Xue_2009_Biological Control

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2009-伏马菌素的研究进展

伏马菌素的研究进展刘书宇,杨美华3　(中国医学科学院药用植物研究所,北京100094)摘要　综述了伏马菌素毒性、测定方法及限量的研究现状。

关键词　伏马菌素;毒性;限量中图分类号　S182 文献标识码　A 文章编号　0517-6611(2009)24-11397-03Research Progress on Fum on isi nsL I U Shu 2yu et a l (Institute of Medicinal Plant,Chinese Academy of Medical Sciences,Beijing 100094)Abstract Summarized the research status on t oxicity,detective method and li m es of fumonisins .Key words Fumonisins;Toxicity;L i mes基金项目　中国医学科学院药用植物研究所中央级公益性科研院所基本科研业务专项(YZ 21220);科技部重大新药创制专项(2009ZX095022025);国家中医药管理局2008年度中医药行业科研专项(200807042)。

作者简介　刘书宇(1986-),女,重庆人,硕士研究生,研究方向:中药材质量分析。

3通讯作者。

收稿日期　2009204223 伏马菌素(Fum onisin )是一组主要由串珠镰刀菌(Fusari 2um verticillioides )产生的水溶性代谢物。

目前发现的伏马菌素有F A 1、F A 2、F B 1、F B 2、F B 3、F B 4、FC 1、FC 2、FC 3、FC 4、FP 等共11种[1]。

1993年,美国食品药品监督管理局(US Food and D rug Ad m inistrati on,USF DA )根据美国兽医实验室诊断协会(American A ss ociati on of Veterinary Laborat ory D iagnosis,AAV 2LD )真菌毒素专业委员会制定的伏马菌素非正式指导水平进行动物饲料检测,至此对串珠镰刀菌伏马菌素产毒株的研究深入到分子遗传学领域。

莲草直胸跳甲短神经肽F受体基因sNPFR的克隆及表达分析

山西农业科学 2024，52（1）：137-144Journal of Shanxi Agricultural Sciences 莲草直胸跳甲短神经肽F受体基因sNPFR的克隆及表达分析王康，赵雪莹，霍楠，胡军，王苑馨，杨军，贾栋（山西农业大学植物保护学院，山西太谷 030801）摘要：为明确莲草直胸跳甲（Agasicles hygrophila）短神经肽F受体AhsNPFR功能及其表达特点，为探索莲草直胸跳甲生防作用奠定理论基础，利用PCR技术克隆鉴定莲草直胸跳甲AhsNPFR基因并进行生物信息学分析，通过实时荧光定量PCR技术分析其在莲草直胸跳甲不同发育时期和组织中的时空表达谱。

结果表明，克隆获得基因AhsNPFR全长1 669 bp，开放阅读框1 257 bp，编码418个氨基酸；预测其蛋白质分子质量为48.11 ku，理论等电点为8.21，AhsNPFR具有7个典型保守跨膜结构域，属于GPCRs家族，系统进化树分析表明，其与玉米根萤叶甲Diabrotica virgifera virgifera sNPFR亲缘关系最近。

AhsNPFR基因在不同发育阶段均有表达，在1龄幼虫中的表达量最高，是卵中表达量的9.06倍；在卵中表达量最低；雌成虫的表达量显著高于雄成虫。

AhsN⁃PFR基因在不同组织中均有表达，在3龄幼虫后肠中显著高表达，是脂肪体表达量的12.21倍；在雌雄成虫后肠显著高表达，并在所有组织中均没有雌雄表达差异。

关键词：莲草直胸跳甲；短神经肽F受体基因sNPFR；基因克隆；发育时期表达；组织表达中图分类号：S476.2　文献标识码：A 文章编号：1002‒2481（2024）01‒0137‒08Cloning and Expression Profiling of Short Neuropeptide F ReceptorGene sNPFR in Agasicles hygrophilaWANG Kang，ZHAO Xueying，HUO Nan，HU Jun，WANG Yuanxin，YANG Jun，JIA Dong （College of Plant Protection，Shanxi Agricultural University，Taigu 030801，China）Abstract：To explore the function and expression characteristics of the short neuropeptide receptor AhsNPFR in Agasicles hygrophila, and to lay a theoretical foundation for exploring the biological control role of Agasicles hygrophila, in this study, the AhsNPFR gene was cloned using PCR technology and analyzed with bioinformatics software. The spatiotemporal expression profiles of AhsNPFR across different developmental stages and tissues were investigated by quantitative real-time PCR(qPCR). The results showed that the complete sequence of 1 669 bp was obtained, which was found to contain a 1 257 bp open reading frame(ORF), encoding 418 amino acids. The predicted molecular weight was 48.11 ku and the theoretical isoelectric point(pI) was 8.21. AhsNPFR had 7 typical conserved transmembrane domains and belonged to the GPCRs family. Phylogenetic tree analysis showed that AhsNPFR was closely related to Diabrotica virgifera virgifera sNPFR. AhsNPFR gene was expressed throughout the developmental stages tested with the highest expression levels at the first instar larva which was 9.06 times higher than those eggs, the stage with the lowest expression level. The expression level in female adult was significantly higher than that in male adult. The AhsNPFR gene was expressed in different tissues and was significantly highly expressed in the hindgut of the 3rd instar larvae, which was 12.21 times higher than those fat body. The AhsNPFR gene was significantly highly expressed in the hindgut of male and female adults, with no difference expression between the sexes across all tissues.Key words：Agasicles hygrophila; short neuropeptide F receptor gene sNPFR; gene cloning; developmental stage expres⁃sion; tissue expression神经肽是一类重要的信号分子，在昆虫的生长发育、繁殖和行为等多种生物学过程中发挥重要调节作用[1]。

2009年诺贝尔化学奖成果简介

2009年诺贝尔化学奖成果简介摘要:主要介绍了2009年诺贝尔化学奖得主文卡特拉曼•拉马克里希南、托马斯•施泰茨和阿达•约纳特在有关核糖体结构和功能领域的研究成果,并阐述其现实意义和发展前景。

关键词核糖体晶体结构抗生素生理功能蛋白质瑞典皇家科学院2009年10月7日宣布,将本年度诺贝尔化学奖授予美国科学家文卡特拉曼•拉马克里希南(Venkatraman Ramakrishnan)、美国科学家托马斯•施泰茨(Thomas A. Steitz)和以色列女科学家阿达•约纳特(Ada E. Yonath),以表彰他们在核糖体结构和功能研究领域作出的突出贡献。

他们以较高的分辨率确定了核糖体的结构以及它在原子水平上的功能机理,并通过建立3D模型展示不同抗生素与核糖体的结合。

本文主要介绍该项研究成果,并阐述其现实意义和发展前景。

1 核糖体简介蛋白质生物合成是把储存在DNA分子上的遗传信息“翻译”成有各种生物功能蛋白质的复杂过程。

所有有机体中,DNA的转录都是在RNA聚合酶的作用下传递给mRNA,而mRNA的翻译过程则需要在核糖体这个平台的作用下进行【1】。

1.1 核糖体的组成细菌(70S)核糖体包含了一大一小2个亚基(30S,50S),S表示超离心沉降系数。

30S亚基由大约20个不同的蛋白质与16S rRNA(含有1600个核苷酸)组成;50S 大亚基由大约33个不同的蛋白质、23S rRNA(含有2900个核苷酸)和5S rRNA(含有120个核苷酸)组成。

