[Supported by the National Natural Science Foundation of China (Nos. U1903201, 31670298, 31771413, 21702100, 21907051), and theProgram for Changjiang Scholars and Innovative Research Team in University from the Ministry of Education of China (No. IRT_14R27)]作者简介: 林红燕，博士，助理研究员，研究方向：药用植物天然产物化学和分子药理。

E-mail:*************.cn王煊，博士研究生，研究方向：植物分子代谢。

E-mail:*******************林红燕和王煊并列第一作者。

通讯作者:杨永华，教授，博士生导师，研究方向：分子代谢与生物技术安全。

E-mail:**************.cn DOI: 10.16288/j.yczz.20-341 网络出版时间: 2021/3/29 11:37:11URI: https:///kcms/detail/11.1913.R.20210326.0956.002.html综 述中药植物紫草天然产物的生物合成及其功能研究进展林红燕，王煊，何聪，周紫玲，杨旻恺，文钟灵，韩洪苇，陆桂华， 戚金亮，杨永华南京大学医药生物技术国家重点实验室，植物分子生物学研究所，生命科学学院，南京 210023摘要: 紫草为我国传统的重要药用植物资源，其根部代谢产生的紫红色萘醌类天然产物—紫草素及其衍生物，临床上常被用于治疗疮疡和皮肤炎症。

