C A T A L O G U E 法学集刊

a b c d e f g h i j k l m n o p q r s t u v w x y z注：(1)r、v两个字母用来拼写普通话和外来语，拼写广州话时不用。

(2)广州话拼音字母有三个附加符号：ê、é、ü，其中é是和汉语拼音字母不同的。

这几个字母是e、u字母的变体，叫不列入表内。

二、声母表b波p婆m摸f科d多t拖n挪l罗g哥k卡ng我h何gu姑ku箍z左c初s梳j知q雌x思y也w华注：(1)z、c、s和j、q、x两组声母，广州话的读音没有区别，只是在拼写韵母时有不同，z、c、s拼写a、o、é及a、o、e、é、ê、u等字母开头的韵母，例如：za渣，ca茶，xa沙。

j、q、x拼写i、ü及i、ü字母开头的韵母，例如：ji知，qi次，xi思。

(2)gu姑、ku箍是圆唇的舌跟音，作为声母使用，不能单独注音，单独注音时是音节，不是声母。

(3)y也，w华拼音时作为声母使用，拼写出来的音节相当于汉语拼音方案的复韵母，但由于广州话当中这些韵母前面不再拼声母，因此只作为音节使用三、韵母表a呀o柯u乌i衣ū于ê(靴) é诶m唔n五ai挨 ei矮 oi哀 ui会éi(非)ao拗 eo欧 ou奥iu妖êu(去)am(监) em庵im淹an晏 en(恩) on安 un碗in烟ūn冤 ên(春)ang(横) eng莺 ong(康) ung瓮ing英êng(香) éng(镜)ab鸭 eb(急) ib叶ad押 ed(不) od(渴) ud活 id热ūd月 êd(律)ag(客) eg(德) og恶 ug屋 ig益êg(约) ég(尺)注：(1)例字外加( )号的，只取其韵母。

