微生物生理学实验指导(圈起来的不做)

微生物生理学实验―――铜绿假单胞菌fabI基因的克隆及功能鉴定目录TOC\o"1-1"\n\p""\h\z\u实验〇铜绿假单胞菌fabI基因鉴定的原理实验一细菌总DNA的提取与检测实验二铜绿假单胞菌fabI基因PCR扩增实验三铜绿假单胞菌pafabI基因表达载体构建实验四质粒的提取与检测实验五大肠杆菌感受态细胞的制备及DNA转化实验六遗传互补大肠杆菌fabI温度敏感突变菌株实验七铜绿假单胞菌烯酯酰ACP还原酶的原核表达实验八铜绿假单胞菌烯酯酰ACP还原酶纯化实验九体外鉴定烯酯酰ACP还原酶的酶活性实验十构建铜绿假单胞菌fabI突变菌株实验十一菌落PCR筛选重组子实验十二铜绿假单胞菌脂肪酸合成分析实验报告

实验〇铜绿假单胞菌fabI基因鉴定的原理脂肪酸的生物合成是一种细胞生物体重要的基础代谢。生物体脂肪酸合成系统（FAS）为磷脂和类脂等生物质膜组分提供重要的前体物质ADDINEN.CITE<EndNote><Cite><Author>Zhang</Author><Year>2008</Year><RecNum>116</RecNum><record><rec-number>116</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Zhang,Y.M.</author><author>Rock,C.O.</author></authors></contributors><auth-address>DepartmentofInfectiousDiseases,St.JudeChildren'sResearchHospital,332N.Lauderdale,Memphis,Tennessee38105,USA.yongmei.zhang@charles.rock@</auth-address><titles><title>Membranelipidhomeostasisinbacteria</title><secondary-title>NatRevMicrobiol</secondary-title><alt-title>Naturereviews</alt-title></titles><pages>222-33</pages><volume>6</volume><number>3</number><keywords><keyword>AcylCarrierProtein/physiology</keyword><keyword>Bacteria/genetics/*metabolism</keyword><keyword>*BacterialPhysiologicalPhenomena</keyword><keyword>CellMembrane/metabolism</keyword><keyword>FattyAcidDesaturases/metabolism</keyword><keyword>FattyAcids/chemistry/metabolism</keyword><keyword>*Homeostasis</keyword><keyword>LipidBilayers/metabolism</keyword><keyword>*LipidMetabolism</keyword><keyword>Phospholipids/chemistry/metabolism</keyword></keywords><dates><year>2008</year><pub-dates><date>Mar</date></pub-dates></dates><isbn>1740-1534(Electronic)</isbn><accession-num>18264115</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18264115</url></related-urls></urls><language>eng</language></record></Cite></EndNote>，同时生物体内一些重要的化合物如硫辛酸、生物素以及细菌群体感应的信号分子等也都需要以脂肪酸合成代谢中的中间产物为前体物质合成。在不同生物体内，脂肪酸的生物合成过程的基本原理是相似的，但是催化反应的蛋白序列和结构则有着很大的差异。根据合成酶系统的这种差异，人们将脂肪酸生物合成系统分为I型脂肪酸合成酶系（FASI）和II型脂肪酸合成酶系（FASII）ADDINEN.CITE<EndNote><Cite><Author>Cronan</Author><Year>2006</Year><RecNum>219</RecNum><record><rec-number>219</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Cronan,J.E.</author></authors></contributors><auth-address>DepartmentofMicrobiology,UniversityofIllinois,Urbana,61801,USA.j-cronan@</auth-address><titles><title>Avantgardefattyacidsynthesisbytrypanosomes</title><secondary-title>Cell</secondary-title><alt-title>Cell</alt-title></titles><pages>641-3</pages><volume>126</volume><number>4</number><keywords><keyword>Animals</keyword><keyword>FattyAcids/*biosynthesis</keyword><keyword>Humans</keyword><keyword>MyristicAcid/*metabolism</keyword><keyword>ProtozoanProteins/genetics/metabolism</keyword><keyword>Trypanosomabruceibrucei/genetics/*metabolism/pathogenicity</keyword></keywords><dates><year>2006</year><pub-dates><date>Aug25</date></pub-dates></dates><isbn>0092-8674(Print)</isbn><accession-num>16923380</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16923380</url></related-urls></urls><language>eng</language></record></Cite></EndNote>(Cronan,2006)。I型脂肪酸合成酶系主要分布在哺乳动物和真菌等生物中，该种脂肪酸合成由一个（或两个）分子量较大的多功能酶蛋白催化。这种多功能酶蛋白有多个不同功能的结构域，脂肪酸延伸循环中的各个反应分别在酶蛋白的不同结构域进行ADDINEN.CITE<EndNote><Cite><Author>White</Author><Year>2005</Year><RecNum>2</RecNum><record><rec-number>2</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>White,S.W.</author><author>Zheng,J.</author><author>Zhang,Y.M.