Biosynthesis of Plant Isoprenoids:Perspectives for Microbial EngineeringJames Kirby 1and Jay D.Keasling 1,2,3,41California Institute of Quantitative Biomedical Research,University of California,Berkeley,California 94720;email:jimkirby@2Departments of Chemical Engineering and Bioengineering,University of California,Berkeley,California 94720;email:keasling@3Physical Biosciences Division,Lawrence Berkeley National Laboratory,Berkeley,California 947204Joint BioEnergy Institute,Emeryville,California 94608Annu.Rev.Plant Biol.2009.60:335–55First published online as a Review in Advance on January 9,2009The Annual Review of Plant Biology is online at This article’s doi:10.1146/annurev.arplant.043008.091955Copyright c2009by Annual Reviews.All rights reserved1543-5008/09/0602-0335$20.00Key Wordsterpenes,P450,yeast,Escherichia coli ,gene discoveryAbstractIsoprenoids are a large and highly diverse group of natural products with many functions in plant primary and secondary metabolism.Isoprenoids are synthesized from common prenyl diphosphate precursors through the action of terpene synthases and terpene-modifying enzymes such as cytochrome P450monooxygenases.Many isoprenoids have impor-tant applications in areas such as human health and nutrition,and much effort has been directed toward their production in microbial hosts.However,many hurdles must be overcome in the elucidation and func-tional microbial expression of the genes responsible for biosynthesis of an isoprenoid of interest.Here,we review investigations into isoprenoid function and gene discovery in plants as well as the latest advances in isoprenoid pathway engineering in both plant and microbial hosts.335A n n u . R e v . P l a n tB i o l . 2009.60:335-355. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b y U N I V E R S I T Y O F F L O R I D A - S m a t h e r s L i b r a r y o n 01/12/10. F o r p e r s o n a l u s e o n l y .Click here for quick links to Annual Reviews content online, including:• Other articles in this volume • Top cited articles• Top downloaded articles • Our comp rehensive searchFurtherANNUAL REVIEWSContentsINTRODUCTION..................336DISCOVERY OF APPLICATIONS FOR ISOPRENOIDS .............340The Role of IndigenousKnowledge.....................340The Role of Plant Investigations....340DISCOVERY OF ISOPRENOIDBIOSYNTHETIC GENES........341T ools for Gene Discovery . (342)GENE EXPRESSION AND PATHWAY ENGINEERINGIN PLANTS ......................343Carotenoids .......................343Menthol...........................344Crossing Plant Compartments (345)GENE EXPRESSION AND PATHWAY ENGINEERINGIN MICROBES...................345Engineering the Mevalonateand DXP Pathways .............345Global Approaches to Improving Pathway Flux...................346Expression of P450sin Microbial Hosts..............347MUTATION OF ISOPRENOIDBIOSYNTHETIC ENZYMES ....348Mutation of T erpene Synthases.....348Mutation of P450s.................348APPROACHING A COMPLETE MICROBIAL SYSTEM ...........349INTRODUCTIONIsoprenoids (also referred to as terpenes)con-stitute one of the most diverse groups of nat-−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 1Overview of the isoprenoid biosynthetic pathways.The 1-deoxy-d -xylulose-5-phosphate (DXP)and mevalonate pathways areresponsible for production of isopentenyl diphosphate (IPP)and dimethylallyl diphosphate (DMAPP)from central metabolites.Key enzymes of each pathway are shown:1-deoxy-d -xylulose-5-phosphate synthase (DXS),1-deoxy-d -xylulose-5-phosphate reductase(DXR),3-hydroxy-3-methylglutaryl-CoA reductase (HMGR),and IPP-DMAPP isomerase (IDI).IPP and DMAPP are then converted to terpene synthase precursors through the action of the prenyltransferases geranyl diphosphate (GPP)synthase (GPPS),farnesyl diphosphate (FPP)synthase (FPPS),and geranylgeranyl diphosphate (GGPP)synthase (GGPPS).One example of a terpene synthase reaction and downstream processing reaction(s)(such as P450oxidation)is given for each category of isoprenoid shown.Multiple steps are indicated by dashed lines.ural products in nature.In plants,isoprenoids range from essential and relatively universal pri-mary metabolites,such as sterols,carotenoids,quinones,and hormones,to more unique and sometimes species-specific secondary metabo-lites that may serve roles such as plant defense and communication (52).Isoprenoids are clas-sified into groups according to the number of carbons they contain;the major groups of in-terest here are monoterpenes (C10),sesquiter-penes (C15),diterpenes (C20),and triterpenes (C30).All isoprenoids are synthesized via the two universal C5building blocks:isopentenyl diphosphate (IPP)and its isomer dimethy-lallyl diphosphate (DMAPP).These univer-sal precursors can be produced by either of two routes:the mevalonate pathway or the 1-deoxy-d -xylulose-5-phosphate (DXP)pathway (Figure 1).These pathways are distributed throughout nature,and as a rule of thumb the mevalonate pathway is prevalent in eukary-otes and archaea,whereas the DXP pathway is widespread in eubacteria.However,there are many exceptions to this pattern;for exam-ple,several eubacteria are known to utilize the mevalonate pathway instead of the DXP path-way,with some species carrying genes from both pathways,whereas the protozoan parasite Plasmodium falciparum relies on the DXP path-way (26).Both pathways are expressed in plants but differ in their localization.The mevalonate pathway enzymes are located in the cytosol,whereas the DXP enzymes are found in the plastid.The exact origin of the DXP pathway genes in plants is not clear from phylogenetic studies because most of the plant DXP genes do not branch with their cyanobacterial coun-terparts (plastids are thought to have evolved336Kirby·KeaslingA n n u . R e v . P l a n tB i o l . 2009.60:335-355. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b y U N I V E R S I T Y O F F L O R I D A - S m a t h e r s L i b r a r y o n 01/12/10. F o r p e r s o n a l u s e o n l y . •Biosynthesis of Plant Isoprenoids 337A n n u . R e v . P l a n tB i o l . 2009.60:335-355. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b y U N I V E R S I T Y O F F L O R I D A - S m a t h e r s L i b r a r y o n 01/12/10. F o r p e r s o n a l u s e o n l y .IPP:isopentenyl diphosphate DMAPP:dimethylallyl diphosphate DXP:1-deoxy-d -xylulose-5-phosphate GPP:geranyl diphosphateFPP:farnesyl diphosphate GGPP:geranylgeranyl diphosphatefrom a cyanobacterium-like progenitor).One theory proposes that the genes currently found in plants were acquired subsequent to the ori-gin of plastids,and lateral transfer of genes from eubacteria played a role (52).The mevalonate pathway consists of six steps that transform acetyl-CoA to IPP ,followed by an IPP isomerase that maintains a balance between IPP and DMAPP .The DXP path-way consists of seven enzymes that transform glyceraldehyde-3-phosphate and pyruvic acid to IPP and DMAPP in a ratio of 5:1.An IPP isomerase gene (idi )is present in only a mi-nority of bacterial species that utilize the DXP pathway (24),and this gene is nonessential in Escherichia coli (35).IDI may play a more im-portant role in plants than in bacteria,how-ever,because gene silencing of IDI in Nicotiana benthamiana resulted in an 80%reduction in pigments (carotenoid and chlorophyll)compared with controls (71).The mevalonate pathway enzymes have received much atten-tion over the past 30years,and several regu-latory mechanisms have been uncovered (96).In contrast,the DXP pathway was fully eluci-dated only in 2002and is less well understood in terms of both requirements and regulation (1).Downstream of both pathways,prenyltrans-ferases convert IPP and DMAPP to longer chain isoprenoid precursors:geranyl diphos-phate (GPP ,the C 10precursor to monoter-penes),farnesyl diphosphate (FPP ,the C 15precursor to sesquiterpenes and triterpenes),and geranylgeranyl diphosphate (GGPP ,the C 20precursor to diterpenes).In plants,syn-thesis of terpenes is compartmentalized such that monoterpenes and diterpenes (as well as carotenoids and chlorophylls)are produced via the DXP pathway in the plastid,whereas sesquiterpenes and triterpenes are made in the cytosol via the mevalonate pathway (Figure 2).Several studies provide evidence for an ex-change of prenyl diphosphate precursors be-tween the cytosol and the plastid.However,the extent of this cross talk is limited and cannot compensate for loss of flux through one path-way,as demonstrated by studies using Arabidop-sis thaliana DXP mutants (14).Many isoprenoids have found applications in medicine and agriculture and as nutraceuti-cals,flavors,and fragrances (31).Some of these natural products are considered to be attractive targets for bioengineering owing to their mar-ket value as commercial products and/or their impact in areas such as human health.For ex-ample,carotenoids have many applications in the food (e.g.,β-carotene),animal feed (e.g.,astaxanthin),cosmetics (e.g.,tocopherols),and health supplement industries.In the midst of a growing awareness of the health benefits of carotenoids as antioxidants and the use of lutein in particular for prevention of age-related mac-ular degeneration disease and related disorders,the total carotenoid market value has been pro-jected to surpass $1billion by the end of this decade (25).Isolation of the heavily decorated diterpene paclitaxel from T axus brevifola (Pacific yew)led to the development of the promising anticancerdrug T axol R,which has since proven to be effective in the treatment of several forms of cancer and has already exceeded a market value of $1billion (93).However,ensuring initial supplies of T axol to meet the growing number of clinical applications met with controversy surrounding the environmental impact on old-growth forests.The bark of 2000–3000T.brevifola trees is estimated to be required for commercial production of 1kg of T axol;this is equivalent to harvesting a 100-year-old tree to extract one dose of the drug (39,44).A semisynthetic route was later devised,involving purification of a T axol precursor from yew tree needles,and this process has now been replaced by production via plant cell culture (/greenchemistry/pubs/pgcc/winners/gspa04.html )(44).Artemisinin,a sesquiterpene produced in the leaves of Artemisia annua (sweet wormwood)is widely considered to be the best treatment for uncomplicated malaria when used as part of a combination therapy (66).The cost of the commercially available drug,however,is pro-hibitive to many people in developing coun-tries who are suffering from malaria.A micro-bial source of artemisinin has been proposed to338Kirby·KeaslingA n n u . R e v . P l a n tB i o l . 2009.60:335-355. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b y U N I V E R S I T Y O F F L O R I D A - S m a t h e r s L i b r a r y o n 01/12/10. F o r p e r s o n a l u s e o n l y .Figure 2Generalized schematic of isoprenoid biosynthesis in plants.Note that although the reactions shown here form the commonly accepted prenyl phosphate pathways,some evidence exists for the expression of a GPPS in the cytosol (15)and for the presence of significant levels of FPP in the plastid (99).The presence of GGPP in the cytosol has also been postulated for the geranylgeranylation of proteins (14).CDP ,copalyl-diphosphate;DMAPP ,dimethylallyl diphosphate;DXP ,1-deoxy-d -xylulose-5-phosphate;FPP ,farnesyl diphosphate;FPPS FPP synthase;GPP ,geranyl diphosphate;GGPP ,geranylgeranyl diphosphate;GPPS,GPP synthase;GGPPS,GGPP synthase;IDI,IPP isomerase;IPP ,isopentenyl diphosphate.Dashed lines indicate multiple steps.ensure a supply of the drug at a significantly lower cost than the current production method of extraction from the plant (36).In many cases such as those of artemisinin and T axol,sev-eral different approaches have been explored to achieve the goal of an economical and reliable source for high-value products with minimal environmental impact.T ypically the different options explored for sourcing plant isoprenoids include extraction from its natural source (16);total or partial chemical synthesis (39);im-provement of existing plant sources through breeding (23),genetic engineering (101),or cell culture (83);and production in a microbial host (3).In the case of commercial production of T axol,all the common routes have been con-sidered and the method used for production has shifted over time.The major challenges associated with the development of a microbial host for plant isoprenoid biosynthesis include engineering of isoprenoid precursor pathways and the discovery and successful expression of the nec-essary biosynthetic genes.One advantage of the microbial system is its adaptability for synthe-sis of analogs through the introduction of new •Biosynthesis of Plant Isoprenoids339A n n u . R e v . P l a n tB i o l . 2009.60:335-355. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b y U N I V E R S I T Y O F F L O R I D A - S m a t h e r s L i b r a r y o n 01/12/10. F o r p e r s o n a l u s e o n l y .genes or mutation of existing pathway genes.For instance,more than 350taxoid products have been detected in T axus spp.,but progress toward clinical evaluation of many of these is limited by the quantities available in the plant (44).Here we review our current understand-ing of isoprenoid biosynthesis and function in plants and how they inform gene discovery and engineering endeavors,as well as more recent advances in microbial platforms for heterolo-gous production.DISCOVERY OF APPLICATIONS FOR ISOPRENOIDSThe Role of Indigenous KnowledgeConsidering the many flavors and fragrances that are available in the world of plant essen-tial oils,it is perhaps not surprising that hu-man knowledge and practical use of these and other sources of isoprenoids date back several millennia.Some of the earliest records of tra-ditional medicine,originating in Mesopotamia more than 4500years ago,report the use of several plant oils,many of which are still in use today (63).Dating from ancient Roman times,one of the first pharmacological manuscripts describes medicinal uses for the latex from the succulent Euphorbia resinifera ;the likely ac-tive component was identified 2000years later as the diterpene derivative resiniferatoxin (8).T oday,the World Health Organization (WHO)estimates that 80%of the world’s population relies on traditional remedies for their pri-mary healthcare,whereas 50%of the most commonly prescribed U.S.drugs either con-tain,or were developed from,natural products (63).The role of indigenous knowledge in the discovery of isoprenoid-based drugs or agri-cultural agents should not be underestimated,and according to the 1992Convention on Bi-ological Diversity,this role should be acknowl-edged in the form of conservation and bene-fits sharing (22).Artemisinin was isolated and demonstrated to be an effective treatment formalaria as a result of a large-scale effort in China,where modern scientists worked along-side practitioners and historians of traditional Chinese medicine.The discovery was made fol-lowing much research into China’s earliest ma-teria medica and in particular a 1700-year-old report from the famous physician Ge Hong en-titled “Emergency Prescriptions Kept Up One’s Sleeve”(41).Ethnobotanical research into cur-rent traditional medicine practices has also led to important discoveries of isoprenoid function.The diterpene 12-deoxyphorbol 13-acetate,or prostratin,was discovered by the U.S.National Cancer Institute (NCI)to display promising activities against HIV following research into current practices by traditional Samoan healers (34).Prostratin is currently in the early stages of drug development following a profit-sharing agreement put in place between the NCI and Samoan healers (21).The Role of Plant InvestigationsWhereas ethnobotanical studies may provide valuable leads for isoprenoids of value to human medicine,studies in plant biology can do the same for agriculturally important compounds.Plant isoprenoids,whether produced constitu-tively or in response to a stimulus,often act as deterrents against assault by pathogens and her-bivores.The optimal defense theory predicts that constitutive accumulation of isoprenoids will be focused on tissues with the highest fit-ness value,such as younger growth and re-productive organs (69).This theory appears to hold true in many cases;for instance,feed-ing on cotton (Gossypium herbaceum )by beet armyworm larvae (Spodopterta exigua )induces terpene production in younger but not older leaves (13).The prediction of a functional role for a particular plant isoprenoid typically in-volves a wide range of disciplines,from ecol-ogy and physiology to analytical chemistry,but may begin with traditional or common knowl-edge of a plant’s natural resistance or toxicity.For example,the neem tree (Azadirachta in-dica )has been known in India for thousands of340Kirby·KeaslingA n n u . R e v . P l a n tB i o l . 