(2)i行的韵母，前面没有声母的时候，写成yi衣，yiu妖，yim淹，yin烟，ying 英，yib叶，yid热，yig益。

Current to June 28, 2010À jour au 28 juin 2010Published by the Minister of Justice at the following address:http://laws-lois.justice.gc.ca Publié par le ministre de la Justice à l’adresse suivante :http://laws-lois.justice.gc.caCANADACONSOLIDATION Hazardous Products (Pacifiers) RegulationsCODIFICATIONRèglement sur les produits dangereux(sucettes)C.R.C., c. 930C.R.C., ch. 930OFFICIAL STATUS OF CONSOLIDATIONS CARACTÈRE OFFICIEL DES CODIFICATIONSSubsections 31(1) and (3) of the Legislation Revision and Consolidation Act, in force on June 1, 2009, provide as follows:Les paragraphes 31(1) et (3) de la Loi sur la révision et la codification des textes législatifs, en vigueur le 1er juin 2009, prévoient ce qui suit :Published consolidation is evidence31. (1) Every copy of a consolidated statute orconsolidated regulation published by the Ministerunder this Act in either print or electronic form is ev-idence of that statute or regulation and of its contentsand every copy purporting to be published by theMinister is deemed to be so published, unless thecontrary is shown.31. (1) Tout exemplaire d'une loi codifiée oud'un règlement codifié, publié par le ministre en ver-tu de la présente loi sur support papier ou sur supportélectronique, fait foi de cette loi ou de ce règlementet de son contenu. Tout exemplaire donné commepublié par le ministre est réputé avoir été ainsi pu-blié, sauf preuve contraire.Codificationscomme élémentde preuve ...[...]Inconsistencies in regulations(3) In the event of an inconsistency between aconsolidated regulation published by the Ministerunder this Act and the original regulation or a subse-quent amendment as registered by the Clerk of thePrivy Council under the Statutory Instruments Act,the original regulation or amendment prevails to theextent of the inconsistency.(3) Les dispositions du règlement d'origine avecses modifications subséquentes enregistrées par legreffier du Conseil privé en vertu de la Loi sur lestextes réglementaires l'emportent sur les dispositionsincompatibles du règlement codifié publié par le mi-nistre en vertu de la présente loi.Incompatibilité— règlementsCHAPTER 930CHAPITRE 930HAZARDOUS PRODUCTS ACT LOI SUR LES PRODUITS DANGEREUX Hazardous Products (Pacifiers) Regulations Règlement sur les produits dangereux (sucettes)REGULATIONS RESPECTING THE ADVERTISING, SALE AND IMPORTATION OF HAZARDOUS PRODUCTS (PACIFIERS)RÈGLEMENT CONCERNANT LA VENTE, L’IMPORTATION ET LA PUBLICITÉ DES SUCETTESSHORT TITLE TITRE ABRÉGÉ1. These Regulations may be cited as the Hazardous Products (Pacifiers) Regulations.1. Le présent règlement peut être cité sous le titre : Règlement sur les produits dangereux (sucettes).INTERPRETATION DÉFINITIONS2. In these Regulations,“Act” means the Hazardous Products Act; (Loi)“product” means a pacifier or similar product included in item 27 of Part II of Schedule I to the Act. (produit)SOR/91-265, s. 2.2. Les définitions qui suivent s’appliquent au présent règlement.« Loi » La Loi sur les produits dangereux. (Act)« produit » Sucette ou produit semblable visé à l’ar-ticle 27 de la partie II de l’annexe I de la Loi. (product) DORS/91-265, art. 2.GENERAL DISPOSITIONS GÉNÉRALES3. A person may advertise, sell or import into Canadaa product only if it meets the requirements of these Reg-ulations.SOR/91-265, s. 3(F).3. La vente, l’importation et la publicité d’un produit sont autorisées à la condition que celui-ci soit conforme aux exigences du présent règlement.DORS/91-265, art. 3(F).ADVERTISING AND LABELLING PUBLICITÉ ET ÉTIQUETAGE4. (1) No reference, direct or indirect, to the Act or to these Regulations shall be made in any written material applied to or accompanying a product or in any adver-tisement thereof.4. (1) Il est interdit de faire tout renvoi direct ou indi-rect à la Loi ou au présent règlement dans les renseigne-ments écrits apposés sur un produit ou l’accompagnant, ainsi que dans la publicité de ce produit.(2) No representation in respect of the use of or modi-fication to a product shall be made in any written materi-al applied to or accompanying the product or in any ad-vertisement thereof, which use or modification would result in the failure of the product to meet a requirement of these Regulations.SOR/91-265, s. 4(F).(2) Il est interdit de donner, dans les renseignements écrits apposés sur un produit ou l’accompagnant, ou dans la publicité du produit, des indications sur un mode d’utilisation ou de modification du produit qui rendrait celui-ci non conforme aux exigences du présent règle-ment.DORS/91-265, art. 4(F).TOXICITY [SOR/92-586, s. 2]TOXICITÉ[DORS/92-586, art. 2]5. (1) [Revoked, SOR/92-586, s. 2] 5. (1) [Abrogé, DORS/92-586, art. 2](2) Every product, including all its parts and compo-nents shall meet the requirements of section 10 of the Hazardous Products (Toys) Regulations.(2) Tout produit, y compris tous ses éléments, doit ré-pondre aux prescriptions de l’article 10 du Règlement sur les produits dangereux (jouets).