</author><author>Rock,</author></authors></contributors><auth-address>DepartmentofStructuralBiology,St.JudeChildren'sResearchHospital,Memphis,Tennessee38105,USA.stephen.white@</auth-address><titles><title>ThestructuralbiologyoftypeIIfattyacidbiosynthesis</title><secondary-title>AnnuRevBiochem</secondary-title></titles><pages>791-831</pages><volume>74</volume><keywords><keyword>3-Oxoacyl-(Acyl-Carrier-Protein)Synthase/chemistry/metabolism</keyword><keyword>Acetyl-CoACarboxylase/chemistry/metabolism</keyword><keyword>AcylCarrierProtein/chemistry/metabolism</keyword><keyword>Acyl-CarrierProteinS-Malonyltransferase</keyword><keyword>Acyltransferases/chemistry/metabolism</keyword><keyword>AlcoholOxidoreductases/chemistry/metabolism</keyword><keyword>CatalyticDomain</keyword><keyword>Enoyl-(Acyl-Carrier-Protein)Reductase(NADH)</keyword><keyword>Escherichiacoli/enzymology/genetics/*metabolism</keyword><keyword>EscherichiacoliProteins/chemistry/metabolism</keyword><keyword>FattyAcids/*biosynthesis/chemistry</keyword><keyword>Hydro-Lyases/chemistry/metabolism</keyword><keyword>Models,Biological</keyword><keyword>Models,Molecular</keyword><keyword>Oxidoreductases/chemistry/metabolism</keyword><keyword>Transferases(OtherSubstitutedPhosphateGroups)/chemistry/metabolism</keyword></keywords><dates><year>2005</year></dates><accession-num>15952903</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15952903</url></related-urls></urls></record></Cite><Cite><Author>Heath</Author><Year>2002</Year><RecNum>187</RecNum><record><rec-number>187</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Heath,R.J.</author><author>Rock,C.O.</author></authors></contributors><auth-address>DepartmentofInfectiousDiseases,StJudeChildren'sResearchHospital,Memphis,Tennessee38105,USA.</auth-address><titles><title>TheClaisencondensationinbiology</title><secondary-title>NatProdRep</secondary-title><alt-title>Naturalproductreports</alt-title></titles><pages>581-96</pages><volume>19</volume><number>5</number><keywords><keyword>Animals</keyword><keyword>BindingSites/genetics</keyword><keyword>Decarboxylation</keyword><keyword>EnzymeInhibitors/pharmacology</keyword><keyword>Enzymes/chemistry/genetics/*metabolism</keyword><keyword>FattyAcids/biosynthesis</keyword><keyword>Humans</keyword><keyword>Models,Molecular</keyword><keyword>Plants/enzymology</keyword></keywords><dates><year>2002</year><pub-dates><date>Oct</date></pub-dates></dates><isbn>0265-0568(Print)</isbn><accession-num>12430724</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12430724</url></related-urls></urls><language>eng</language></record></Cite></EndNote>(Heathetal,2002a;Whiteetal,2005)。II型脂肪酸合成酶系主要分布在细菌，植物和原生动物中，该类生物的脂肪酸合成由一组独立存在的酶催化完成，该系统中各个独立的酶蛋白分别行使一种功能ADDINEN.CITE<EndNote><Cite><Author>Cronan</Author><Year>2006</Year><RecNum>219</RecNum><record><rec-number>219</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Cronan,J.E.</author></authors></contributors><auth-address>DepartmentofMicrobiology,UniversityofIllinois,Urbana,61801,USA.j-cronan@</auth-address><titles><title>Avantgardefattyacidsynthesisbytrypanosomes</title><secondary-title>Cell</secondary-title><alt-title>Cell</alt-title></titles><pages>641-3</pages><volume>126</volume><number>4</number><keywords><keyword>Animals</keyword><keyword>FattyAcids/*biosynthesis</keyword><keyword>Humans</keyword><keyword>MyristicAcid/*metabolism</keyword><keyword>ProtozoanProteins/genetics/metabolism</keyword><keyword>Trypanosomabruceibrucei/genetics/*metabolism/pathogenicity</keyword></keywords><dates><year>2006</year><pub-dates><date>Aug25</date></pub-dates></dates><isbn>0092-8674(Print)</isbn><accession-num>16923380</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16923380</url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>White</Author><Year>2005</Year><RecNum>2</RecNum><record><rec-number>2</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>White,S.W.