2009.60:335-355. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b y U N I V E R S I T Y O F F L O R I D A - S m a t h e r s L i b r a r y o n 01/12/10. F o r p e r s o n a l u s e o n l y .years to possess insecticidal properties—it was used to protect stored grain from pests,among other applications.The many constituents of neem oil are still under evaluation,with a wide range of proposed uses,but a highly modified triterpene constituent,azadirachtin,has been clearly demonstrated to have in-sect antifeedant and growth regulating activity (16).Correlations between isoprenoid levels in a plant and resistance to attack have been discovered in many ways.Another study of the response of cotton to feeding by beet armyworm larvae revealed that the number of leaf glands (which are typically rich in isoprenoids)increased in density after feed-ing.High-performance liquid chromatography (HPLC)analysis showed a significant increase in specific sesquiterpene heliocides,which was correlated with deterrence of further feeding (60).T o demonstrate a protective role for iso-prenoids in Norway spruce (Picea abies ),trees were treated with methyl jasmonate (MeJA,a common signal molecule in plant defense sys-tems),and an increase in both terpene levels and resistance to the spruce bark beetle were observed (28).If a gene responsible for biosynthesis of an isoprenoid is already known,its overexpression or deletion in the plant can provide useful func-tional information.Expression of linalool syn-thase (FaNES1)in A.thaliana led to a large increase in levels of the monoterpene and its derivatives and also a significant increase in re-sistance to the aphid Myzus persicae (2).Con-versely,oat (Avena strigosa )mutants deficient in triterpene saponins displayed increased sensi-tivity to a range of fungal pathogens (73).DISCOVERY OF ISOPRENOID BIOSYNTHETIC GENESElucidation of genes required for biosynthesis of an isoprenoid not only facilitates functional studies in the plant,but also paves the way for engineering higher production levels in plant or microbial hosts.Following biosynthesis ofMeJA:methyl jasmonate TPS:terpene synthaseP450:cytochrome P450monooxygenasethe universal isoprenoid prenyl diphosphate precursors,the next step in an isoprenoid path-way entails conversion of the prenyl diphos-phate into a cyclic product by a terpene synthase (TPS).In the majority of cases a car-bocation is generated following cleavage of the diphosphate group and the synthase then acts as a chaperone to guide a cascade of molecular events that ultimately results in a terpene olefin product.However,in the case of a few (class II)TPSs a carbocation is generated through protonation of a double bond,and the cyclic product retains the diphosphate group.A com-mon example of this second mechanism is the biosynthesis of copalyl-diphosphate,precursor to gibberellins and phytoalexins (Figure 2).The mechanisms,versatility,and diversity of terpene synthases were explored in recent re-views (14,20).Although a TPS cyclization reaction may occasionally constitute the terminal step in a plant secondary metabolism pathway,it is much more common for further modifications of the terpene olefin to take place.These modifica-tions may at later points in the pathway involve the addition of acyl-,aryl-,or sugar moieties,but usually begin with oxidation of the terpene olefin through the action of cytochrome P450monooxgenases (P450s).P450s are capable of catalyzing a wide variety of chemical reactions,from hydroxylation and epoxidation to ring formation,aryl migration,and carbon-carbon bond cleavage (61).Plant P450s,belonging to Class II of the P450group,are found in the en-doplasmic reticulum,where they act in concert with an NADPH-dependent cytochrome P450reductase (CPR).P450s are categorized into families and subfamilies depending on the de-gree of protein sequence identity;for example,enzymes from family CYP1(cytochrome P450family 1)share more than 40%identity,whereas those from subfamily CYP1A share more than 55%identity (87).The fact that plants typi-cally encode several hundred P450s (272are found in Arabidopsis ,458in rice)can make gene discovery projects particularly challenging (87). •Biosynthesis of Plant Isoprenoids 341A n n u . R e v . P l a n tB i o l . 2009.60:335-355. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b y U N I V E R S I T Y O F F L O R I D A - S m a t h e r s L i b r a r y o n 01/12/10. F o r p e r s o n a l u s e o n l y .Tools for Gene DiscoverySome useful tools for the discovery of new TPS and P450genes have arisen from insights gained from plant biology studies.Although the availability of many P450and TPS gene sequences has in many cases facilitated the suc-cessful design of PCR primers for gene iso-lation,this approach when used alone can be limiting in its scope.For instance,there are higher numbers of terpene-modifying P450s within certain CYP families,but any correla-tion postulated between a terpene structure and a P450family is by no means reliable.Even if the assumption of the target family proves to be correct,there may be many representa-tives from that family in the plant in question,and other connections between the terpene and the target gene must be made to narrow the field.The isolation of the gene CYP71AV1from Artemisia annua ,encoding the P450responsi-ble for conversion of the sesquiterpene amor-phadiene into artemisinic acid,provides a good example (82).For this work,CYP71-specific PCR primers were designed on the basis that in other plants of the Asteraceae family sesquiter-penes similar to amorphadiene are oxidized by CYP71enzymes.Isolation of the correct gene,however,required the use of cDNA made from a glandular trichome-enriched cell preparation as a template for the PCR.Thus,knowledge of the site of biosynthesis of the terpene in question proved to be invaluable.Glandular tri-chomes,specialized structures found on the leaf surfaces of many plants,are frequently the pro-duction and storage sites for volatile mono-and sesquiterpenes (86).The use of glandular trichome preparations played a central role in the pioneering work of Rodney Croteau and coworkers (55),including the isolation of two regiospecific limonene hydroxylase genes from tex is also known to be a site for ter-pene biosynthesis in many plants;in the case of Hevea brasiliensis the use of cDNA isolated from latex has been instrumental in elucidat-ing genes involved in rubber biosynthesis (19,67,79),and several other members of the Eu-phorbiaceae are known to accumulate a varietyof isoprenoid secondary metabolites in their la-tex (43),although none of the P450s involved in the synthesis of these compounds have yet been isolated.In many cases an elicitor known to in-duce isoprenoid biosynthesis is used to un-cover new genes.Infection of soybean (Glycine max )with the fungus Phytophythora sojae was found to induce expression of several P450s involved in isoflavonoid biosynthesis (90,92).One gene that is also induced under the same conditions,although it belongs to the same family as the isoflavonoid P450s (CYP93),was later found to encode a hydroxylase that acts on the related triterpenes β-amyrin and sophoradiol (90).T riterpenes derived from β-amyrin and sophoradiol,known as soyas-aponins,are thought to protect plants from phytopathogenic fungi by interacting with sterols in the fungal cell membrane to cause a loss in membrane integrity (98).The tomato linalool synthase gene (LeMTS1)was isolated from plants infested with spider mites and subsequent studies showed that gene expression was induced in stem trichomes by jasmonic acid (95).In fact,plant hormones,and particularly jasmonates,have been commonly used as elicitors of isoprenoid biosynthesis.Nine full-length TPS genes were recovered from Norway spruce (Picea abies L.Karst)using needles collected from trees treated with MeJA (57).Perhaps the most impressive demonstration of the use of elicitors for gene discovery is the case of a MeJA-induced cDNA library from a T axus cuspidata cell culture (45).Following up on previous studies that demonstrated that MeJA significantly increases T axol production in T axus cell cultures (49),a cDNA library was made from pretreated cultures and approxi-mately 8,500clones were randomly selected and sequenced.Within this relatively small sample size,expressed sequence tags (EST s)representing all members of the DXP pathway were identified along with a GGPP synthase,whereas the T axol pathway TPS gene (taxadi-ene synthase)was present in the library at an abundance of almost 5%.Further analysis of342Kirby·KeaslingA n n u . R e v . P l a n tB i o l . 2009.60:335-355. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b y U N I V E R S I T Y O F F L O R I D A - S m a t h e r s L i b r a r y o n 01/12/10. F o r p e r s o n a l u s e o n l y .the sequences revealed 10new putative P450taxoid hydroxylases and six new acyl/aroyl transferases that are most likely also involved in the T axol pathway (45).This work also serves to illustrate the likelihood of a higher incidence of sequence homology between proteins of the same metabolic pathway:The new putative P450s share >75%sequence sim-ilarity with previously identified P450s from the pathway,compared with <35%similarity with most other plant P450s,whereas the acyl/aroyl transferases share >65%similarity as a group.Although several examples exist of likely convergent evolution in plant secondary metabolic genes,the genes of a particular path-way,sharing temporal and spatial expression patterns,more likely have arisen through gene duplication and divergence (77).More high-throughput technologies may play an increasing role in gene discovery as the need arises;one example is the use of robotics for tandem gene expression coupled with arrays of potential substrates.The prediction of P450function based on molecular modeling may also prove fruitful for plant genes,following in the footsteps of work done with bacterial and mammalian P450s (87).GENE EXPRESSION AND PATHWAY ENGINEERING IN PLANTSPlants may be engineered for increased iso-prenoid production with an aim to purify the isoprenoid component,as a means of increas-ing the nutritional value of food crops,or to en-hance the fitness of the plant itself by increasing resistance to herbivores,pests,or pathogens.Of course,gene expression and pathway engineering studies can also advance our un-derstanding of both the regulation and function of isoprenoid biosynthesis in plants.Although microbial engineering offers many advantages over plant systems for product synthesis and pu-rification,many plant isoprenoid biosynthetic pathways are only partially elucidated and are therefore not currently amenable to microbial transformation.CarotenoidsPerhaps the most high-profile early work on engineering higher isoprenoid levels in plants came about as a means of tackling malnutri-tion due to vitamin A deficiency in developing countries.The carotenoids α-and β-carotene serve as precursors to vitamin A in humans,but are scarce in most staple crops of the de-veloping world and absent in polished (white)rice.Successful engineering of phytoene syn-thase,phytoene desaturase,and lycopene β-cyclase into rice resulted in formation of β-carotene and β-xanthophylls,and carotenoid concentrations reached 1.6μg g −1in the rice endosperm (101).Similar results were obtained in rice transformed with only the phytoene syn-thase and desaturase genes (101),and it was later demonstrated that lycopene β-cyclase and downstream genes are constitutively expressed in rice endosperm (85).Improvements were made over the original strain through a sys-tematic evaluation of phytoene synthase genes from carotenoid-rich plant sources,culminat-ing in a 23-fold increase in carotenoid levels (37μg g −1)in a line harboring the phytoene synthase gene from maize (72).However,be-cause lutein and zeaxanthin levels were actu-ally much lower in many of the improved lines compared with the original golden rice,further engineering to boost levels of these important carotenoids would be beneficial.The discovery of a cauliflower gene involved in the differentiation of noncolored plastids into chromoplasts provides a complementary approach to engineering higher carotenoid lev-els in plants.Chromoplasts,which sequester excess carotenoids produced in the chloro-plast,were found to be more numerous in cauliflower plants that express the Orange (Or )gene (53).Expression of Or in potato,under control of the granule-bound starch synthase (GBSS)promoter,resulted in a sixfold increase in carotenoid levels in tubers (54).In addi-tion,increases in certain carotenoids upon Or overexpression may provide insights into rate-limiting steps in the potato carotenoid pathway;for example,the increase in phytoene from zero •Biosynthesis of Plant Isoprenoids 343A n n u . R e v . P l a n tB i o l . 2009.60:335-355. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b y U N I V E R S I T Y O F F L O R I D A - S m a t h e r s L i b r a r y o n 01/12/10. F o r p e r s o n a l u s e o n l y .。