(3) No product or any part or component of the prod-uct shall contain more than 10 micrograms per kilogram total volatile N-nitrosamines, as determined by dichloromethane extraction.SOR/84-272, s. 1; SOR/85-478, s. 1; SOR/92-586, s. 2.(3) Aucun produit, y compris chaque élément, ne doit contenir plus de 10 microgrammes de N-nitrosamines volatiles totales par kilogramme, tel que déterminé par extraction au dichlorométhane.DORS/84-272, art. 1; DORS/85-478, art. 1; DORS/92-586, art. 2.DESIGN AND CONSTRUCTION CONCEPTION ET CONSTRUCTION6. Every product shall(a) be designed and constructed in such a manner as to protect the user, under reasonably foreseeable con-ditions of use, from(i) obstruction of the pharyngeal orifice,(ii) strangulation,(iii) ingestion or aspiration of the product or any part or component thereof, and(iv) wounding;(b) be designed and constructed so that,(i) the nipple is attached to a guard or shield of such dimensions that it cannot pass through the opening in the template illustrated in Schedule I when the nipple is centered on the opening and a load of 2.2 pounds is applied axially to the nipple in such a way as to induce the guard or shield to pull through the opening in the template,(ii) any loop of cord or other material attached to the product is not more than 14 inches in circumfer-ence,(iii) when tested in accordance with the procedure described in Schedule II(A) the nipple remains attached to the guard orshield described in subparagraph (i), and(B) no part or component is separated or brokenfree from the product that will fit, in a non-com-pressed state, into the small parts cylinder illus-trated in Schedule III, and 6. Tout produit doit êtrea) conçu et construit de façon à protéger l’utilisateur, dans les conditions d’utilisation raisonnablement pré-visibles, contre les dangers suivants :(i) obstruction de l’orifice pharyngien,(ii) strangulation,(iii) ingestion ou aspiration du produit ou d’un élé-ment du produit, et(iv) lésion;b) conçu et construit de façon(i) que la tétine soit fixée à une garde assez grande pour que celle-ci ne puisse passer par l’ouverture du gabarit indiqué à l’annexe I, lorsque la tétine est centrée sur l’ouverture et qu’une charge de 2,2 livres est appliquée à la tétine suivant l’axe de celle-ci de façon à entraîner la garde à travers l’ou-verture du gabarit,(ii) que toute boucle de corde ou d’autre matière at-tachée au produit ne mesure pas plus de 14 pouces de circonférence,(iii) que lorsque le produit est soumis à un essai conformément à la méthode exposée à l’annexe II,(A) la tétine reste fixée à la garde mentionnée ausous-alinéa (i), et(B) aucun élément qui s’insère, à l’état non com-primé, dans le cylindre pour petites pièces illustréà l’annexe III ne se détache ni ne se dégage; et(iv) any ring or handle is hinged, collapsible or flexible.SOR/2004-65, s. 1.(iv) que tout anneau ou poignée soit articulé, souple ou flexible.DORS/2004-65, art. 1.SCHEDULE I(s. 6)ANNEXE I (art. 6)GUARD TEMPLATE GABARIT DE LA GARDESCHEDULE II(s. 6)ANNEXE II (art. 6)TESTING PROCEDURE MÉTHODE D’ESSAI1. Hold the nipple of the pacifier in a fixed position. Apply a load 10 ± 0.25 pounds in the plane of the axis of the nipple to the handle of the pacifier at a rate of 1 ± 0.25 pounds per second and maintain the final load for 10 ± 0.5 seconds.1. Tenir la tétine de la sucette en position fixe. Appliquer à la poi-gnée une charge de 10 ± 0,25 livres sur le plan de l’axe de la tétine au rythme de 1 ± 0,25 livre par seconde et maintenir la tension définitive durant 10 ± 0,5 secondes.2. Hold the guard or shield of the pacifier in a fixed position. Ap-ply a load of 10 ± 0.25 pounds in the plane normal to the axis of the nipple to the handle of the pacifier at a rate of 1 ± 0.25 pounds per second and maintain the final load for 10 ± 0.5 seconds.2. Tenir la garde de la sucette en position fixe. Appliquer à la poi-gnée une charge de 10 ± 0,25 livres sur un plan normal par rapport àl’axe de la tétine au rythme de 1 ± 0,25 livre par seconde et maintenir la tension définitive durant 10 ± 0,5 secondes.3. Repeat the procedure described in section 2 with the load ap-plied to the nipple of the pacifier.3. Répéter l’opération de l’article 2, la charge étant appliquée à la tétine de la sucette.4. Immerse the pacifier in boiling water for 10 ± 0.5 minutes. Re-move the pacifier from the boiling water and allow to cool in air at 70 ± 5 degrees Fahrenheit for 15 ± 0.5 minutes. Repeat the tests de-scribed in sections 1, 2 and 3.4. Plonger la sucette dans de l’eau bouillante pour une période de 10 ± 0,5 minutes. Retirer la sucette de l’eau bouillante et laisser re-froidir à l’air à 70 ± 5 degrés Fahrenheit durant 15 ± 0,5 minutes. Ré-péter les essais des articles 1, 2 et 3.5. Repeat the entire procedure described in section 4 nine times. 5. Répéter neuf fois toute l’opération de l’article 4.SCHEDULE III (Clause 6(b)(iii)(B))ANNEXE III (division 6b)(iii)(B))SMALL PARTS CYLINDERCYLINDRE POUR PETITES PIÈCESNotes:– Not to scale– All dimensions in mmSOR/2004-65, s. 2.Remarques :– Pas à l’échelle– Dimensions en mmDORS/2004-65, art. 2.。