</author><author>Zheng,J.</author><author>Zhang,Y.M.</author><author>Rock,</author></authors></contributors><auth-address>DepartmentofStructuralBiology,St.JudeChildren'sResearchHospital,Memphis,Tennessee38105,USA.stephen.white@</auth-address><titles><title>ThestructuralbiologyoftypeIIfattyacidbiosynthesis</title><secondary-title>AnnuRevBiochem</secondary-title></titles><pages>791-831</pages><volume>74</volume><keywords><keyword>3-Oxoacyl-(Acyl-Carrier-Protein)Synthase/chemistry/metabolism</keyword><keyword>Acetyl-CoACarboxylase/chemistry/metabolism</keyword><keyword>AcylCarrierProtein/chemistry/metabolism</keyword><keyword>Acyl-CarrierProteinS-Malonyltransferase</keyword><keyword>Acyltransferases/chemistry/metabolism</keyword><keyword>AlcoholOxidoreductases/chemistry/metabolism</keyword><keyword>CatalyticDomain</keyword><keyword>Enoyl-(Acyl-Carrier-Protein)Reductase(NADH)</keyword><keyword>Escherichiacoli/enzymology/genetics/*metabolism</keyword><keyword>EscherichiacoliProteins/chemistry/metabolism</keyword><keyword>FattyAcids/*biosynthesis/chemistry</keyword><keyword>Hydro-Lyases/chemistry/metabolism</keyword><keyword>Models,Biological</keyword><keyword>Models,Molecular</keyword><keyword>Oxidoreductases/chemistry/metabolism</keyword><keyword>Transferases(OtherSubstitutedPhosphateGroups)/chemistry/metabolism</keyword></keywords><dates><year>2005</year></dates><accession-num>15952903</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15952903</url></related-urls></urls></record></Cite></EndNote>(Cronan,2006;Whiteetal,2005)。细菌脂肪酸的合成过程中，酰基链通过硫脂键连接在酰基载体蛋白（ACP）上。ACP是一种小分子量蛋白，一般由70-80个氨基酸残基组成，是FASII的核心成员之一。以大肠杆菌为例，说明脂肪酸合成的路径。1.脂肪酸合成的起始：脂肪酸合成的起始反应是以细菌体内的乙酰辅酶A（acyl-CoA）及CO2合成起初的四碳的短链脂肪酸的过程，该四碳的短链脂肪酸产物是酰基链延长反应的基础ADDINEN.CITE<EndNote><Cite><Author>White</Author><Year>2005</Year><RecNum>2</RecNum><record><rec-number>2</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>White,S.W.</author><author>Zheng,J.</author><author>Zhang,Y.M.</author><author>Rock,</author></authors></contributors><auth-address>DepartmentofStructuralBiology,St.JudeChildren'sResearchHospital,Memphis,Tennessee38105,USA.stephen.white@</auth-address><titles><title>ThestructuralbiologyoftypeIIfattyacidbiosynthesis</title><secondary-title>AnnuRevBiochem</secondary-title></titles><pages>791-831</pages><volume>74</volume><keywords><keyword>3-Oxoacyl-(Acyl-Carrier-Protein)Synthase/chemistry/metabolism</keyword><keyword>Acetyl-CoACarboxylase/chemistry/metabolism</keyword><keyword>AcylCarrierProtein/chemistry/metabolism</keyword><keyword>Acyl-CarrierProteinS-Malonyltransferase</keyword><keyword>Acyltransferases/chemistry/metabolism</keyword><keyword>AlcoholOxidoreductases/chemistry/metabolism</keyword><keyword>CatalyticDomain</keyword><keyword>Enoyl-(Acyl-Carrier-Protein)Reductase(NADH)</keyword><keyword>Escherichiacoli/enzymology/genetics/*metabolism</keyword><keyword>EscherichiacoliProteins/chemistry/metabolism</keyword><keyword>FattyAcids/*biosynthesis/chemistry</keyword><keyword>Hydro-Lyases/chemistry/metabolism</keyword><keyword>Models,Biological</keyword><keyword>Models,Molecular</keyword><keyword>Oxidoreductases/chemistry/metabolism</keyword><keyword>Transferases(OtherSubstitutedPhosphateGroups)/chemistry/metabolism</keyword></keywords><dates><year>2005</year></dates><accession-num>15952903</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15952903</url></related-urls></urls></record></Cite><Cite><Author>Revill</Author><Year>2001</Year><RecNum>238</RecNum><record><rec-number>238</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Revill,W.P.