新疆医科大学 硕士学位论文 大蒜辣素生物合成、稳定性研究及相关物质分析 姓名：林守峰 申请学位级别：硕士 专业：药物化学 指导教师：李新霞;陈坚 2010-04摘 要　大蒜辣素生物合成、稳定性研究及相关物质分析研究生：林守峰 导师：李新霞 陈坚 教授摘要目的 ： 本研究以大蒜中提取的蒜氨酸和蒜酶为原料，生物合成制备大蒜辣素。

在前期建立的蒜酶活力测定方法和蒜氨酸、大蒜辣素测定方法基础上，研究蒜酶催 化裂解蒜氨酸的配比，从而为蒜氨酸/蒜酶二元制剂的研究建立基础；考察大蒜辣素 溶液的稳定性，研究和探讨影响稳定性因素。

由于生物合成大蒜辣素所使用的蒜氨 酸和蒜酶原料为中试生产得到，原料中的杂质可能会对制备得到的大蒜辣素溶液的 纯度造成影响，通过液质联用方法，对于大蒜辣素溶液中的杂质和相关物质及降解 产物进行结构解析，分析杂质来源，从而提出改进和优化生物合成大蒜辣素的工艺 条件。

方法：1.综合考虑所使用蒜氨酸原料的含量和经济性，通过 HPLC 对反应产物 进行测定，确定蒜酶催化蒜氨酸生物合成大蒜辣素工艺中的原料配比、反应温度、 投料方式等。

2.参考欧洲药典中潜在大蒜辣素含量测定方法，HPLC 测定考察三个温 度条件下的大蒜辣素溶液的稳定性，计算半衰期作为评价稳定性的标准。

3.通过液质 联用测定大蒜辣素溶液和蒜氨酸原料，建立 HPLC-MS 分析方法，进行相应物质的结 构解析和确证。

结果： 1.确定大蒜辣素生物合成工艺中的蒜氨酸和蒜酶配比为 1mg 蒜氨酸需要 1.3U 蒜酶，综合蒜酶活性和大蒜辣素稳定性确定 2５℃为反应温度，由于 蒜酶为自杀式酶，生成的大蒜辣素抑制蒜酶活力，故经考察分次将蒜酶溶液滴加到 蒜氨酸中用最少的蒜酶量制备大蒜辣素，结合膜分离技术滤除蒜酶等大分子物质。

2.影响大蒜辣素稳定性的因素除了温度原因之外， 还有空气中的氧气对于亚砜基氧化 作用等，采用隔绝空气等方法提高稳定性。

3.HPLC-MS 确定了大蒜辣素溶液中的杂 质为甲基丙烯基硫代亚硫酸脂；并且不同含量的蒜氨酸原料合成的大蒜辣素溶液含 有不等量的蒜氨酸和大蒜辣素的同分异构体；确定了大蒜辣素的部分降解产物。