《澳門法學》稿約一、《澳門法學》是澳門大學法學院高級法律研究所編輯出版的法學學術刊物。

二、《澳門法學》以促進法學研究以及建立和豐富澳門特別行政區法學理論體系為宗旨。

三、本刊著重發表專門研究或比較研究澳門法律的學術論文，也發表研究中國內地、近鄰地區、歐盟各國、葡語國家和英美法系國家法律的學術論文以及有關國際法、法哲學和法制史方面的學術論文。

歡迎本澳和外地專家學者惠賜論文。

四、本刊採用的稿件均經過匿名評審。

五、本刊堅持學術自由原則，文責自負。

六、本刊只接受通過電子郵件（本刊編輯部電子郵箱為：macaulawreview@umac.mo）以文字檔傳來或寄來軟盤的稿件，手寫稿件一律不收。

文稿發表與否，編輯部將及時通知作者。

七、本刊僅接受中文稿件。

每篇稿件字數（包括註腳和章節附註）原則在8,000字至20,000字。

優秀的外文法學論文的中文譯本也可在本刊發表。

八、本刊編輯部對選用文稿有權進行必要體例規範工作，倘作者有所保留，請在來稿中說明。

九、作者需連同以下項目一併提交文稿：題目：中文及英文題目；摘要：100字之內中英文摘要；關鍵字：4至5個中文及英文關鍵字。

附：稿件體例要求1.文稿採用現代漢語規範標點符號（半號），引號“”（不用直引號「」）；逗號用，（置於中位，不用下落的逗號）；書名號用《》（不宜混用引號）。

2.文稿採用新細明體：內文：11點，間隔為單行間距；題目：18點加粗居中列印，副標題另起一行，16點加粗居中列印；標題：章節標題位於居中位置，14點加粗居中列印。

正文一級標題位於頁面左端，頂格放置，序號後加冒號“、”12點加粗列印。

例：一、標題內容正文二級標題，位於頁面左端，空兩格放置，序號加小括孤，後空一格接標題內容，本尾不加標題，11點加粗居中列印。

例：（一）標題內容第三級標題空兩格放置，序號加“.”後空一格接標題內容。

例：1. 正文內容第四級以下單獨占行的標題均空兩格放置序數，後空一格接標題內容。

法学期刊发表哪些
法学期刊是法学领域内学术研究成果的重要发表平台，它涵盖了法学理论研究、法律实务经验、法律制度改革等多个方面的内容。

那么，法学期刊都会发表哪些内容呢？
首先，法学期刊会发表法学理论研究成果。

这类文章通常是对法学理论进行深
入探讨和分析，包括对法律概念、法律原理、法律逻辑等方面的研究，旨在推动法学理论的发展和完善。

其次，法学期刊还会发表法律实务经验。

这类文章通常是从法律实践中总结出
的经验和教训，包括案例分析、诉讼经验、法律实务技巧等内容，旨在为从业者提供实用的指导和借鉴。

此外，法学期刊还会发表法律制度改革方面的研究成果。

这类文章通常是针对
法律制度的现状和存在的问题进行分析，并提出改革建议和方案，旨在促进法律制度的完善和进步。

另外，法学期刊还会发表法学评论和综述。

这类文章通常是对法学领域内热点
问题进行评论和总结，包括对法学理论、法律实务、法律制度改革等方面的评论和综述，旨在为学术界和从业者提供理论交流和思想碰撞的平台。

总的来说，法学期刊发表的内容涵盖了法学理论研究、法律实务经验、法律制
度改革以及评论和综述等多个方面，旨在推动法学领域的学术研究和实践发展。

因此，对于法学研究者和从业者来说，关注法学期刊的最新发表成果，对于提升自身学术水平和实践能力都具有重要意义。

法律哲学与政治哲学L E G A L A N D P OLI TIC AL PHILOSOPHYJeremy Waldron著，庞正+译法律和政治或许法律哲学可以被理解为政治哲学的一个分支?这两者之间显然存在着关联。