</author><author>Bibb,M.J.</author><author>Scheu,A.K.</author><author>Kieser,H.J.</author><author>Hopwood,D.A.</author></authors></contributors><auth-address>JohnInnesCentre,NorwichResearchPark,Colney,NorwichNR47UH,UnitedKingdom.revill@</auth-address><titles><title>Beta-ketoacylacylcarrierproteinsynthaseIII(FabH)isessentialforfattyacidbiosynthesisinStreptomycescoelicolorA3(2)</title><secondary-title>JBacteriol</secondary-title><alt-title>Journalofbacteriology</alt-title></titles><pages>3526-30</pages><volume>183</volume><number>11</number><keywords><keyword>3-Oxoacyl-(Acyl-Carrier-Protein)Synthase/*genetics/*metabolism</keyword><keyword>CultureMedia</keyword><keyword>FattyAcids/*biosynthesis/genetics</keyword><keyword>GeneDeletion</keyword><keyword>Genes,Bacterial</keyword><keyword>*Genes,Essential</keyword><keyword>MultigeneFamily</keyword><keyword>Streptomyces/*enzymology/genetics/growth&development</keyword></keywords><dates><year>2001</year><pub-dates><date>Jun</date></pub-dates></dates><isbn>0021-9193(Print)</isbn><accession-num>11344162</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11344162</url></related-urls></urls><language>eng</language></record></Cite></EndNote>(Revilletal,2001;Whiteetal,2005)。此过程通过羧化、转移、缩合三步来完成：第一步，羧化：乙酰CoA在乙酰辅酶A羧化酶（ACC）作用下转化为丙二酸单酰辅酶A（Mal-CoA）。ACC有4个独立的蛋白构成（AccA，AccB，AccC，AccD），其中AccB的行使功能需要生物素作为共价结合的辅因子ADDINEN.CITE<EndNote><Cite><Author>Cronan</Author><Year>2002</Year><RecNum>228</RecNum><record><rec-number>228</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Cronan,J.E.,Jr.</author><author>Waldrop,G.L.</author></authors></contributors><auth-address>DepartmentofMicrobiology,B103ChemicalandLifeSciencesLaboratory,UniversityofIllinois,601S.GoodwinAvenue,Urbana,IL61801,USA.j-cronan@</auth-address><titles><title>Multi-subunitacetyl-CoAcarboxylases</title><secondary-title>ProgLipidRes</secondary-title><alt-title>Progressinlipidresearch</alt-title></titles><pages>407-35</pages><volume>41</volume><number>5</number><keywords><keyword>Acetyl-CoACarboxylase/chemistry/genetics/*physiology</keyword><keyword>CarrierProteins/chemistry</keyword><keyword>Catalysis</keyword><keyword>Escherichiacoli/enzymology</keyword><keyword>Humans</keyword><keyword>Mutation</keyword><keyword>Plants/enzymology</keyword><keyword>Structure-ActivityRelationship</keyword></keywords><dates><year>2002</year><pub-dates><date>Sep</date></pub-dates></dates><isbn>0163-7827(Print)</isbn><accession-num>12121720</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12121720</url></related-urls></urls><language>eng</language></record></Cite></EndNote>(Cronanetal,2002)。第二步，转移：Mal-CoA在丙二酰转酰基酶（FabD）的作用下将丙二酸单酰基团转移至ACP上，形成Mal–ACPADDINEN.CITE<EndNote><Cite><Author>Jackowski</Author><Year>1987</Year><RecNum>275</RecNum><record><rec-number>275</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Jackowski,S.</author><author>Rock,C.O.