DOI: 10.1126/science.1191652, 70 (2010);330 Science , et al.Parayil Kumaran Ajikumar Escherichia coliin Isoprenoid Pathway Optimization for Taxol Precursor OverproductionThis copy is for your personal, non-commercial use only.clicking here.colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to othershere.following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles): August 4, 2011 (this infomation is current as of The following resources related to this article are available online at/content/330/6000/70.full.html version of this article at:including high-resolution figures, can be found in the online Updated information and services, /content/suppl/2010/09/27/330.6000.70.DC1.htmlcan be found at:Supporting Online Material /content/330/6000/70.full.html#related found at:can be related to this article A list of selected additional articles on the Science Web sites /content/330/6000/70.full.html#ref-list-1, 4 of which can be accessed free:cites 33 articles This article 1 article(s) on the ISI Web of Science cited by This article has been /content/330/6000/70.full.html#related-urls 1 articles hosted by HighWire Press; see:cited by This article has been/cgi/collection/chemistry Chemistrysubject collections:This article appears in the following registered trademark of AAAS.is a Science 2010 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science o n A u g u s t 4, 2011w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mIsoprenoid Pathway Optimizationfor Taxol Precursor Overproductionin Escherichia coliParayil Kumaran Ajikumar,1,2Wen-Hai Xiao,1Keith E.J.Tyo,1Yong Wang,3Fritz Simeon,1 Effendi Leonard,1Oliver Mucha,1Too Heng Phon,2Blaine Pfeifer,3*Gregory Stephanopoulos1,2* Taxol(paclitaxel)is a potent anticancer drug first isolated from the Taxus brevifolia Pacific yew tree. Currently,cost-efficient production of Taxol and its analogs remains limited.Here,we report a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene—the first committed Taxol intermediate—approximately1gram per liter(~15,000-fold)in an engineered Escherichia coli strain.Our approach partitioned the taxadiene metabolic pathwayinto two modules:a native upstream methylerythritol-phosphate(MEP)pathway forming isopentenyl pyrophosphate and a heterologous downstream terpenoid–forming pathway.Systematic multivariate search identified conditions that optimally balance the two pathway modules so as to maximize the taxadiene production with minimal accumulation of indole,which is an inhibitory compound found here. We also engineered the next step in Taxol biosynthesis,a P450-mediated5a-oxidation of taxadieneto taxadien-5a-ol.More broadly,the modular pathway engineering approach helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products.T axol(paclitaxel)and its structural analogs are among the most potent and commer-cially successful anticancer drugs(1).Taxol was first isolated from the bark of the Pacific yew tree(2),and early-stage production methods required sacrificing two to four fully grown trees to secure sufficient dosage for one patient(3). Taxol’s structural complexity limited its chemical synthesis to elaborate routes that required35to 51steps,with a highest yield of0.4%(4–6).Asemisynthetic route was later devised in whichthe biosynthetic intermediate baccatin III,isolatedfrom plant sources,was chemically converted toTaxol(7).Although this approach and subse-quent plant cell culture–based production effortshave decreased the need for harvesting the yewtree,production still depends on plant-based pro-cesses(8),with accompanying limitations onproductivity and scalability.These methods ofproduction also constrain the number of Taxolderivatives that can be synthesized in the searchfor more efficacious drugs(9,10).Recent developments in metabolic engineer-ing and synthetic biology offer new possibilitiesfor the overproduction of complex natural productsby optimizing more technically amenable micro-bial hosts(11,12).The metabolic pathway forTaxol consists of an upstream isoprenoid pathwaythat is native to Escherichia coli and a het-erologous downstream terpenoid pathway(fig.S1).The upstream methylerythritol-phosphate(MEP)or heterologous mevalonic acid(MV A)pathwayscan produce the two common building blocks,isopentenyl pyrophosphate(IPP)and dimethyl-allyl pyrophosphate(DMAPP),from which Taxoland other isoprenoid compounds are formed(12).Recent studies have highlighted the engi-neering of the above upstream pathways to sup-port the biosynthesis of heterologous isoprenoidssuch as lycopene(13,14),artemisinic acid(15,16),and abietadiene(17,18).The downstream taxadienepathway has been reconstructed in E.coli andSaccharomyces cerevisiae together with the over-expression of upstream pathway enzymes,but todate titers have been limited to less than10mg/liter(19,20).The above rational metabolic engineering ap-proaches examined separately either the upstreamor the downstream terpenoid pathway,implicitlyassuming that modifications are additive(a linearbehavior)(13,17,21).Although this approachcan yield moderate increases in flux,it generallyignores nonspecific effects,such as toxicity of in-termediate metabolites,adverse cellular effects ofthe vectors used for expression,and hidden path-ways and metabolites that may compete with themain pathway and inhibit the production of thedesired binatorial approaches canovercome such problems because they offer theopportunity to broadly sample the parameter spaceand bypass these complex nonlinear interactions(21–23).However,combinatorial approaches re-quire high-throughput screens,which are often notavailable for many desirable natural products(24).Considering the lack of a high-throughputscreen for taxadiene(or other Taxol pathwayintermediate),we resorted to a focused combi-1Department of Chemical Engineering,Massachusetts Institute of Technology(MIT),Cambridge,MA02139,USA.2Chemical and Pharmaceutical Engineering Program,Singapore-MIT Alli-ance,117546Singapore.3Department of Chemical and Bio-logical Engineering,Tufts University,4Colby Street,Medford, MA02155,USA.*To whom correspondence should be addressed.E-mail: gregstep@(G.S.);blaine.pfeifer@(B.P.)Upstream moduleFig.1.isoprenoid pathwaythe flux through thewe targeted reported(dxs,idi,ispD,andexpression by anTo channel theversal isoprenoidtoward Taxolsynthetic operon of downstream genes GGPP synthase(G)and taxadienesynthase(T)(37).Both pathways were placed under the control of induciblepromoters in order to control their relative gene expression.In the E.colimetabolic network,the MEP isoprenoid pathway is initiated by the con-densation of the precursors glyceraldehyde-3phosphate(G3P)and pyruvate(PYR)from glycolysis.The Taxol pathway bifurcation starts from the universalisoprenoid precursors IPP and DMAPP to form geranylgeranyl diphosphate,and then the taxadiene.The cyclic olefin taxadiene undergoes multiple roundsof stereospecific oxidations,acylations,and benzoylation to form the lateintermediate Baccatin III and side chain assembly to,ultimately,form Taxol. REPORTS1OCTOBER2010VOL330SCIENCE 70onAugust4,211www.sciencemag.orgDownloadedfromnatorial approach,which we term “multivariate-modular pathway engineering.”In this approach,the overall pathway is partitioned into smaller modules,and the modules ’expression are varied simultaneously —a multivariate search.This ap-proach can identify an optimally balanced path-way while searching a small combinatorial space.Specifically,we partition the taxadiene-forming pathway into two modules separated at IPP,which is the key intermediate in terpenoid bio-synthesis.The first module comprises an eight-gene,upstream,native (MEP)pathway of which the expression of only four genes deemed to be rate-limiting was modulated,and the second mod-ule comprises a two-gene,downstream,heterolo-gous pathway to taxadiene (Fig.1).This modular approach allowed us to efficiently sample the main parameters affecting pathway flux without the need for a high-throughput screen and to unveil the role of the metabolite indole as in-hibitor of isoprenoid pathway activity.Addition-ally,the multivariate search revealed a highly nonlinear taxadiene flux landscape with a global maximum exhibiting a 15,000-fold increase in taxadiene production over the control,yielding 1.02T 0.08g/liter (SD)taxadiene in fed-batch bioreactor fermentations.We have further engineered the P450-based oxidation chemistry in Taxol biosynthesis in E.coli to convert taxadiene to taxadien-5a -ol and provide the basis for the synthesis of sub-sequent metabolites in the pathway by means of similar cytochrome P450(CYP450)oxida-tion chemistry.Our engineered strain improved taxadiene-5a -ol production by 2400-fold over the state of the art with yeast (25).These ad-vances unlock the potential of microbial pro-cesses for the large-scale production of Taxol or its derivatives and thousands of other valuable terpenoids.The multivariate-modular approach in which various promoters and gene copy-numbers are combined to modulate diverse expression levels of upstream and downstream pathways of taxadiene synthesis is schematically described in fig.S2.A total of 16strains were constructed in order to widen the bottleneck of the MEP pathway as well as optimally balance it with the downstream tax-adiene pathway (26).The dependence of tax-adiene accumulation on the upstream pathway for constant values of the downstream pathway is shown in Fig.2A,and the dependence on the downstream pathway for constant upstream path-way strength is shown in Fig.2B (table S1,cal-culation of the upstream and downstream pathway strength from gene copy number and promoter strength).As the upstream pathway expression increases in Fig.2A from very low levels,tax-adiene production also rises initially because of increased supply of precursors to the overall path-way.However,after an intermediate value further upstream pathway increases cannot be accom-modated by the capacity of the downstream path-way.For constant upstream pathway expression (Fig.2B),a maximum in downstream expressionwas similarly observed owing to the rising edge to initial limiting of taxadiene production by low expression levels of the downstream pathway.At high (after peak)levels of downstream pathway expression,we were probably observing the neg-ative effect on cell physiology of the high copy number.These results demonstrate that dramatic changes in taxadiene accumulation can be obtained fromchanges within a narrow window of expression levels for the upstream and downstream path-ways.For example,a strain containing an ad-ditional copy of the upstream pathway on its chromosome under Trc promoter control (strain 8)(Fig.2A)produced 2000-fold more taxadiene than one expressing only the native MEP path-way (strain 1)(Fig.2A).Furthermore,changing the order of the genes in the downstreamsyn-Fig.2.Optimization of taxadiene production through regulating the expression of the upstream and downstream modular pathways.(A )Response in taxadiene accumulation to changes in upstream pathway strengths for constant values of the downstream pathway.(B )Dependence of taxadiene on the down-stream pathway for constant levels of upstream pathway strength.(C )Taxadiene response from strains (17to 24)engineered with high upstream pathway overexpressions (6to 100a.u.)at two different down-stream expressions (31a.u.and 61a.u.).(D )Modulation of a chromosomally integrated upstream pathway by using increasing promoter strength at two different downstream expressions (31a.u.and 61a.u.).(E )Genotypes of the 32strain constructs whose taxadiene phenotype is shown in Fig.2,A to D.E,E.coli K12MG1655D recA D endA ;EDE3,E.coli K12MG1655D recA D endA with DE3T7RNA polymerase gene in the chromosome;MEP,dxs-idi-ispDF operon;GT,GPPS-TS operon;TG,TS-GPPS operon;Ch1,1copy in chromosome;Trc,Trc promoter;T5,T5promoter;T7,T7promoter;p5,pSC101plasmid;p10,p15A plasmid;and p20,pBR322plasmid. SCIENCEVOL 3301OCTOBER 201071REPORTSo n A u g u s t 4, 2011w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mthetic operon from GT (GGPS-TS)to TG (TS-GGPS)resulted in a two-to threefold increase (strains 1to 4as compared with strains 5,8,11,and 14).Altogether,the engineered strains estab-lished that the MEP pathway flux can be substan-tial if an appropriate range of expression levels for the endogenous upstream and synthetic down-stream pathway are searched simultaneously.To provide ample downstream pathway strength while minimizing the plasmid-born metabolic bur-den (27),two new sets of four strains each were engineered (strains 17to 20and 21to 24),in which the downstream pathway was placed un-der the control of a strong promoter (T7)while keeping a relatively low number of five and 10plasmid copies,respectively.The taxadiene maxi-mum was maintained at high downstream strength (strains 21to 24),whereas a monotonic response was obtained at the low downstream pathway strength (strains 17to 20)(Fig.2C).This ob-servation prompted the construction of two addi-tional sets of four strains each that maintained the same level of downstream pathway strength as before but expressed very low levels of the up-stream pathway (strains 25to 28and 29to 32)(Fig.2D).Additionally,the operon of the up-stream pathway of the latter strain set was chro-mosomally integrated (fig S3).Not only was the taxadiene maximum recovered in these strains,albeit at very low upstream pathway levels,but a much greater taxadiene maximum was attained (~300mg/liter).We believe that this significant increase can be attributed to a decrease in the cell ’s metabolic burden.We next quantified the mRNA levels of 1-deoxy-D -xylulose-5-phosphate synthase (dxs)and taxadiene synthase (TS)(representing the up-stream and downstream pathways,respectively)for the high-taxadiene-producing strains (25to 32and 17and 22)that exhibited varying up-stream and downstream pathway strengths (fig.S4,A and B)to verify our predicted expression strengths were consistent with the actual pathway levels.We found that dxs expression level cor-relates well with the upstream pathway strength.Similar correlations were found for the other genes of the upstream pathway:idi ,ispD ,and ispF (fig.S4,C and D).In downstream TS gene expres-sion,an approximately twofold improvement was quantified as the downstream pathway strength increased from 31to 61arbitrary units (a.u.)