法律体系是政治体系的一个组成部分，而奇怪的是政治学的研究者们竟始终对它的实施不感兴趣。

法律、法律适用、法律构成、立法机关、法院、司法判决、法律推理、法治，如此等等，都是政治学研究的重要课题。

立法机关和法院是政治机构，法治是政治理念，司法判决和法律推理是一定社会的政治文化的组成部分，是人们展示的政治实践和政治技能。

的确，它们都不是政治学的研究者们感兴趣的主题。

政治学研究者的兴趣点，在于像政党这样的非法律的组织，像自由与繁荣这样的非法律的理想，像选举和游说这样的非法律的实践活动，还包括像权力和战争这样的非法律现象。

然而，法律学者们所研究的课题，是政治学的研究者必须关注的一个非常丰富的子集。

因此，既然事实上法律制度作为政治制度的组成部分必须被研究政治制度的人所考虑，那么就没有理由不认为法律理论是——或者说应当被归为——一般政治理论中的实质组成部分。

法律是政治的一个方面，这种主题之间的关系似乎就规定了学科之间的关系——法律理论必然是政治理论的一个分支或者说是一个子集。

当我在法律、法律机构、法律理念和法律实践前面冠以“政治”的时候，我并没有挑衅的意思。

有时，当人们说“法律是政治的”这句话时，他们的意思是案件的判决是以法官在公共政治问题上的党派观点或法官对政治意识形态的忠诚为基础的。

换个稍微抽象一点的说法，他们的这种非难旨在试图逃避政治机构的力量，以确保有争议的案件是由立法机关或是合乎宪法的会议而不是由法官之类的官员做出最终裁决。

他们的意思也可能是，为了满足所有中立和客观的要求，法律原则和法律教义掩盖了实际存在的、有争议的政治承诺。

所有这些都不是我在开篇中所讲的意思(尽管这些说法想必可能是对的)。

法学类外文核心期刊这里总共列有31种期刊简介（主要包括英文和日文期刊），供参考。

刊名：Law and Society Review中图刊号：336B0152 ISSN：0023-9216 期数：4 出版状态：正常出版出版者：Law and Society Association, 出版地：美国译名简介：《法律与社会评论》刊载研究社会与法律的关系，包括法律、法律制度在社会、心理、政治、经济和文化等方面应用的文章、评论和札记。

刊名：American Journal of International Law.中图刊号：340B0001 ISSN：0002-9300 期数：4 出版状态：正常出版出版者：American Society of International Law, 出版地：美国译名简介：《美国国际法杂志》刊载有关国际法、国际间的条约和协定及案例裁决等方面的文章、评论、资料、书评和札记。

刊名：Harvard Law Review.中图刊号：340B0003 ISSN：0017-811X 期数：8 出版状态：正常出版出版者：Harvard Law Review Association, 出版地：美国译名简介：《哈佛法律评论》刊载法律研究论文、札记、案例分析和书刊名：Yale Law Journal.中图刊号：340B0004 ISSN：0044-0094 期数：8 出版状态：正常出版出版者：Yale Journal Co. Inc., 出版地：美国译名简介：《耶鲁法律杂志》刊载法学研究论文、评论和札记。

刊名：Business Lawyer.中图刊号：340B0008 ISSN：0007-6899 期数：4 出版状态：正常出版出版者：American Bar Association, 出版地：美国译名简介：《商业律师》刊载商业法和金融法，包括会计、仲裁、合同、雇佣、税务、专利、股票和投资等法律问题的论文、评论和文摘。

a b c d e f g h i j k l m n o p q r s t u v w x y z注：(1)r、v两个字母用来拼写普通话和外来语，拼写广州话时不用。

(2)广州话拼音字母有三个附加符号：ê、é、ü，其中é是和汉语拼音字母不同的。

这几个字母是e、u字母的变体，叫不列入表内。

二、声母表b波 p婆 m摸 f科 d多 t拖 n挪 l罗 g哥 k卡 ng我 h何 gu姑 ku箍 z左 c初 s梳j知 q雌 x思 y也 w华注：(1)z、c、s和j、q、x两组声母，广州话的读音没有区别，只是在拼写韵母时有不同，z、c、s拼写a、o、é及a、o、e、é、ê、u等字母开头的韵母，例如：za渣，ca茶，xa沙。

j、q、x拼写i、ü及i、ü字母开头的韵母，例如：ji知，qi次，xi思。

(2)gu姑、ku箍是圆唇的舌跟音，作为声母使用，不能单独注音，单独注音时是音节，不是声母。

(3)y也，w华拼音时作为声母使用，拼写出来的音节相当于汉语拼音方案的复韵母，但由于广州话当中这些韵母前面不再拼声母，因此只作为音节使用三、韵母表a呀 o柯 u乌 i衣ū于ê(靴) é诶 m唔 n五ai挨ei矮oi哀ui会éi(非)ao拗eo欧ou奥 iu妖êu(去)am(监) em庵 im淹an晏en(恩) on安un碗 in烟ūn冤 ên(春)ang(横) eng莺ong(康) ung瓮 ing英êng(香) éng(镜)ab鸭eb(急) ib叶ad押ed(不) od(渴) ud活id热ūd月 êd(律)ag(客) eg(德) og恶ug屋ig益êg(约) ég(尺)注：(1)例字外加( )号的，只取其韵母。