尽管真核生物的核糖体比原核生物的更大更复杂,但核糖体的总体结构却相似【2】。

对于tRNA,核糖体有3个结合位点:A位点、P位点和E位点(见图1)。

而mRNA定位于30S亚基颈部的通道上,在新生肽链的延伸过程中它以梯状排列的方式穿过通道。

水稻稻米品质的分子设计育种研究进展

分子植物育种(网络版), 2011年, 第9卷, 第1134-1151页 Fenzi Zhiwu Yuzhong (Online), 2011, Vol.9, 1134-1151 http://mpb. 5th 评述与展望Reviews and Progress水稻稻米品质的分子设计育种研究进展易俊良1,2,3, 周少川1,2, 唐晓艳3, 周向阳4, 彭琼5, 陈立云1, 王海斌61.湖南农业大学水稻科学研究所, 长沙, 4101282.广东省农业科学院水稻科学研究所, 广州, 5106403.深圳热带亚热带作物分子设计育种研究中心, 深圳, 5180404.深圳市农作物良种引进中心, 深圳, 5180405.湖南农业大学生物技术学院, 长沙, 4101286.衡阳市现代农业示范园,衡阳, 421200 通讯作者: xxs123@ 作者分子植物育种, 2011年, 第9卷, 第19篇 doi: 10.5376/.2011.09.0019收稿日期：2010年11月02日接受日期：2011年02月11日发表日期：2011年02月23日这是一篇采用Creative Commons Attribution License 进行授权的开放取阅论文。

引用格式：易俊良等, 2011, 水稻稻米品质的分子设计育种研究进展, 分子植物育种 V ol.9 No.19 (doi: 10.5376/.2011.09.0019)摘要随着水稻基因组测序及其分析技术和功能基因组学研究的深入发展，大量与稻米品质有关的基因或QTL 在染色体上精确定位并克隆。

新一代分子标记技术的发展，高通量、高效、简便的检测技术平台的建立，为水稻在品质的分子设计育种奠定了基础。

本文从水稻品质遗传的分子生物学基础、品质分子设计育种相关基础研究，影响未来品质分子设计育种的限制因子等几个方面进行了概述，提出高通量、高效、简便的检测技术平台的建立与完善，是未来品质分子设计育种的主要研究方向。

四川大学2009年博士研究生入学考试《生物学综合》试题

四川大学 2009年博士研究生入学考试《生物学综合》试题
1、试述植物激素的种类及其主要的生理功能。

2、植物是怎样适应逆境条件的？
3、微生物的发展方向及其在现代农业中的作用。

4、比较基因组学在微生物领域中的作用。

5、你认为人类基因组计划的意义是什么？今近年基因组研究有哪些重要进展？
6、遗传学有哪些重要的分支学科？为什么说遗传学即使生命科学的基础学科，又是生命科学的领头羊？
7、阐述真核细胞基因表达调控的基本环节、主要调控分子和调控方式。

8、蛋白质的结构层次、维持的因素及其测定方法。

9、请从不同层析不同角度阐述你所了解的组学概念，包括基因组学、蛋白质组学、作用组学和代谢组学。

它们通常采用何种技术进行研究，分别用于解决何种科学问题？10、一个物种的全基因组测序后，你应该怎么使用生物信息学方法对其进行研究？请从基因的功能研究、进行等角度进行阐述。

11、外来物种怎样成为入侵物种的？
12、请你就实验室工作的一个方向谈谈生物安全问题和措施。

13、生命科学的发展过程中有哪些重要的历史事件，请
用 3 个例子来说明它们对生命科学的发展所起到的重要作用。

14、为什么核糖核酸（RNA）已经开始成为现代生物学研究的热点领域？试从核糖核酸的生物学功能来分析它的科学意义及其重要性。

西北大学考研真题生化演示教学

A.3+2+1B.4+5C.2+1+3D.2+3+1E.1+2+3
4、上述9肽中含有的碱性氨基酸是：________。
A.组氨酸，精氨酸B.精氨酸，赖氨酸C.谷氨酸，赖氨酸D.丝氨酸，酪氨酸
5、下列哪组的物质与颜色反应的组合是错误的？______。
A. Arg-坂口反应B.多肽-双缩脲反应C.氨基酸-茚三酮反应D. Phe-偶氮反应
2、Diabetes, the inability to assimilate and metabolize blood glucose, afflicts millions of people. People with type 1 diabetes are unable to synthesize and secrete insulin. On the other hand, people with type 2 diabetes make sufficient insulin, but the molecular pathways that respond to insulin are defective. Many type 2 diabetic people exhibit a condition termed “insulin resistance”before the onset of diabetes. Gluconeogenesis inhibitors may be the next wave in type 2 diabetes therapy. Drugs targeting one of the enzyme unique to gluconeogenesis can blocks this pathway without affecting glycolysis.

英夫利昔治疗克罗恩病进展

疗效…。另一对照实验显示单次使用英夫利昔４周
后患者不仅临床症状得到改善，内镜检查也证实获得明显的改善¨一。１．２瘘管型克罗恩病的治疗目的：主要是减少活动期瘘管引流，促进瘘管完全或部分闭合。适应证：活动期肠皮肤瘘或肛瘘经抗生素单独
（３６％）瘘管完全闭合，而对照组中，９８例中仅有１９
例（１９％）完全闭合（Ｐ＝０．００９）。该研究表明英夫利昔维持疗法可有效预防新的活动期瘘的发生¨１。以肠道克罗恩病患者为研究对象的两个安慰剂一对照研究显示英夫利昔联合免疫抑制剂（硫唑嘌呤、６一巯基嘌呤、甲氨喋呤）维持治疗疗效最
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收稿日期：２００８—１２—１４；修回日期：２００９—０２—１０作者简介：耿学斯（１９５９一）男，湖北人，教授。
疗效：一个以１０８例传统疗法无效的克罗恩病患者为研究对象的单盲一安慰剂对照试验证实了英夫利昔单次给药诱导缓解的临床疗效…。该研究
万方数据
大连医科大学学报中２７例患者以推荐剂量（５ｍｇ／ｋｇ）治疗。剂量更高时（１０—２０ｍｇ／ｋｇ）疗效反而不明显。研究过程中发现药物在两周之内开始起效，疗效最显著时间在治疗４周后，１２周之后，将近一半患者仍可观察到
的症状为头痛、恶心、胸痛、头晕、荨麻疹、其他类型
皮疹或瘙痒以及呼吸短促；急性荨麻疹、低血压和支气管痉挛发生率远低于其他症状；过敏性休克、喉头水肿、喘鸣等严重的不良反应罕见【ｌ４｜。单次治疗后

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抗菌肽ｂｕｆｏｒｉｎＩＩ的研究进展冯泽猛等・６５ｌ・２配ＲＧＫ掣茹砥琢Ａ嚣Ａ蓄ｒＲｓｓＲＡＧＬ口ＦＰＶＧ鼠Ⅵ强ＬＬＲｚ＠Ｈ…一３９３～——～・－—・—・—－—・—－－・－ＴＲＳＳＲＡＧＬＱＦＰＶＧＲ’，＂ＭＲＬＬＲＫ－－——－－－—－—・－－２一’ｌＳＧＲＧ篮ＱＧＧＫＴＲＡＫＡ盯ＲｓｓＲＡＧ碥ＦＰＶＧＲ促ＲＬＬ赋伽ＡＥ虱ｖ４３●＿●●－－●－－－★●－＿＿●ｔ●自●－＾Ｉ：ｘｅｎｏｐｕｓ组蛋白Ｈ，Ａ的Ｎ末端；２：ｂｕｆｏｒｉｎＩ，３：ｂｕｆｏｒｉｎＩＩ图１组蛋白Ｈ２ＡＮ末端、ｂｕｆｏｒｉｎ１和ｂｕｆｏｒｉｎ１１氨基酸序列比较，序列来源参考文献“。