(fig.S4B).Metabolomic analysis of the previous strains led to the identification of a distinct metabolite by-product that inversely correlated with taxadiene accumulation (figs.S5and S6).The corresponding peak in the gas chromatography –mass spectrom-etry (GC-MS)chromatogram was identified as indole through GC-MS,1H,and 13C nuclear magnetic resonance (NMR)spectroscopy studies (fig.S7).We found that taxadiene synthesis by strain 26is severely inhibited by exogenous in-dole at indole levels higher than ~100mg/liter (fig.S5B).Further increasing the indole concen-tration also inhibited cell growth,with the level ofinhibition being very strain-dependent (fig.S5C).Although the biochemical mechanism of indole interaction with the isoprenoid pathway is pres-ently unclear,the results in fig.S5suggest a possible synergistic effect between indole and terpenoid compounds of the isoprenoid pathway in inhibiting cell growth.Without knowing the specific mechanism,it appears that strain 26has mitigated the indole ’s effect,which we carried forward for further study.In order to explore the taxadiene-producing potential under controlled conditions for the en-gineered strains,fed-batch cultivations of the three highest taxadiene accumulating strains (~60mg/liter from strain 22;~125mg/liter from strain 17;and ~300mg/liter from strain 26)were carried out in 1-liter bioreactors (Fig.3).The fed-batch cultivation studies were carried out as liquid-liquid two-phase fermentation using a 20%(v/v)dodecane overlay.The organic solvent was intro-duced to prevent air stripping of secreted tax-adiene from the fermentation medium,as indicated by preliminary findings (fig.S8).In defined media with controlled glycerol feeding,taxadiene pro-ductivity increased to 174T 5mg/liter (SD),210T 7mg/liter (SD),and 1020T 80mg/liter (SD)for strains 22,17,and 26,respectively (Fig.3A).Additionally,taxadiene production significantly affected the growth phenotype,acetate accumu-lation,and glycerol consumption [Fig.3,B and D,and supporting online material (SOM)text].Clearly,the high productivity and more robustgrowth of strain 26allowed very high taxadiene accumulation.Further improvements should be possible through optimizing conditions in the bio-reactor,balancing nutrients in the growth medi-um and optimizing carbon delivery.Having succeeded in engineering the bio-synthesis of the “cyclase phase ”of Taxol for high taxadiene production,we turned next to engineer-ing the oxidation-chemistry of Taxol biosynthesis.In this phase,hydroxyl groups are incorporated by oxygenation at seven positions on the taxane core structure,mediated by CYP450-dependent monooxygenases (28).The first oxygenation is the hydroxylation of the C5position,followed by seven similar reactions en route to Taxol (fig.S1)(29).Thus,a key step toward engineering Taxol-producing microbes is the development of CYP450-based oxidation chemistry in vivo.The first oxygenation step is catalyzed by a CYP450,taxadiene 5a -hydroxylase,which is an unusual monooxygenase that catalyzes the hydroxylation reaction along with double-bond migration in the diterpene precursor taxadiene (Fig.1).In general,functional expression of plant CYP450in E.coli is challenging (30)because of the inherent limitations of bacterial platforms,such as the absence of electron transfer machin-ery and CYP450-reductases (CPRs)and trans-lational incompatibility of the membrane signal modules of CYP450enzymes because of the lack of an endoplasmic reticulum.Recently,through transmembrane (TM)engineering and the gener-24487296120T a x a d i e n e (m g /L )Time (h)1234024487296120N e t g l y c e r o l a d d e d (g /L )Time (h)A BC DC e l l g r o w t h (OD 600 n m )Time (h)24487296120A c e t i c a c i d (g /L )Time (h)Fig.3.Fed-batch cultivation of engineered strains in a 1-liter bioreactor.Time courses of (A )taxadiene accumulation,(B )cell growth,(C )acetic acid accumulation,and (D )total substrate (glycerol)addition for strains 22,17,and 26during 5days of fed-batch bioreactor cultivation in 1-liter bioreactor vessels under controlled pH and oxygen conditions with minimal media and 0.5%yeast extract.After glycerol depletes to ~0.5to 1g/liter in the fermentor,3g/liter of glycerol was introduced into the bioreactor during the fermentation.Data are mean of two replicate bioreactors.1OCTOBER 2010VOL 330SCIENCE72REPORTSo n A u g u s t 4, 2011w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mation of chimera enzymes of CYP450and CPR,some plant CYP450s have been expressed in E.coli for the biosynthesis of functional mole-cules (15,31).Still,every plant CYP450has distinct TM signal sequences and electron transfer characteristics from its reductase counterpart (32).Our initial studies were focused on optimizing the expression of codon-optimized synthetic tax-adiene 5a -hydroxylase by N-terminal TM engi-neering and generating chimera enzymes through translational fusion with the CPR redox partner from the Taxus species,Taxus CYP450reductase (TCPR)(Fig.4A)(29,31,33).One of the chi-mera enzymes generated,At24T5a OH-tTCPR,was highly efficient in carrying out the first oxi-dation step,resulting in more than 98%taxadiene conversion to taxadien-5a -ol and the byproduct 5(12)-Oxa-3(11)-cyclotaxane (OCT)(fig.S9A).Compared with the other chimeric CYP450s,At24T5a OH-tTCPR yielded twofold higher (21mg/liter)production of taxadien-5a -ol (Fig.4B).Because of the functional plasticity of taxadiene 5a -hydroxylase with its chimeric CYP450’s en-zymes (At8T5a OH-tTCPR,At24T5a OH-tTCPR,and At42T5a OH-tTCPR),the reaction also yields a complex structural rearrangement of taxadiene into the cyclic ether OCT (fig.S9)(34).The by-product accumulated in approximately equal amounts (~24mg/liter from At24T5a OH-tTCPR)to the desired product taxadien-5a -ol.The productivity of strain 26-At24T5a OH-tTCPR was significantly reduced relative to that of taxadiene production by the parent strain 26(~300mg/liter),with a concomitant increase in indole accumulation.No taxadiene accumulation was observed.Apparently,the introduction of an additional medium copy plasmid (10-copy,p10T7)bearing the At24T5a OH-tTCPR construct dis-turbed the carefully engineered balance in the up-stream and downstream pathway of strain 26(fig S10).Small-scale fermentations were carried out in bioreactors so as to quantify the alcohol production by strain 26-At24T5a OH-tTCPR.The time course profile of taxadien-5a -ol accumulation (Fig.4C)indicates alcohol production of up to 58T 3mg/liter (SD)with an equal amount of the OCT by-product produced.The observed alcohol production was approximately 2400-fold higher than previous production in S.cerevisiae (25).The MEP pathway is energetically balanced and thus overall more efficient in converting either glucose or glycerol to isoprenoids (fig.S11).Yet,during the past 10years many attempts at en-gineering the MEP pathway in E.coli in order to increase the supply of the key precursors IPP and DMAPP for carotenoid (21,35),sesquiterpenoid (16),and diterpenoid (17)overproduction met with limited success.This inefficiency was at-tributed to unknown regulatory effects associated specifically with the expression of the MEP path-way in E.coli (16).Here,we provide evidence that such limitations are correlated with the accumu-lation of the metabolite indole,owing to the non-optimal expression of the pathway,which inhibits the isoprenoid pathway activity.Taxadiene over-production (under conditions of indole-formation suppression),establishes the MEP pathway as a very efficient route for biosynthesis of pharma-ceutical and chemical products of the isoprenoid family (fig.S11).One simply needs to carefully balance the modular pathways,as suggested by our multivariate-modular pathway –engineering approach.For successful microbial production of Taxol,demonstration of the chemical decoration of the taxadiene core by means of CYP450-based oxi-dation chemistry is essential (28).Previous ef-forts to reconstitute partial Taxol pathways in yeast found CYP450activity limiting (25),making the At24T5a OH-tTCPR activity levels an im-portant step to debottleneck the late Taxol path-way.Additionally,the strategies used to create At24T5a OH-tTCPR are probably applicable for the remaining monooxygenases that will require expression in E.coli .CYP450monooxygenases constitute about one half of the 19distinct en-zymatic steps in the Taxol biosynthetic pathway.These genes show unusually high sequence sim-ilarity with each other (>70%)but low similarity (<30%)with other plant CYP450s (36),implying that these monooxygenases are amenable to similar engineering.To complete the synthesis of a suitable Taxol precursor,baccatin III,six more hydroxylation reactions and other steps (including some that have not been identified)need to be effectively engineered.Although this is certainly a daunting task,the current study shows potential by provid-ing the basis for the functional expression of two key steps,cyclization and oxygenation,in Taxol biosynthesis.Most importantly,by unlocking the potential of the MEP pathway a new more ef-ficient route to terpenoid biosynthesis is capable of providing potential commercial production of microbially derived terpenoids for use as chem-icals and fuels from renewable resources.References and Notes1.D.G.Kingston,Phytochemistry 68,1844(2007).2.M.C.Wani,H.L.Taylor,M.E.Wall,P.Coggon,A.T.McPhail,J.Am.Chem.Soc.93,2325(1971).3.M.Suffness,M.E.Wall,in Taxol:Science and 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.(A )TM engineering and construction of chimera protein from taxadien-5a -ol hydroxylase (T5a OH)and Taxus cytochrome P450reductase (TCPR).The labels 1and 2represent the full-length proteins of T5a OH and TCPR identified with 42and 74amino acid TM regions,respectively,and 3represents chimera enzymes generated from three different TM en-gineered T5a OH constructs [At8T5a OH,At24T5a OH,and At42T5a OH constructed by fusing an 8-residue synthetic peptide MALLLAVF (A)to 8,24,and 42AA truncated T5a OH]through a translational fusion with 74AA truncated TCPR (tTCPR)by use of linker peptide GSTGS.(B )Functional activity of At8T5a OH-tTCPR,At24T5a OH-tTCPR,and At42T5a OH-tTCPR constructs transformed into taxadiene producing strain 26.Data are mean T SD for three replicates.(C )Time course profile of taxadien-5a -ol accumulation and growth profile of the strain 26-At24T5a OH-tTCPR fermented in a 1-liter bioreactor.Data are mean of two replicate bioreactors.SCIENCEVOL 3301OCTOBER 201073REPORTSo n 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gratefully acknowledge support by the Singapore-MIT Alliance (SMA-2)and NIH,grant 1-R01-GM085323-01A1.B.P.acknowledges the Milheim Foundation Grant for Cancer Research 2006-17.A patent application that is based on the results presented here has been filed by MIT.P.K.A.designed the experiments and performed the engineering and screening of the strains;W-H.X.performed screening of the strains,bioreactorexperiments,and GC-MS analysis;F.S.carried out the quantitative PCR measurements;O.M.performed the extraction and characterization of taxadiene standard;E.L.,Y.W.,and B.P.supported with cloning experiments;P.K.A.,K.E.J.T.,T.H.P.,B.P.and G.S.analyzed the data;P.K.A.,K.E.J.T.,and G.S.wrote the manuscript;G.S.supervised the research;and all of the authors contributed to discussion of the research and edited and commented on the manuscript.Supporting Online Material/cgi/content/full/330/6000/70/DC1Materials and Methods SOM TextFigs.S1to S11Tables S1to S4References29April 2010;accepted 9August 201010.1126/science.1191652Reactivity of the Gold/Water Interface During Selective Oxidation CatalysisBhushan N.Zope,David D.Hibbitts,Matthew Neurock,Robert J.Davis *The selective oxidation of alcohols in aqueous phase over supported metal catalysts is facilitated by high-pH conditions.We have studied the mechanism of ethanol and glycerol oxidation to acids over various supported gold and platinum beling experiments with 18O 2and H 218O demonstrate that oxygen atoms originating from hydroxide ions instead of molecular oxygen are incorporated into the alcohol during the oxidation reaction.Density functional theory calculations suggest that the reaction path involves both solution-mediated and metal-catalyzed elementary steps.Molecular oxygen is proposed to participate in the catalytic cycle not by dissociation to atomic oxygen but by regenerating hydroxide ions formed via the catalytic decomposition of a peroxide intermediate.The selective oxidation of alcohols with mo-lecular oxygen over gold (Au)catalysts in liquid water offers a sustainable,envi-ronmentally benign alternative to traditional pro-cesses that use expensive inorganic oxidants and harmful organic solvents (1,2).These catalytic transformations are important to the rapidly de-veloping industry based on the conversion of bio-renewable feedstocks to higher-valued chemicals (3,4)as well as the current production of petro-chemicals.Although gold is the noblest of metals (5),the water/Au interface provides a reaction en-vironment that enhances its catalytic performance.We provide here direct evidence for the predomi-nant reaction path during alcohol oxidation at high pH that includes the coupling of both solution-mediated and metal-catalyzed elementary steps.Alcohol oxidation catalyzed by Pt-group metals has been studied extensively,although the precisereaction path and extent of O 2contribution are still under debate (4,6–8).The mechanism for the selective oxidation of alcohols in liquid water over the Au catalysts remains largely un-known (6,9),despite a few recent studies with organic solvents (10–12).In general,supported Au nanoparticles are exceptionally good catalysts for the aerobic oxidation of diverse reagents ranging from simple molecules such as CO and H 2(13)to more complex substrates such as hy-drocarbons and alcohols (14).Au catalysts are also substrate-specific,highly selective,stable against metal leaching,and resistant to overoxidation by O 2(6,15,16).The active catalytic species has been suggested to be anionic Au species (17),cat-ionic Au species (18,19),and neutral Au metal particles (20).Moreover,the size and structure of Au nanoparticles (21,22)as well as the interface of these particles with the support (23)have also been claimed to be important for catalytic ac-tivity.For the well-studied CO oxidation reaction,the presence of water vapor increases the observed rate of the reaction (24–26).Large metallic Au particles and Au metal powder,which are usually considered to be catalytically inert,have consider-able oxidation activity under aqueous conditions at high pH (27,28).We provide insights into the active intermediates and the mechanism for al-cohol oxidation in aqueous media derived from experimental kinetic studies on the oxidation of glycerol and ethanol with isotopically labeled O 2and H 2O over supported Au and Pt catalysts,as well as ab initio density functional theory calcu-lations on ethanol oxidation over metal surfaces.Previous studies indicate that alcohol oxida-tion over supported metal catalysts (Au,Pt,and Pd)proceeds by dehydrogenation to an aldehyde or ketone intermediate,followed by oxidation to the acid product (Eq.1)RCH 2OH À!O 2,catalyst RCH ¼O À!O 2,catalystRCOOH(1)Hydroxide ions play an important role during oxidation;the product distribution depends on pH,and little or no activity is seen over Au cat-alysts without added base.We studied Au par-ticles of various sizes (average diameter ranging from 3.5to 10nm)on different supports (TiO 2and C)as catalysts for alcohol oxidation and com-pared them to Pt and Pd particles supported on C.The oxidation of glycerol (HOCH 2CHOHCH 2OH)to glyceric (HOCH 2CHOHCOOH)and glycolic (HOCH 2COOH)acids occurred at a turnover frequency (TOF)of 6.1and 4.9s −1on Au/C and Au/TiO 2,respectively,at high pH (>13)whereas the TOF on supported Pt and Pd (1.6and 2.2s −1,respectively)was slightly lower at otherwise iden-tical conditions (Table 1).For these Au catalysts,particle size and support composition had negligi-ble effect on the rate or selectivity.In the absence of base,the glycerol oxidation rate was much lower over the Pt and Pd catalysts and no conver-sion was observed over the Au catalysts (Table 1).Moreover,the products detected over Pt and Pd in the absence of base are primarily the intermediate aldehyde and ketone,rather than acids.Department of Chemical Engineering,University of Virginia,102Engineers ’Way,Post Office Box 400741,Charlottesville,VA,22904–4741,USA.*To whom correspondence should be addressed.E-mail:rjd4f@1OCTOBER 2010VOL 330SCIENCE74REPORTSo n A u g u s t 4, 2011w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m。