</author></authors></contributors><titles><title>Acetoacetyl-acylcarrierproteinsynthase,apotentialregulatoroffattyacidbiosynthesisinbacteria</title><secondary-title>JBiolChem</secondary-title><alt-title>TheJournalofbiologicalchemistry</alt-title></titles><pages>7927-31</pages><volume>262</volume><number>16</number><keywords><keyword>Acyltransferases/isolation&purification/*metabolism</keyword><keyword>Escherichiacoli/*enzymology/genetics</keyword><keyword>FattyAcids/*biosynthesis</keyword><keyword>Homeostasis</keyword><keyword>Kinetics</keyword><keyword>Mutation</keyword></keywords><dates><year>1987</year><pub-dates><date>Jun5</date></pub-dates></dates><isbn>0021-9258(Print)</isbn><accession-num>3294837</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=3294837</url></related-urls></urls><language>eng</language></record></Cite></EndNote>(Jackowskietal,1987)。第三步，缩合：在β酮脂酰ACP合成酶III（KASIII，FabH）的作用下将乙酰CoA所携带的酰基链合缩合到Mal-ACP上，生成β酮丁酰ACP，起始新的酰基链的生成ADDINEN.CITE<EndNote><Cite><Author>Revill</Author><Year>2001</Year><RecNum>238</RecNum><record><rec-number>238</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Revill,W.P.</author><author>Bibb,M.J.</author><author>Scheu,A.K.</author><author>Kieser,H.J.</author><author>Hopwood,D.A.</author></authors></contributors><auth-address>JohnInnesCentre,NorwichResearchPark,Colney,NorwichNR47UH,UnitedKingdom.revill@</auth-address><titles><title>Beta-ketoacylacylcarrierproteinsynthaseIII(FabH)isessentialforfattyacidbiosynthesisinStreptomycescoelicolorA3(2)</title><secondary-title>JBacteriol</secondary-title><alt-title>Journalofbacteriology</alt-title></titles><pages>3526-30</pages><volume>183</volume><number>11</number><keywords><keyword>3-Oxoacyl-(Acyl-Carrier-Protein)Synthase/*genetics/*metabolism</keyword><keyword>CultureMedia</keyword><keyword>FattyAcids/*biosynthesis/genetics</keyword><keyword>GeneDeletion</keyword><keyword>Genes,Bacterial</keyword><keyword>*Genes,Essential</keyword><keyword>MultigeneFamily</keyword><keyword>Streptomyces/*enzymology/genetics/growth&development</keyword></keywords><dates><year>2001</year><pub-dates><date>Jun</date></pub-dates></dates><isbn>0021-9193(Print)</isbn><accession-num>11344162</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11344162</url></related-urls></urls><language>eng</language></record></Cite></EndNote>(Revilletal,2001)。图1大肠杆菌脂肪酸合成途径2.酰基链的延长：FASII中有四种关键酶行使碳链延长的功能，该延长过程是以循环的形式进行，每次循环都经过缩合、还原、脱水、再还原四个步骤，使脂肪酸碳链延长两个碳原子ADDINEN.CITE<EndNote><Cite><Author>Zhang</Author><Year>2008</Year><RecNum>116</RecNum><record><rec-number>116</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Zhang,Y.M.</author><author>Rock,C.O.</author></authors></contributors><auth-address>DepartmentofInfectiousDiseases,St.JudeChildren'sResearchHospital,332N.Lauderdale,Memphis,Tennessee38105,USA.yongmei.zhang@charles.rock@</auth-address><titles><title>Membranelipidhomeostasisinbacteria</title><secondary-title>NatRevMicrobiol</secondary-title><alt-title>Naturereviews</alt-title></titles><pages>222-33</pages><volume>6</volume><number>3</number><keywords><keyword>AcylCarrierProtein/physiology</keyword><keyword>Bacteria/genetics/*metabolism</keyword><keyword>*BacterialPhysiologicalPhenomena</keyword><keyword>CellMembrane/metabolism</keyword><keyword>FattyAcidDesaturases/metabolism</keyword><keyword>FattyAcids/chemistry/metabolism</keyword><keyword>*Homeostasis</keyword><keyword>LipidBilayers/metabolism</keyword><keyword>*LipidMetabolism</keyword><keyword>Phospholipids/chemistry/metabolism</keyword></keywords><dates><year>2008</year><pub-dates><date>Mar</date></pub-dates></dates><isbn>1740-1534(Electronic)</isbn><accession-num>18264115</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18264115</url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>White</Author><Year>2005</Year><RecNum>2</RecNum><record><rec-number>2</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>White,S.W.