溶液中，Ｖａｌｌ２至Ａｒ９２０肽段呈现常规仅螺旋结构，Ｇｌｙ７至Ｐｒｏ¨呈现扭曲的ａ螺旋结构。

在Ａｒ９５至Ｌｙｓ２１的Ｃ末端显示出两亲性特征。

与其他阳离子抗菌肽相似，两亲性结构可能是ｂｕｆｏｒｉｎＩＩ拥有抗菌活性的主要因素。

ＢｕｆｏｒｉｎＩＩ由中等疏水性和两亲结构所构成的Ｎ末端卷曲区域（残基１－４）、扭曲螺旋区域（残基５．１０）、铰链（残基１１）和Ｃ端常规螺旋区域（残基１２－２１）构成，具有一个螺旋铰链螺旋结构，两螺旋由位于１１位的脯氨酸残基隔开，具体如图２所示。

多肽的Ｃ端区域即便在两亲性环境中也有形成仅螺旋的趋势，而Ｎ端另外区域则困环境的不同具有不同的结构。

Ｃ端区域是组蛋白Ｈ：ＡＤＮＡ结合基序的一部分∞Ｊ，在与ＤＮＡ的相互作用中有着重要意义。

图２丝带模型代表ｂｕｆｏｒｉｎＩＩ在５０％ＴＦＥ溶液中的骨架结构。

６］ＢｕｆｏｒｉｎＩＩ的结构决定了它的抗菌活性。

ＢｕｆｏｒｉｎＩＩＮ端无规卷曲区域（残基１至４）的删除能使其抗细菌活性增加至２倍左右，但不影响其抗真菌活性。

Ｎ端残基的进一步删除，残基６至２１，７至２ｌ，８至２１，９至２ｌ，１０至２１，１１至２１使ｂｕｆｏｒｉｎＩＩ的抗菌和抗真菌活性逐步降低，而从ｂｕｆｏｒｉｎＩＩＣ端去除四氨基酸将导致其抗菌活性的完全丧失。

2009 plant physilolgy

BioinformaticsTriFLDB:ADatabaseofClusteredFull-LengthCodingSequencesfromTriticeaewithApplicationstoComparativeGrassGenomics[C][W][OA]

KeiichiMochida,TakuhiroYoshida,TetsuyaSakurai,YasunariOgihara,andKazuoShinozaki*PlantScienceCenter,RIKEN,Yokohama230–0045,Japan(K.M.,T.Y.,T.S.,K.S.);andKiharaInstituteforBiologicalResearch,YokohamaCityUniversity,Yokohama710–0046,Japan(Y.O.)

TheTriticeaeFull-LengthCDSDatabase(TriFLDB)containsavailableinformationregardingfull-lengthcodingsequences(CDSs)oftheTriticeaecropswheat(Triticumaestivum)andbarley(Hordeumvulgare)andincludesfunctionalannotationsandcomparativegenomicsfeatures.TriFLDBprovidesasearchinterfaceusingkeywordsforgenefunctionandrelatedGeneOntologytermsandasimilaritysearchforDNAanddeducedtranslatedaminoacidsequencestoaccessannotationsofTriticeaefull-lengthCDS(TriFLCDS)entries.AnnotationsconsistofsimilaritysearchresultsagainstseveralsequencedatabasesanddomainstructurepredictionsbyInterProScan.ThededucedaminoacidsequencesinTriFLDBaregroupedwiththeproteomedatasetsforArabidopsis(Arabidopsisthaliana),rice(Oryzasativa),andsorghum(Sorghumbicolor)byhierarchicalclusteringinstepwisethresholdsofsequenceidentity,providinghierarchicalclusteringresultsbasedonfull-lengthproteinsequences.ThedatabasealsoprovidessequencesimilarityresultsbasedoncomparativemappingofTriFLCDSsontothericeandsorghumgenomesequences,whichtogetherwithcurrentannotationscanbeusedtopredictgenestructuresforTriFLCDSentries.Toprovidethepossiblegeneticlocationsoffull-lengthCDSs,TriFLCDSentriesarealsoassignedtothegeneticallymappedcDNAsequencesofbarleyanddiploidwheat,whicharecurrentlyaccommodatedintheTriticeaeMappedESTDatabase.Theserelationaldataaresearchablefromthesearchinterfacesofbothdatabases.ThecurrentTriFLDBcontains15,871full-lengthCDSsfrombarleyandwheatandincludesputativefull-lengthcDNAsforbarleyandwheat,whicharepubliclyaccessible.ThisinformativecontentprovidesaninformaticsgatewayforTriticeaegenomicsandgrasscomparativegenomics.TriFLDBispubliclyavailableathttp://TriFLDB.psc.riken.jp/.