(2)i行的韵母，前面没有声母的时候，写成yi衣，yiu妖，yim淹，yin烟，ying 英，yib叶，yid热，yig益。

鲁东大学中文学术期刊分类目录
一、人文、社科类
二、理工科类
说明：
1．A类、B类期刊若不在《中国科学引文数据库（CSCD）》(最新版)核心库、《中文社会科学引文索引（CSSCI）》(最新版)来源期刊中，将自动降为C类期刊。

2．C类期刊指未在本目录A类、B类期刊中，被《中国科学引文数据库（CSCD）》(最新版，含核心库与扩展库）、《中文社会科学引文索引（CSSCI）》(最新版，含来源期刊、来源集刊与扩展版来源期刊）所收录的学术性期刊；中国社会科学（内参）、人民日报（内参）、光明日报（内参）、求是（内参）、《经济日报》、《中国教育报》理论版（3000字以上）；《山东社会科学界学术年会文集》。

3．D类期刊为最新北京大学版《中文核心期刊要目总览》期刊中除已列为A、B、C类以外的学术性期刊。

4．E类期刊为中华人民共和国新闻出版总署批准公开出版的除A、B、C、D类以外的学术性期刊。

注：《目录》自2011年1月1日起实施。

0301法学一级学科简介一级学科（中文）名称：法学（英文）名称： Science of Law一、学科概况法学是高等教育中最早的专业之一。

世界上早期的大学，如博洛尼亚、巴黎、牛津、剑桥等，均有法学专业。

随着法治理念的成熟和全球性推广，法学教育始终处于稳定发展的状态。

中国的法学教育历史悠久，源远流长。

早在2000多年前的春秋战国时期就有了私塾性质的法学教育，至汉唐时期已经相当发展。

不过，正规的、职业化的法学教育则出现于清末民初。

近代中国法学始于19世纪中叶，继受了诸多西方法律传统。

新中国成立后，中国法学经历了引进初创（1949-1957）、遭受挫折（1958-1966）、恢复重建（1978-1991）、改革发展（1992—）的发展历程。

改革开放以来，我国法学教育飞跃发展，形成了从大专、本科到法学硕士研究生、法律硕士专业学位研究生、法学博士研究生、博士后的完整的法学教育体系，以及包括法学学士、法律硕士、法学硕士、法学博士在内的多层次的高等法学教育学位制度体系；法学学科日臻完善，师资队伍不断壮大，培养了一大批高素质的法学法律人才，成为我国社会主义现代化建设的重要力量；法学研究繁荣发展，形成了一大批高质量的研究成果，在我国社会主义法治建设中发挥了重要作用；法学教学质量提高，法学专业课程设置日益系统化，培养方法不断改进，不同层级、类型的法学学位定位逐渐清晰、衔接日益合理，法学教学活动与科研、司法考试、法律实践、职业发展等联系更为紧密，案例教学、讨论式教学、诊所式教学和模拟法庭等多种教学方式得到逐步推广。

二、学科内涵法学是研究法、法的现象以及与法相关问题的专门学问，是关于法律问题的知识和理论体系，是社会科学的一门重要学科。

（1）法学的研究对象首先是法。

这里的“法”包括通常所说各种意义的法：从法的形式角度说，包括宪法、法律、法规以及其他各种形式的成文法和不成文法；从法的体系角度说，包括宪法、行政法、民商法、经济法、社会法、刑法等法律部门；从时间角度说，包括古代法、近代法、现代法和当代法；从空间角度说，包括本国法、外国法、本地法、外地法；从历史类型角度说，包括奴隶制法、封建制法、资本主义法、社会主义法；从一般分类角度说，包括国内法和国际法、根本法和普通法、一般法和特别法、实体法和程序法；从表现形态角度说，包括动态法和静态法、具体法和抽象法、纸面法和生活中的法、理想法（如自然法）和现实法（如实际生效的法）等等。