贵州黄花蒿内生真菌多样性及其产抗病活性物质菌株的筛选曾茜;陈旭;田维毅;王平;雷帮星【摘要】为了解贵州黄花蒿植株内生真菌多样性,挖掘农作物病害生物防治新资源,采集贵州生物多样性丰富地区的黄花蒿(Artemisia annua.Linn.)植物13株,分离获得内生真菌;通过形态学和5.8S-ITSrDNA分子系统发育分析得出分类;以小麦赤霉、水稻纹枯、番茄黑斑等9种农作物病原真菌作为靶标菌,采用平板对峙法对产抗病活性物质的菌株进行筛选.结果表明:分离获得内生真菌122株,鉴定得到子囊菌门真菌共10目8科12属,担子菌门2目2科2属,接合菌门1目1科1属.共计18株(14.8％)黄花蒿内生真菌对病原菌具有较强的拮抗作用,其中以青霉属GZUCCZY0012菌株的抗病活性最为广谱,对6种病原真菌菌丝生长均具有较强的抑制作用,抑制效果为72.60％～94.86％.【期刊名称】《贵州农业科学》【年(卷),期】2015(043)009【总页数】6页(P164-169)【关键词】黄花蒿;内生真菌;分类鉴定;抗病活性;贵州【作者】曾茜;陈旭;田维毅;王平;雷帮星【作者单位】贵阳中医学院基础部微生物研究室,贵州贵阳550025;贵州省农业科学院农业资源与环境研究所,贵州贵阳550025;贵阳中医学院基础部微生物研究室,贵州贵阳550025;贵阳中医学院基础部微生物研究室,贵州贵阳550025;贵州大学贵州省生化工程中心,贵州贵阳550025【正文语种】中文【中图分类】Q939.96植物内生真菌指生活在植物组织内或生活史中的某一阶段生活在植物组织内，对植物组织未引起明显病害症状的一类真菌[1]。