Tatsuno-2009-Extensive mutational

Extensive Mutational Analysis of Modular-IterativeMixed Polyketide Biosynthesis of Lankacidin in Streptomyces rocheiSatoshi T ATSUNO ,Kenji A RAKAWA ,and Haruyasu K INASHI yDepartment of Molecular Biotechnology,Graduate School of Advanced Sciences of Matter,Hiroshima University,1-3-1Kagamiyama,Higashi-Hiroshima,Hiroshima 739-8530,JapanReceived August 7,2009;Accepted September 9,2009;Online Publication,December 7,2009[doi:10.1271/bbb.90591]Extensive mutations of lankacidin synthase genes were carried out to analyze the modular-iterative mixed polyketide biosynthesis of lankacidin.Three ketoreduc-tase domains (lkcC -KR ,lkcF -KR1,and lkcF -KR2)were inactivated by in-frame deletion and site-directed muta-genesis of their active sites.The mutants ceased or diminished lankacidin production,indicating that the three KR domains are functional in lankacidin biosyn-thesis.However,all of the KR mutants failed to accumulate the expected unreduced metabolites.Muta-tional analysis of two tandemly aligned acyl carrier protein domains (lkcC -ACP1and lkcC -ACP2)revealed that either ACP is suﬃcient for lankacidin production.Disruption and complementation experiments on three unique genes/domain (lkcD for acyltransferase,lkcB for dehydratase,and lkcC-MT for a C-methyltransferase domain)suggested that their gene products function iteratively during lankacidin biosynthesis.Key words:antibiotic;polyketide synthase;Streptomy-ces rochei ;biosynthesisLankacidin (Fig.1),1)a unique 17-membered carbo-cyclic antibiotic,is produced by Streptomyces rochei strain 7434AN4,which carries three linear plasmids,pSLA2-L,-M,and -S.2)The complete nucleotide sequencing of the largest plasmid pSLA2-L (210,614bp)together with gene disruption experiments have revealed that the lankacidin synthase (lkc )gene cluster (lkcA -lkcO )is located on pSLA2-L.3,4)Lankacidin belongs to a group of reduced polyketides including macrolides and polyene macrolides,which are usually biosynthesized by modular type-I polyketide synthases (PKSs).Typical modular PKSs contain sev-eral ketosynthase (KS)and ketoreductase (KR)domains,whose numbers exactly coincide with the numbers of chain extension and reduction reactions;that is,a colinear relationship is observed between domains and reactions.5–8)However,the lkc cluster contains ﬁve KS domains although eight condensation reactions are necessary for the synthesis of the lankacidin skeleton.Hence,it was speculated that one (or some)of the ﬁve lkc -PKS modules may function iteratively instead of modularly.To analyze the modular-iterative mixed biosynthesis of lankacidin,we have carried out heterologous ex-pression and gene fusion experiments.9)The introduc-tion of the whole lkc cluster into Streptomyces lividans resulted in the production of lankacidinol A (Fig.1).Thus the lkcA -lkcO genes are suﬃcient for synthesis of the lankacidin skeleton,but not for the ﬁnal steps,including the oxidation of the lactate moiety and the hydrolysis of the C-7acetoxy group.These ﬁnal steps might be done by enzymes coded on the S.rochei chromosome.On the other hand,the gene fusant of two PKS genes,lkcF and lkcG ,produced lankacidin at a comparable level to the parent strain 51252.This result suggested a modular function of the LkcF and LkcG proteins,because gene fusion of two modular PKSs did not aﬀect antibiotic production,10)while that including an iterative PKS greatly reduced it.11)All of these results led us to propose a hypothesis,that LkcC is used 4times and the remaining three PKS proteins (LkcA,LkcF,and LkcG)are used modularly to accomplish eight condensation cycles in lankacidin biosynthesis (Fig.1).This hypothesis exactly agrees with the chemical structure of lankacidin.The nonribosomal peptide synthetase (NRPS)/PKS hybrid protein LkcA recognizes glycine as a starter and condenses it with malonyl coenzyme A (CoA).Then LkcC extends the polyketide chain 4times,from C-14to C-7.Finally,two modules in LkcF and one in LkcG catalyze three rounds of chain extension and modiﬁcation reactions to create the lankacidin skeleton.Three KR domains (lkcC-KR ,lkcF-KR1,and lkcF-KR2)are arranged in the cluster to catalyze seven rounds of reduction of -keto groups during polyketide assembly.Similar iterative use of modular PKSs has been proposed for stigmatellin biosynthesis in Stigmatella aurantiaca ,12)borrelidin in Streptomyces parvulus ,11)aureothin in Streptomyces thioluteus ,13)and neocarzilin in Streptomyces carzinostaticus .14)It was suggested that either StiI or StiJ is used twice for stigmatellin biosyn-thesis,BorA53times for borrelidin,AurA twice for aureothin,and Orf6twice for neocarzilin,but no metabolites conﬁrming their iterative use have been isolated.To isolate key metabolites supporting the modular-iterative polyketide biosynthesis of lankacidin,we carried out extensive mutations of the lkc -PKS genes in this study.The target genes for mutations were threeyTo whom correspondence should be addressed.Tel/Fax:+81-82-424-7869;E-mail:kinashi@hiroshima-u.ac.jpAbbreviations :PKS,polyketide synthase;KS,ketosynthase;KR,ketoreductase;CoA,coenzyme A;ACP,acyl carrier protein;AT,acyltransferase;DH,dehydratase;MT,C-methyltransferase;nt,nucleotide;SDR,short-chaindehydrogenase/reductaseBiosci.Biotechnol.Biochem.,73(12),2712–2719,2009KR domains (lkcC-KR ,lkcF-KR1,and lkcF-KR2)and two tandemly aligned acyl carrier protein (ACP)domains (lkcC-ACP1and lkcC-ACP2).In addition,three unique genes/domain in the cluster,lkcD for acyltransferase (AT),lkcB for dehydratase (DH),and lkcC-MT for a C-methyltransferase (MT)domain,were mutated to analyze their iterative function.Materials and MethodsBacterial strains and culture conditions.All the strains used in this study are listed in Table 1.Designed oligonucleotides are listed in Table 2.The plasmids constructed or used in this study are listed in Supplemental Table S1,and the construction schemes of gene disruptants are shown in Supplemental Figs.S1–S15(see Biosci.Biotechnol.Biochem.Web site).Site-directed mutations were intro-duced by the Altered Sites II in vitro Mutagenesis System (Promega,Madison,WI).S.rochei strain 51252,which carries only pSLA2-L,2)was used as the parent strain.DNA manipulations for E.coli 15)and Streptomyces 16)were performed according to the standard protocols.YEME liquid medium 16)was used in the preparation of protoplasts.Protoplasts were regenerated on R1M solid medium.17)Thiostrepton (10m g/ml)was used in the selection of plasmid-integrated Strepto-myces strains.E.coli strain XL1-Blue was used in routine cloning and construction of targeting plasmids.Ampicillin (100m g/ml),apramycinTable 1.S.rochei Strains Used in This StudyStrains DescriptionSource/ref.7434AN4Wild type (pSLA2-L,M,S)Ref.251252pSLA2-LRef.2FS16141-bp Stu I-Nru I fragment eliminated from lkcC-KRThis study KAFS13-1387-bp Ava I fragment eliminated from lkcF-KR1Ref.4FS13-2456-bp Hin cII fragment eliminated from lkcF-KR2This study STKR04lkcC-KR S362A This study KA14lkcF-KR1S330A This study STKR01lkcF-KR2S1523A This study KA35lkcF-KR1Y343F This study STKR02lkcF-KR2Y1536F This study STACP01lkcC-ACP1S851A This study STACP02lkcC-ACP2S956AThis study STACP03lkcC-ACP1S851A .lkcC-ACP2S956A This study STACP04315-bp Bst EII fragment eliminated from lkcC-ACP2ThisstudySTAT010.52-kb Bsi WI-Age I fragment eliminated from lkcD This study STDH01lkcB ::kanThis study KA03135-bp Bgl II-Bam HI fragment eliminated from lkcC-MTThis studyTable 2.Oligonucleotides Used in This StudyOligonucleotides Sequences (50–30)KAR-1301S01TGCACTTCAGCGCTCTGACGAGCGTCA KAR-1301Y01GCCAGGCGGCCTTCGCTGCAGCCAACG Ampicillin Repair a GTTGCCATTGCTGCAGGCATCGTGGTG ST1000AAGAATTCATTGGGCGCGGTGTAGCCGTTC ST1001GATCGATGCATTGATCTCGATGGAGTST1002AGATCAATGCATCGATCCCCATGTCACGGA ST1003ATTCTAGACTCACCTACGAGATCTGCGAC ST1006CTGCGTCTTCTCCGCGATATCCAGCGTCAT ST1007CACCGGGCAGAGCGAATTCATCGCCGST1008TCTTCTTCTCCGCTGTACAGGCCTGCATGC ORF15-1ATCATATGATGGCCGGCGATGTORF15-3ATAGATCTTCACACGCCCATGAATTCC ORF17-1N ATTCTAGAATGACGACCCGAGCC ORF17-2NATGGATCCTCAAAGAGCGGGTTaSupplied by PromegaMutational Analysis of Lankacidin