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 Applications ,M.Suffness,Ed.(CRC,Boca Raton,FL,1995),pp.3–26.4.K.C.Nicolaou et al .,Nature 367,630(1994).5.R.A.Holton et al .,J.Am.Chem.Soc.116,1597(1994).6.A.M.Walji,D.W.C.MacMillan,Synlett 18,1477(2007).7.R.A.Holton,R.J.Biediger,P.D.Boatman,in Taxol:Science and Applications ,M.Suffness,Ed.(CRC,Boca Raton,FL,1995),pp.97–119.8.D.Frense,Appl.Microbiol.Biotechnol.73,1233(2007).9.S.C.Roberts,Nat.Chem.Biol.3,387(2007).10.J.Goodman,V.Walsh,The Story of Taxol:Nature andPolitics in the Pursuit of an Anti-Cancer Drug .(Cambridge Univ.Press,Cambridge,2001).11.K.E.Tyo,H.S.Alper,G.N.Stephanopoulos,TrendsBiotechnol.25,132(2007).12.P.K.Ajikumar et al .,Mol.Pharm.5,167(2008).510152025T a x a d i e n -5α-o l p r o d u c t i o n (m g e q u i v a l e n t o f t a x a d i e n e /L )BC048121620020406020406080100C e l l g r o w t h (OD 600n m )T a x a d i e n e -5α-o l p r o d u c t i o n (m g e q u i v a l e n t o f t a x a d i e n e /L )Time (h)Fig.4.Engineering Taxol P450oxidation chemistry in E.coli .(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 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 m13.W.R.Farmer,J.C.Liao,Nat.Biotechnol.18,533(2000).14.H.Alper,K.Miyaoku,G.Stephanopoulos,Nat.Biotechnol.23,612(2005).15.M.C.Chang,J.D.Keasling,Nat.Chem.Biol.2,674(2006).16.V.J.Martin,D.J.Pitera,S.T.Withers,J.D.Newman,J.D.Keasling,Nat.Biotechnol.21,796(2003).17.D.Morrone et al .,Appl.Microbiol.Biotechnol.85,1893(2010).18.E.Leonard et al .,Proc.Natl.Acad.Sci.U.S.A.107,13654(2010).19.Q.Huang,C.A.Roessner,R.Croteau,A.I.Scott,Bioorg.Med.Chem.9,2237(2001).20.B.Engels,P.Dahm,S.Jennewein,Metab.Eng.10,201(2008).21.L.Z.Yuan,P.E.Rouvière,rossa,W.Suh,Metab.Eng.8,79(2006).22.Y.S.Jin,G.Stephanopoulos,Metab.Eng.9,337(2007).23.H.H.Wang et al .,Nature 460,894(2009).24.D.Klein-Marcuschamer,P.K.Ajikumar,G.Stephanopoulos,Trends Biotechnol.25,417(2007).25.J.M.Dejong et al .,Biotechnol.Bioeng.93,212(2006).26.Materials and methods are available as supportingmaterial on Science Online.27.K.L.Jones,S.W.Kim,J.D.Keasling,Metab.Eng.2,328(2000).28.R.Kaspera,R.Croteau,Phytochem.Rev.5,433(2006).29.S.Jennewein,R.M.Long,R.M.Williams,R.Croteau,Chem.Biol.11,379(2004).30.M.A.Schuler,D.Werck-Reichhart,Annu.Rev.Plant Biol.54,629(2003).31.E.Leonard,M.A.Koffas,Appl.Environ.Microbiol.73,7246(2007).32.D.R.Nelson,Arch.Biochem.Biophys.369,1(1999).33.S.Jennewein et al .,Biotechnol.Bioeng.89,588(2005).34.D.Rontein et al .,J.Biol.Chem.283,6067(2008).35.W.R.Farmer,J.C.Liao,Biotechnol.Prog.17,57(2001).36.S.Jennewein,M.R.Wildung,M.Chau,K.Walker,R.Croteau,Proc.Natl.Acad.Sci.U.S.A.101,9149(2004).37.K.Walker,R.Croteau,Phytochemistry 58,1(2001).38.We thank R.Renu for extraction,purification,andcharacterization of metabolite Indole;C.Santos for providing the pACYCmelA plasmid,constructivesuggestions during the experiments,and preparation of the manuscript;D.Dugar,H.Zhou,and X.Huang for helping with experiments and suggestions;and K.Hiller for data analysis and comments on the manuscript.We 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。