Stierle[2]首次从短叶红豆杉植物的韧皮部分离得到内生真菌安德紫衫菌(Taxomyces andreanae)具有生产抗癌物质紫杉醇(Taxol)的能力，这一发现引起了各相关领域研究者对内生真菌的关注，并为天然产物来源药物的开发利用和药用植物资源的保护提供了新的思路和方法。

紫杉醇生物合成研究历史、现状及展望邱德有;张彬;杨艳芳;邵芬娟;滕文静【摘要】Taxol is a diterpene isolated from Taxus. It is one of the most widely used compounds in the treatment of lung, ovarian and breast cancer. In this paper, the history of taxol biosynthesis research, its current status and the present research progress are reviewed. The future directions as well as the prospects of taxol biosynthesis research are also discussed.%紫杉醇是从红豆杉中分离出来的一种二萜类化合物，是目前临床上使用最广泛的抗癌药物之一。

回顾了紫杉醇生物合成的研究历史，主要包括参与红豆杉紫杉醇生物合成的结构基因、调控基因、红豆杉代谢组、内生真菌紫杉醇生物合成研究历史以及国际上紫杉醇生物合成专利情况等。

介绍了近年来红豆杉和内生真菌紫杉醇生物合成研究的现状。

最后探讨了本领域未来的研究方向并对其前景进行展望。

【期刊名称】《生物技术通报》【年(卷),期】2015(000)004【总页数】9页(P56-64)【关键词】紫杉醇;生物合成;现状;前景【作者】邱德有;张彬;杨艳芳;邵芬娟;滕文静【作者单位】中国林业科学研究院林业研究所林木遗传育种国家重点实验室，北京 100091;国家知识产权局专利局专利审查协作北京中心，北京 100190;中国林业科学研究院林业研究所林木遗传育种国家重点实验室，北京 100091;中国林业科学研究院林业研究所林木遗传育种国家重点实验室，北京 100091;国家知识产权局专利局专利审查协作北京中心，北京 100190【正文语种】中文紫杉醇（Taxol）是红豆杉属植物中的一种二萜类化合物。