Biosynthesis 2713(50m g/ml),or tetracycline(10m g/ml)was added to Luria-Bertani (LB)medium when necessary.DIG DNA labeling and detection kit (Roche Diagnostics GmbH,Mannheim,Germany)was used for Southern hybridization analysis.Construction of targeting plasmids.In-frame deletion of lkcC-KR:A2.9-kb Sal I fragment(nt28,208–31,118of pSLA2-L)from cosmid B103)was cloned into pUC19to give pFSU16-01.The141-bp Stu I-Nru I fragment in the targeted lkcC-KR portion was eliminated by double digestion and self-ligation to give pFSU16-02.The vector part of pFSU16-02was replaced with an E.coli-Streptomyces shuttle vector pRES1818)to give a targeting plasmid,pFSU16-03.In-frame deletion of lkcF-KR1:A2.4-kb Fsp I-Eco RI fragment(nt 19,975–22,371)from cosmid B10was cloned into pUC19predigested with Sma I and Eco RI to give pFSU13-01.The387-bp Ava I fragment in the targeted lkcF-KR1portion was eliminated to give pFSU13-03.The vector part of pFSU13-03was replaced by pRES18to give a targeting plasmid,pFSU13-05.In-frame deletion of lkcF-KR2:A 3.2-kb Pst I-Stu I(nt16,621–19,851)fragment from cosmid B10was cloned into pUC19predi-gested with Pst I and Sma I to give pFSU13-02.The456-bp Hin cII fragment in the targeted lkcF-KR2portion was eliminated to give pFSU13-04.The vector part of pFSU13-04was replaced by pRES18to give a targeting plasmid,pFSU13-06.S362A mutation of lkcC-KR:Plasmid pFSU16-01was digested with Bam HI and Hin dIII,and a2.1-kb fragment was recloned into pUC57 (Fermentas,Vilnius,Lithuania)to give pST16KR-01.The 2.1-kb Bam HI-Stu I fragment from pST16KR-01was cloned into pAlter-1 (Promega)to give pST16KR-02.The lkcC-KR S362A mutation was introduced by oligonucleotide ST1008to give a mutated plasmid, pST16KR-03.The2.1-kb Bam HI-Stu I fragment from pST16KR-03 was substituted for the original fragment in pST16KR-01to give pST16KR-04,the vector of which was replaced by pRES18to give a targeting plasmid,pST16KR-06.S330A mutation of lkcF-KR1:Plasmid pFSU13-01was digested with Eco RI and Pst I,and the insert was recloned into pBluescript SK(+)to give pKAR1010.The1.4-kb Eco RI-Sph I fragment from pKAR1010was cloned into pAlter-1to give pKAR1011.The lkcF-KR1S330A mutation was introduced by oligonucleotide KAR-1301S01 to give a mutated plasmid,pKAR1012.The 1.4-kb EcoR I-Sph I fragment from pKAR1012was substituted for the original fragment in pKAR1010to give pKAR1013,the vector of which was replaced by pRES18to give a targeting plasmid,pKAR1014.S1523A mutation of lkcF-KR2:Plasmid pFSU13-02was digested with Eco RI and Pst I,and the insert was recloned into pAlter-1to give pST13KR2-01.The lkcF-KR2S1523A mutation was introduced by oligonucleotide ST1006to give a mutated plasmid,pST13KR2-02. The vector part of pST13KR2-02was replaced by pRES18to give a targeting plasmid,pST13KR2-03.Y343F mutation of lkcF-KR1:The lkcF-KR1Y343F mutation was introduced into pKAR1011by oligonucleotide KAR-1301Y01to give a mutated plasmid,pKAR1016.The0.31-kb Age I-Sph I fragment from pKAR1016was substituted for the original fragment in pKAR1010to give pKAR1018,the vector of which was replaced by pRES18to give a targeting plasmid,pKAR1020.Y1536F mutation of lkcF-KR2:The lkcF-KR2Y1536F mutation was introduced into pST13KR2-01by oligonucleotide ST1007to give a mutated plasmid,pST13KR2-04.The vector part of pST13KR2-04was replaced by pRES18to give a targeting plasmid,pST13KR2-05.S851A mutation of lkcC-ACP1:The1.4-kb PCR fragment contain-ing the30-portion of lkcC-ACP1S851A was ampliﬁed using cosmid B10 as a template and primers ST1000and ST1001.The0.54-kb PCR fragment containing the50-portion of lkcC-ACP1S851A was ampliﬁed using cosmid B10and primers ST1002and ST1003.The1.4-kb PCR fragment was digested with Eco RI and Nsi I and cloned into Litmus28i(New England Biolabs,Ipswich,MA)to give pSTACP05.The0.54-kb PCR fragment digested with Nsi I and Bgl II was cloned into pSTACP05to give pSTACP06.The1.6-kb Apa I-Bgl II fragment from pSTACP06was substituted for the original fragment of pKAR1006to give pSTACP07,the vector of which was replaced by pRES18to give a targeting plasmid,pSTACP08.S956A mutation of lkcC-ACP2:The1.1-kb PCR fragment contain-ing the30-portion of lkcC-ACP2S956A was ampliﬁed using cosmid B10and primers ST1000and ST1001.The0.85-kb PCR fragment containing the50-portion of lkcC-ACP2S956A was ampliﬁed using cosmid B10and primers ST1002and ST1003.The1.1-kb PCR product was digested with Eco RI and Nsi I and cloned into Litmus28i to give pSTACP01.The0.85-kb PCR fragment digested with Nsi I and Bgl II was cloned into pSTACP01to give pSTACP02.The1.6-kb Apa I-Bgl II fragment from pSTACP02was substituted for the original fragment in pKAR1006to give pSTACP03, the vector of which was replaced by pRES18to give a targeting plasmid,pSTACP04.S851A-S956A double mutation of lkcC-ACP1and lkcC-ACP2:A 315-bp Bst EII fragment from pSTACP04,which carries the lkcC-ACP2S956A mutation,was substituted for the original fragment in pSTACP08to give a targeting plasmid,pSTACP09.In-frame deletion of lkcC-ACP2:The315-bp Bst EII fragment was eliminated from pSTACP04,and the resulting DNA was ligated to give a targeting plasmid,pSTACP10.Deletion of lkcD:A 2.3-kb Bsp EI-Bgl II fragment(nt22,755–25,038)from cosmid B10was cloned into Litmus28i to give pSTAT01.This plasmid was digested with Bgl II and Sal I,and recloned into pET32b(+)(Novagen,Darmstadt,Germany)to give pSTAT02. The0.52-kb Bsi WI-Age I fragment in the middle of lkcD was eliminated from pSTAT02,and the resulting DNA wasﬁlled in and self-ligated to give pSTAT03.The1.0-kb Bgl II-Sal I fragment from pSTAT03was substituted for the original fragment in pSTAT01to give pSTAT04,the vector of which was replaced by pRES18to give a targeting plasmid,pSTAT05.Insertional mutation of lkcB:A1.9-kb Not I fragment(nt30,023–31,932)from cosmid B10was cloned into pBluescript SK(+)to give pSTDH01.This plasmid was digested with Xba I and Sac I,and the insert was recloned into pUC19to give pSTDH02.A1.2-kb Sma I fragment containing the kanamycin resistance gene cassette19)was inserted into the Sma I site in the middle of lkcB to give pSTDH03.The vector part of pSTDH03was replaced by pRES18to give a targeting plasmid,pSTDH04.In-frame deletion of lkcC-MT:A3.8-kb Nco I fragment(nt25,693–29,514)from cosmid B10was cloned into pRSET-B(Invitrogen, Carlsbad,CA)to give pKAR1005.The3.8-kb Eco RI-Pst I insert in pKAR1005was cloned into pUC19to give pKAR1006.The135-bp Bam HI-Bgl II fragment was eliminated from pKAR1006to give pKAR1007,the vector of which was replaced by pRES18to give a targeting plasmid,pKAR1008.Construction of various PKS mutants.Targeting plasmids were transformed into S.rochei51252protoplasts.The plasmid-integrated strains were selected with thiostrepton(10m g/ml),and were serially cultured in the absence of thiostrepton to facilitate the second crossover.The double-crossovered strains were identiﬁed by Southern hybridization analysis.Analysis of metabolites.S.rochei strains were cultured at28 C for 2d in500-ml Sakaguchiﬂasks.Cultured broth was extracted twice with ethyl acetate,and the combined organic phase was dried (Na2SO4),ﬁltered,and concentrated to dryness.The lankacidin production of the mutants was analyzed by high performance liquid chromatography(HPLC)and electrospray-ionization mass spectrom-etry(ESI-MS).The crude extract was applied on a COSMOSIL5C18-MS-II column(4:6Â250mm,Nakarai Tesque,Kyoto,Japan)and eluted with a mixture of acetonitrile-10m M sodium phosphate buﬀer (pH8.2)(3:7,v/v)at aﬂow rate of1.0ml/min.The eluate was monitored at230nm with a MD-2010multiwavelength photodiode array detector(Jasco,Tokyo,Japan).Lankacidin C and lankacidinol A were detected at8.5min and16.9min,respectively.The relative yield of lankacidin C in various mutants was determined from peak intensities on a chromatogram in comparison with the parent strain, 51252.ESI-MS spectra were recorded on an ALLIANCE2690/ ZQ2000mass spectrometer(Waters,Milford,MA).Complementation by intact lkcD and lkcB.The lkcD gene was ampliﬁed by PCR using cosmid B10as template and primers ORF15-1 and ORF15-3.The ampliﬁed