</author><author>Zheng,J.</author><author>Zhang,Y.M.</author><author>Rock,</author></authors></contributors><auth-address>DepartmentofStructuralBiology,St.JudeChildren'sResearchHospital,Memphis,Tennessee38105,USA.stephen.white@</auth-address><titles><title>ThestructuralbiologyoftypeIIfattyacidbiosynthesis</title><secondary-title>AnnuRevBiochem</secondary-title></titles><pages>791-831</pages><volume>74</volume><keywords><keyword>3-Oxoacyl-(Acyl-Carrier-Protein)Synthase/chemistry/metabolism</keyword><keyword>Acetyl-CoACarboxylase/chemistry/metabolism</keyword><keyword>AcylCarrierProtein/chemistry/metabolism</keyword><keyword>Acyl-CarrierProteinS-Malonyltransferase</keyword><keyword>Acyltransferases/chemistry/metabolism</keyword><keyword>AlcoholOxidoreductases/chemistry/metabolism</keyword><keyword>CatalyticDomain</keyword><keyword>Enoyl-(Acyl-Carrier-Protein)Reductase(NADH)</keyword><keyword>Escherichiacoli/enzymology/genetics/*metabolism</keyword><keyword>EscherichiacoliProteins/chemistry/metabolism</keyword><keyword>FattyAcids/*biosynthesis/chemistry</keyword><keyword>Hydro-Lyases/chemistry/metabolism</keyword><keyword>Models,Biological</keyword><keyword>Models,Molecular</keyword><keyword>Oxidoreductases/chemistry/metabolism</keyword><keyword>Transferases(OtherSubstitutedPhosphateGroups)/chemistry/metabolism</keyword></keywords><dates><year>2005</year></dates><accession-num>15952903</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15952903</url></related-urls></urls></record></Cite></EndNote>(Whiteetal,2005;Zhangetal,2008a)。第一步，缩合：碳链的延长是由β酮脂酰ACP合成酶（-ketoactyl-ACPsynthetase，KAS）来催化完成的。延伸反应中β酮脂酰ACP合成酶有KASI（FabB）和KASII（FabF）两种。每次缩合反应，碳链延长两个C原子，同时在酰基链的β位引入一个酮基ADDINEN.CITE<EndNote><Cite><Author>Bergler</Author><Year>1992</Year><RecNum>75</RecNum><record><rec-number>75</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Bergler,H.</author><author>Hogenauer,G.</author><author>Turnowsky,F.</author></authors></contributors><auth-address>InstitutfurMikrobiologie,Karl-Franzens-UniversitatGraz,Austria.</auth-address><titles><title>SequencesoftheenvMgeneandoftwomutatedallelesinEscherichiacoli</title><secondary-title>JGenMicrobiol</secondary-title><alt-title>Journalofgeneralmicrobiology</alt-title></titles><pages>2093-100</pages><volume>138</volume><number>10</number><keywords><keyword>AlcoholDehydrogenase/genetics</keyword><keyword>Alleles</keyword><keyword>AminoAcidSequence</keyword><keyword>AzaCompounds/pharmacology</keyword><keyword>BacterialProteins/*genetics</keyword><keyword>BaseSequence</keyword><keyword>BoronCompounds/pharmacology</keyword><keyword>ChromosomeMapping</keyword><keyword>Cloning,Molecular</keyword><keyword>DrugResistance,Microbial/genetics</keyword><keyword>Enoyl-(Acyl-Carrier-Protein)Reductase(NADH)</keyword><keyword>Escherichiacoli/drugeffects/*genetics</keyword><keyword>EscherichiacoliProteins</keyword><keyword>Genes,Bacterial/*genetics</keyword><keyword>MolecularSequenceData</keyword><keyword>Oxidoreductases/*genetics</keyword><keyword>SequenceHomology,AminoAcid</keyword><keyword>SequenceHomology,NucleicAcid</keyword></keywords><dates><year>1992</year><pub-dates><date>Oct</date></pub-dates></dates><isbn>0022-1287(Print)</isbn><accession-num>1364817</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1364817</url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>D'Agnolo</Author><Year>1975</Year><RecNum>258</RecNum><record><rec-number>258</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>D'Agnolo,G.