product was digested with Nde I and Bgl II and cloned into pIJ8600,an E-coli-Streptomyces shuttle vector carrying a thiostrepton-inducible promoter,20)to give plasmid pSTAT06N.This plasmid was transformed into protoplasts of the2714S.T ATSUNO et al.lkcD mutant STAT01.The transformant was cultured at28 C in YM liquid medium supplemented with apramycin(20m g/ml)for24h,and then thiostrepton(10m g/ml)was added to induce expression of lkcD. After further cultivation for2d,the supernatant was extracted with ethyl acetate.Plasmid pIJ8600was also transformed as a control.The lkcB gene was ampliﬁed by PCR using cosmid B10as template and primers ORF17-1N and ORF17-2N.The ampliﬁed product was digested with Xba I and Bam HI and cloned into pIJ8600to give plasmid plementation of lkcB in the lkcB mutant STDH01 was carried out in a way similar to lkcD.ResultsThree ketoreductase domains,LkcC-KR,LkcF-KR1, and LkcF-KR2,were functional in lankacidin biosyn-thesisIn erythromycin biosynthesis,a mutant carrying an in-frame deletion in the eryAIII-KR5domain produced theexpected unreduced5-keto compound,5,6-dideoxy-3- -L-mycarosyl-5-oxoerythronolide B.21)The lkc cluster contains three KR domains,lkcC-KR,lkcF-KR1,andlkcF-KR2.Based on our hypothesis(Fig.1),it was speculated that the lkcF-KR1and lkcF-KR2domains are involved in the reduction of the two ketone groups at C-7and C-5respectively.To obtain unreduced lanka-cidin derivatives,mutational analysis of these three KR domains was done.First,KR mutants were constructed by in-framedeletion.The lkcC-KR mutant FS16(ÁlkcC-KR)wasconstructed by deletion of a141-bp DNA.TheÁlkcF-KR1mutant KSFS13-1was constructed by deletion of a 387-bp DNA,and theÁlkcF-KR2mutant FS13-2by deletion of a456-bp DNA.The detailed construction schemes of the mutants are shown in Supplemental data (see Biosci.Biotechnol.Biochem.Web site).Metabolites of these KR deletion mutants were analyzed by high performance liquid chromatography(HPLC)of their ethyl acetate extracts.The parent strain,51252,showed two peaks,lankacidin C at8.5min and lankacidinol A at 14.8min(Fig.2-I).On the other hand,three deletion mutants did not show these peaks(Fig.2-II–IV).We also searched for5-keto and7-keto compounds and other unreduced metabolites by TLC,HPLC,and ESI-MS based on their predicted conjugated double bonds and molecular sizes,but did not detect them in any KR deletion mutants.It is likely that the three deletion mutants did not produce lankacidin or its derivatives due to wrong folding of the mutated proteins with a large deletion.To minimize this possibility,we next mutated the KR domains by amino acid substitution.The KR domains contain conserved lysine,serine,and tyrosine residues, which form a catalytic triad.22)Hence,the serine and tyrosine residues were replaced with alanine and phenylalanine respectively.At the same time,a new restriction site was introduced to conﬁrm the mutation. Consequently,an lkcC-KR S362A mutant with a Bsr GI site (strain STKR04),an lkcF-KR1S330A mutant with an Eco47III site(strain KA14),and an lkcF-KR2S1523A mutant with an Eco RV site(strain STKR01)were obtained.The lkcC-KR S362A mutant did not produce lankacidin,while the lkcF-KR1S330A and lkcF-KR2S1523A mutants produced38%and2.2%of lankacidin C as compared with strain51252(Fig.2-V–VII).The iden-tity of the metabolite with lankacidin C was established by ESI-MS[ðMþNaÞþ¼482].Another series of mutants with replacement of tyrosine by phenylalanine were also constructed:an lkcF-KR1Y343F mutant with a Pst I site(strain KA35)and an lkcF-KR2Y1536F mutant with an Eco RI site(strain STKR02).Neither mutant produced lankacidin at all(Fig.2-VIII and IX).None of theﬁve KR substitution mutants accumulated unreduced lankacidinderivatives.Either of the two tandemly aligned ACP domains was suﬃcient for lankacidin biosynthesisThe LkcC protein carries two tandemly aligned ACP domains (LkcC-ACP1and LkcC-ACP2).Similar tandem alignments of two ACP domains have been found in several bacterial and fungal type-I PKSs,for example,albicidin PKS of Xanthomonas albilineans ,23)mupirocin PKS of Pseudomonas ﬂuorescens ,24)and naphtopyrone and sterigmatocystin PKSs of Aspergillus nidulans .25,26)To determine the function of LkcC-ACP1and LkcC-ACP2,three types of substitution mutants were con-structed by site-directed mutagenesis.Since the lkcC-ACP1and lkcC-ACP2domains have identical nucleotide sequences,PCR ampliﬁcation using a pair of a mutated ACP primer and an outside primer gave two ampliﬁed products of diﬀerent size,each containing a mutation in ACP1and ACP2.In this way,a Ser-to-Ala mutation and an Nsi I site were introduced into ACP1and ACP2.Proper combinations of two PCR products were ligated to construct targeting plasmids for mutation.Thus an lkcC-ACP1S851A mutant (strain STACP01),an lkcC-ACP2S956A mutant (strain STACP02),and an lkcC-ACP1S851A .lkcC-ACP2S956A double mutant (strain STACP03)were obtained.Both the lkcC-ACP1S851A and the lkcC-ACP2S956A mutant produced 54%of lankacidin C as compared with strain 51252,while the lkcC-ACP1S851A .lkcC-ACP2S956A double mutant did not produce lankacidin (Fig.3).We also constructed a solo ACP mutant (strain STACP04,ÁlkcC-ACP2)by eliminating a 315-bp Bst EII fragment covering the ACP2region.The ÁlkcC-ACP2mutant produced 48%of lankacidin C as compared with strain 51252,a comparable level to the lkcC-ACP1S851A and lkcC-ACP2S956A mutants (Fig.3-VI).However,no ring-contracted lankacidin derivatives were detected in any of the four ACP mutants.Acyltransferase LkcD,dehydratase LkcB,and the C-methyltransferase domain LkcC-MT act iteratively The lkc cluster contains three unique genes/domain for lankacidin biosynthesis;two discrete genes (lkcD and lkcB )for acyltransferase (AT)and dehydratase (DH),and one domain (lkcC-MT )for C-methyltransfer-ase (MT).Since no other genes or domains with AT,DH,and MT activities are found in the cluster,their gene products might act iteratively to accomplish polyketide assembly.The discrete AT protein,LkcD,contains a catalytic serine residue in the GHSXG motif (nt 24,295–24,281of pSLA2-L),27)which is conserved in all the AT proteins/domains (Fig.4A).Gene inactivation of lkcD was carried out by deletion of a 0.52-kb Age I-Bsi WI frag-ment containing this motif.The disruptant obtained,STAT01(ÁlkcD ),completely abolished lankacidin production (Fig.5-II),and this defect was complement-ed by the introduction of an intact lkcD gene (Fig.5-III).The discrete dehydratase gene lkcB was inactivated by insertion of a kanamycin resistance gene cassette into the Sma I site in the middle of the gene.The resulting mutant,STDH01(lkcB::kan ),did not produce any lankacidin derivatives (Fig.5-V),and this defect was also restored by the introduction of an intact lkcB gene (Fig.5-VI).Feeding experiments on labeled S-adenosyl-L -methio-nine (SAM)revealed that four methyl groups,at C-2,C-4,C-10,and C-16,of the lankacidin skeleton were introduced by C-methylation.28)The unique LkcC-MT domain in the cluster contains an SAM-binding EXGXG motif (nt 28,275–28,261)characteristic of C-methyl-transferases.29)Disruption of lkcC-MT was carried out by in-frame deletion of a 135-bp Bam HI-Bgl II fragment containing this motif.The disruptant,KA03(ÁlkcC-MT ),did not produce lankacidin (Fig.5-VIII),andFig.3.HPLC Analysis of Various Mutants of the Tandemly Aligned lkcC-ACP1and lkcC-ACP2Domains.I,strain 51252(parent);II,strain STACP01(lkcC-ACP1S851A );III,strain STACP02(lkcC-ACP2S956A );IV,strain STACP03(lkcC-ACP1S851A .lkcC-ACP2S956A );V,strain STACP04(ÁlkcC-ACP2).2716S.T ATSUNO et al.contrary to our expectations,the mutant did not produce desmethyl lankacidin derivatives.We have not yet done complementation experiments due to the domain struc-ture of lkcC-MT .DiscussionIn this study,we carried out extensive mutations of the lkc -PKS genes to isolate key metabolites supporting the modular-iterative mixed biosynthesis of lankacidin.Although we have not succeeded in isolating the expected lankacidin derivatives from any mutants,analysis of their metabolic abilities revealed several features of the Lkc-PKS enzymes.Crystallographic analysis of tropinone reductase-II,a short-chain dehydrogenase/reductase (SDR)from the plant Datura stramonium ,showed that the conserved serine residue stabilizes the substrate by hydrogen bonding,while the catalytic tyrosine residue has a proton-donating function to a -keto group of the substrate.30)The KR domains in actinomycete modular PKSs showed end-to-end sequence similarities to SDRs.Hence,we mutated the serine and tyrosine residues in the lkcC-KR ,lkcF-KR1,and lkcF-KR2domains to obtain unreduced metabolites.In the case of the Ser-to-Ala substitution,the lkcF-KR1and lkcF-KR2mutants pro-duced decreased levels of lankacidin (38%and 2.2%as compared to strain 51252),while the lkcC-KRmutantBA parison of LkcD with Typical Acyltransferases for Polyketide Synthesis.A,Partial amino acid alignments of LkcD and typical acyltransferases.Solid box indicates the conserved active site motif,GHSXG.Bold letters indicate the consensus motifs for recognition of malonyl and methylmalony CoA.LkcD (accession no.BAC76473,S.rochei 7434AN4);LnmG (AAN85520,Streptomyces atroolivaceus );MmpIII (AAM12912,Pseudomonas ﬂuorescens );MlnA (YP 001421027,Bacillus amyloliquefaciens FZB42);PedD (AAS47563,symbiont bacterium of Paederus fuscipes );PedC (AAS47559);RapA (CAA60460);RapB (CAA60459);RifA (AAC01710);LkmAI (BAC76493);EryAI (AAV51820);and AveAI (NP 822113).The numerals following protein names indicate module numbers.B,Phylogenetic tree of acyltransferases.The unrooted radial tree was drawn with TreeView1.6.6software (/rod/treeview.html).ceased lankacidin production.Thus the serine residue in the KR domains is important but not critical for the enzyme activity.It is reasonable that the Ser-to-Ala mutation of the lkcC-KR domain resulted in nonproduc-tion of lankacidin,because LkcC might function iter-atively in the early steps of biosynthesis.In contrast to the Ser-to-Ala mutation,the Tyr-to-Phe mutation of the lkcF-KR1and lkcF-KR2domains abolished lankacidin production,indicating that the proton-donating function of the tyrosine residue has a critical role in ketoreduction. In this study,we could not isolate any unreduced metabo-lites of lankacidin from any of the KR mutants.This is in contrast to typical modular polyketide biosynthesis,in which the KR mutants accumulate unreduced metabo-lites of erythromycin.21,31)Thus the Lkc-PKS enzymes might strictly recognize the structures of unidentiﬁed intermediates,including their reductive states.All of the three mutants carrying one active LkcC-ACP domain produced about half the amount of lankacidin as compared with strain51252,but not any ring-contracted derivatives.Thus,either ACP is suﬃcient for lankacidin biosynthesis,and the tandem alignment of two ACPs is not essential for iterative condensation.Accumulated examples also suggest no relationship between tandem alignment and iterative condensation.24–26)The copy numbers of ACP genes/domains rather than their tandem alignment appears to function in overall PKS activity.32,33)Introduction of the tetracenomycin ACP gene on a high-copy vector into the wild-type strain of Streptomyces glaucescens caused30-fold production of tetracenomycin D3.32)Shen et al.analyzed the function of sextet ACP domains in the eicosapentaenoic acid (EPA)synthase of Shewanella japonica.34)They con-structed many mutants in which one to six ACP domains were inactivated.Mutants with the same number of active ACPs produced similar amounts of EPA,indicat-ing a correlation between the number of active ACPs and the titer of EPA.Thus the tandem alignment of two ACP domains in LkcC might also increase the ACP concen-tration and aﬀect the titer of lankacidin.The acyltransferase(AT)activity in modular PKSs is integrated into each module as a domain and functions once in elongation cycles(named cis-AT).5)Cis-AT domains contain consensus recognition sequences for malony or methylmalonyl CoA(Fig.4A),which gives structure diversity to polyketides.On the other hand, discrete AT genes were foundﬁrst in the pksX cluster for a cryptic polyketide of Bacillus subtilis,35)and then in the clusters for mupirocin of Pseudomonasﬂuores-cens,24)leinamycin of Streptomyces atroolivaceus,36) and pederin of a bacterial symbiont of the beetle Paederus fuscipes.37)In all these cases,only malonyl CoA was transferred in trans iteratively to other PKSs (named trans-ATs).Disruption and complementation of the discrete lkcD gene in the lkc cluster indicated that LkcD belongs to trans-ATs and that it transfers eight malonyl CoA molecules to LkcA,LkcC,LkcF,and LkcG.Reﬂecting these diﬀerences,three types of AT domains/proteins are grouped into diﬀerent phyloge-netic clusters(Fig.4B).Very recently,in silico analysis of the kirromycin cluster from Streptomyces collinus suggested that KirCII,one of the two trans-ATs, functions in the transfer of ethylmalony CoA.38)The lkc cluster does not possess a dehydratase(DH)domain,instead containing a discrete DH gene,lkcB. Disruption and complementation experiments have sug-gested that LkcB functions in trans iteratively to generate all of the four double bonds,at C-8=C-9,C-10=C-11, C-14=C-15,and C-16=C-17.A similar lack of DH domains in type-I PKS modules was found in the biosynthetic clusters for chalcomycin in Streptomyces bikiniensis,39)leinamycin in S.atroolivaceus,36)and macrolactin in Bacillus amyloliquefaciens.40)Modules 3,5,and7of the leinamycin cluster do not possess a DH domain,which is required for biosynthesis.Since discrete DH genes are not present in the cluster,it is likely that the unique DH domain in module4functions in trans iteratively.To our knowledge,the biosynthetic gene cluster for coronafacic acid from Pseudomonas syringae41)is the only example in which a discrete DH gene has been identiﬁed in type-I PKSs.However,in this case,the discrete DH enzyme functions on a pentanone moiety,which is formed after completion of the poly-ketide backbone.Thus the present study provides theﬁrst evidence that a discrete DH enzyme functions iteratively in trans in type-I polyketide biosynthesis.Disruption of the lkcC-MT domain in this study together with previous heterologous expression of the lkc cluster in S.lividans9)indicates that this domain iteratively introduces four methyl groups at C-2,C-4, C-10,and C-16of the lankacidin skeleton.Heterologous expression of alt5,an iterative type-I PKS gene with a MT domain from Alternaria solani,also produced a correctly methylated product,alternapyrone,in Asper-gillus oryzae.42)Contrary to our expectations,the lkcC-MT mutant did not accumulate desmethyl lankacidin, suggesting that the Lkc-PKS modules do not process intermediates lacking methyl substituents.On the other hand,disruption of the discrete MT genes in the type-II PKS clusters for mithramycin in Streptomyces argilla-ceus43)and coumermycin in Streptomyces rishiriensis44) gave desmethyl derivatives.In both cases the MT enzymes function only once on a speciﬁc position of the intermediate.Although disruption and complementation of the discrete genes/domain,lkcD,lkcB,and lkcC-MT,sug-gested an iterative use of their gene products,the possi-bility that other enzymes are involved in the iterative reactions cannot be ruled out.However,this possibility appears extremely low,because the lankacidin skeleton was synthesized by heterologous expression of the lkc cluster in S.lividans.9)Thus the results of this study indicate the modular-iterative mixed polyketide biosyn-thesis of lankacidin in a diﬀerent way from that which we anticipated atﬁrst.Moreover,they suggest challenging questions as to how LkcB and LkcC-MT select the reaction sites for dehydration and C-methylation from multiple candidate sites.The lasting question how modular and iterative condensations can be distinguished by LkcA,LkcC,LkcF,and LkcG remains to be solved. AcknowledgmentsThis work was supported by Grant-in-Aids for Scientiﬁc Research on Priority Areas18018028(to H.K.)and for Young Scientists(B)20780083(to K.A.) from the Ministry of Education,Culture,Sports, Science,and Technology of Japan.2718S.T ATSUNO et al.。