</author><author>Rosenfeld,I.S.</author><author>Vagelos,P.R.</author></authors></contributors><titles><title>Beta-Ketoacyl-acylcarrierproteinsynthetase.Characterizationoftheacyl-enzymeintermediate</title><secondary-title>JBiolChem</secondary-title><alt-title>TheJournalofbiologicalchemistry</alt-title></titles><pages>5283-8</pages><volume>250</volume><number>14</number><keywords><keyword>3-Oxoacyl-(Acyl-Carrier-Protein)Synthase/*metabolism</keyword><keyword>AcetylCoenzymeA/metabolism</keyword><keyword>Acyltransferases/*metabolism</keyword><keyword>Alanine/metabolism</keyword><keyword>Caproates/metabolism</keyword><keyword>Caprylates/metabolism</keyword><keyword>CarbonRadioisotopes</keyword><keyword>Chromatography,Gel</keyword><keyword>Chromatography,ThinLayer</keyword><keyword>CircularDichroism</keyword><keyword>CoenzymeA/metabolism</keyword><keyword>Dithiothreitol/metabolism</keyword><keyword>Escherichiacoli/enzymology</keyword><keyword>FattyAcids/biosynthesis</keyword><keyword>Hydrogen-IonConcentration</keyword><keyword>HydroxamicAcids/metabolism</keyword><keyword>Malonates/pharmacology</keyword><keyword>Mercaptoethanol/metabolism</keyword><keyword>Tritium</keyword><keyword>Urea/pharmacology</keyword></keywords><dates><year>1975</year><pub-dates><date>Jul25</date></pub-dates></dates><isbn>0021-9258(Print)</isbn><accession-num>237913</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=237913</url></related-urls></urls><language>eng</language></record></Cite></EndNote>(Bergleretal,1992;D'Agnoloetal,1975)。第二步，还原：KAS在延长碳链的同时引入了β位酮基，该基团在β酮酯酰ACP还原酶（-ketoactyl-ACPreductase，KAR，FabG）的作用下加氢还原，转化为羟基集团，将β酮酯酰ACP转化成为β羟酯酰ACP，同时将还原剂NADPH氧化ADDINEN.CITE<EndNote><Cite><Author>Price</Author><Year>2004</Year><RecNum>142</RecNum><record><rec-number>142</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Price,A.C.</author><author>Zhang,Y.M.</author><author>Rock,C.O.</author><author>White,S.W.</author></authors></contributors><auth-address>DepartmentofStructuralBiology,StJudeChildren'sResearchHospital,Memphis,TN38105USA.</auth-address><titles><title>Cofactor-inducedconformationalrearrangementsestablishacatalyticallycompetentactivesiteandaprotonrelayconduitinFabG</title><secondary-title>Structure</secondary-title></titles><pages>417-28</pages><volume>12</volume><number>3</number><keywords><keyword>AlcoholOxidoreductases/chemistry/*metabolism</keyword><keyword>BindingSites</keyword><keyword>Calcium/metabolism</keyword><keyword>*CatalyticDomain</keyword><keyword>Crystallography,X-Ray</keyword><keyword>Escherichiacoli/enzymology</keyword><keyword>NADP/*metabolism</keyword><keyword>ProteinBinding</keyword><keyword>ProteinConformation</keyword><keyword>*Protons</keyword></keywords><dates><year>2004</year><pub-dates><date>Mar</date></pub-dates></dates><isbn>0969-2126(Print)</isbn><accession-num>15016358</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15016358</url></related-urls></urls><language>eng</language></record></Cite><Cite><Author>Price</Author><Year>2001</Year><RecNum>155</RecNum><record><rec-number>155</rec-number><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Price,A.C.</author><author>Zhang,Y.M.</author><author>Rock,C.O.</author><author>White,S.W.</author></authors></contributors><auth-address>DepartmentofStructuralBiology,St.JudeChildren'sResearchHospital,Memphis,Tennessee381