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http://www.elsevier.com/copyrightAuthor's personal copyEvaluationofthestrainsofAcinetobacterandEnterobacteraspotentialbiocontrolagentsagainstRalstoniawiltoftomato

Qing-YunXuea,1,YuChenb,1,Shi-MoLic,Li-FengChena,Guo-ChunDinga,Da-WeiGuod,Jian-HuaGuoa,*

aCollegeofPlantProtection,NanjingAgriculturalUniversity,KeyLaboratoryofMonitoringandManagementofCropDiseasesandPestInsects,

MinistryofAgriculture,Nanjing,Jiangsu210095,ChinabCollegeofHorticulture,NanjingAgriculturalUniversity,Nanjing,Jiangsu210095,China

cHuaiyinTearcher’scollege,JiangsuKeyLaboratoryofEco-AgriculturalBiotechnologyaroundHongzeLake,Huaian,Jiangsu223001,China

dLongyanAcademyofAgriculturalSciences,Longyan,Fujian364000,China

articleinfoArticlehistory:Received19July2008Accepted12November2008Availableonline25November2008

Keywords:AntagonistRalstoniasolanacearumBiologicalcontrolAcinetobactersp.Root-dippingDrenchingEnterobactersp.

abstractBacterialwilt(Ralstoniasolanacearum)oftomato,Lycopersiconesculentum,causesaconsiderableamountofdamagetotomatoinSouthernChina.Biologicalcontrolisoneofthemorepromisingapproachestoreducethediseaseincidenceandyieldlossescausedbythisdisease.BasedonantagonisticactivityagainstR.solanacearumandthreesoil-bornefungalpathogensaswellasbiocontrolefﬁcacyinthegreen-house,twobacterialstrainsXa6(Acinetobactersp.)andXy3(Enterobactersp.)wereselectedoutoffour-teencandidatesaspotentialbiocontrolagents.Inordertoﬁndasuitableantagonistinoculationmethod,wecomparedthemethodsofroot-dippingwithsoil-drenchingintheaspectsincludingrhizocompetence,biocontrolefﬁcacy,andeffectofpromotingplantgrowthundergreenhouseconditions.Thedrenchingtreatmentresultedinahigherbiocontrolefﬁcacyandplant-yieldincrease,andthismethodwasalsoeas-iertooperateintheﬁeldonalargescale.Fieldtrialswereconductedforfurtherevaluationofthesetwoantagonisticstrains.Inbothgreenhouseandﬁeldexperiments,thestrainXy3hadabettercontroleffectagainstbacterialwiltthanXa6did,whileXa6causedhigherbiomassoryieldincreases.Asrecordedonthe75thdayaftertreatmentintwoﬁeldexperiments,biocontrolefﬁcacyofXy3wasabout65%inbothﬁeldtrials,andtheyieldincreasescausedbyXa6were32.4and40.7%,respectively,inthetwotrials.ThisistheﬁrstreportofanAcinetobactersp.strainusedasaBCAagainstRalstoniawiltoftomato.Ó2008ElsevierInc.Allrightsreserved.

1.IntroductionBacterialwiltcausedbyRalstoniasolanacearum(Yabuuchietal.,1995)isoneofthemostdevastatingplantdiseasesworldwide.R.solanacearumaffectsawiderangeofplantsinmorethan50fami-lies(Hayward,1995).InChina,itshostsincludeeconomicallyimportantcropssuchaspotato(SolanumtuberossumL.),tomato(LycopersiconesculentumMiller),tobacco(NicotianatabacumL.),eggplant(SolanummelongenaL.),pepper(CapsicumannuumL.),peanut(ArachishypogaeaL.)andginger(ZingiberofﬁcinaleRoscoe).Thisdiseasehascausedgreatlossesinagricultureandhorticulture(Lietal.,2004;Liuetal.,2005).Ralstoniawiltcontrolapproaches,includingﬁeldsanitation,croprotation,andapplicationofresistantvarieties,haveprovenlimitedsuccess(Ciampi-Pannoetal.,1989).Althoughusingresis-tantcultivarsisanimportantpartoftheintegrateddiseaseman-agement,breedingfordiseaseresistanceisalong-termtaskthat

isbothexpensiveanddifﬁcult.Inaddition,diseaseresistanceofacultivarisusuallynotstableand/ordurable(Hayward,1991;Boucheretal.,1992).Inspeciﬁcregions,inﬂuenceofenvironmen-talfactorsonhost–pathogeninteractionsoftenrestrictstheexpressionofdiseaseresistance(Hayward,1991).Applyingchem-icalpesticidesisgenerallyconsideredasthemosteffectiveandfasteststrategyforplantdiseasemanagement,however,noeffec-tivechemicalproductisavailableforRalstoniawilt.Althoughstreptomycinisregardedasasuitablebactericidetocontrolthedisease,Chinesefarmersarereluctanttouseitbecauselargedos-agesarerequiredfortheexpectedeffectiveness,probablyduetothebactericideresistancethatdevelopedduringitsrepeatedandabusiveuseforlastseveraldecadesinthiscountry.Therefore,moreeffortsneedtobedevotedtobiologicalcontrolwithlivingmicrobes.Severallivingmicrobialproductshavebeencommercializedasbiologicalcontrolagents(BCAs).Someoftheseproductsareawet-tablepowderofBacillussubtilis(Cohn)Y1336,awatersuspensionofPseudomonasﬂuorescens(Migula),andamixtureofwettablepowderandgranuleofPaenibacilluspolymyxa(Ash,PriestandCol-lins)(Sunetal.,2004).However,livingmicrobialBCAshavenot