力达霉素生物合成调控基因sgcR3功能研究及其与sgcR1和sgcR2调控关系的研究

【作者】 王丽非；

【导师】 洪斌； 李元； 姜威；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2008， 博士

【摘要】 力达霉素(lidamycin)是一种新型的烯二炔类抗肿瘤抗生素,又名C-1027,由球孢链霉菌C-1027(S.globisporus C-1027)产生。经过十余年的新药研究和开发,目前力达霉素已经进入临床Ⅱ期研究。力达霉素生物合成基因簇已被克隆和测序,其基因簇中一些生物合成酶的功能和生物合成步骤已经阐明,但其生物合成的调控机制尚未见报道。根据生物信息学分析的结果,力达霉素的生物合成基因簇中至少存在三个调控基因sgcR1、sgcR2和sgcR3。sgcR3基因的产物SgcR3蛋白与泰乐菌素生物合成途径特异性正调控蛋白TylR具有较高的同源性。sgcR1基因的产物SgcR1蛋白与S.griseus链霉素生物合成的途径特异性激活因子StrR高度同源。蛋白SgcR2属于AraC/XylS转录因子家族。sgcR2与sgcR1串联,两基因间相隔25个碱基,推测其与sgcR1位于同一个操纵子。sgcR3和sgcR2基因内部各含有一个TTA稀有密码子。在三个已知的九元环类烯二炔基因簇(C-1027,新制癌菌素和maduropeptin)中,sgcR1、sgcR2和sgcR3及它们的同源基因均围绕在烯二炔聚酮合酶基因(PKSE)及其相关基因附近,并且基因排列组成也非常相似,推测sgcR1、sgcR2和sgcR3对这类抗生素生物合成具有重要的调控作用。本工作采用同源重组双交换的方法将sgcR3基因阻断,研究sgcR3的生物学功能。分别以阿普霉素和硫链丝菌素抗性基因片段作为插入片段构建了sgcR3阻断突变株S.globisporus R3KO1和S.globisporus R3KO,阻断突变株不产生力达霉素。用三种质粒pKCR3、pSETR3和pLR3分别导入在自身启动子控制下和强启动子控制下的sgcR3对阻断突变株S.globisporus R3KO进行互补,均能恢复力达霉素的产生。在野生菌株中过量表达sgcR3可提高力达霉素的产量。以上结果说明SgcR3是力达霉素生物合成的正调控因子。为了确定sgcR3所调控的下游基因,本工作在大肠杆菌BL21(DE3)中表达并纯化了SgcR3蛋白。凝胶阻滞实验结果表明SgcR3可结合于sgcR1R2基因上游片段。将带有自身启动子和/或强启动子ermE~*p的sgcR1、sgcR2以及sgcR1R2分别克隆至载体pKC1139的多克隆位点,导入野生菌株中进行过表达研究,结果发现均能不同程度的提高力达霉素的产量,提示sgcR1和sgcR2对力达霉素的产生有促进作用。交叉互补实验结果表明,sgcR3的导入不能使sgcR1R2阻断突变株S.globisporus R1R2KO恢复力达霉素的产生,sgcR1、sgcR1R2的导入则可使S.globisporus R3KO恢复力达霉素的产生,表明sgcR3在力达霉素生物合成途径特异性调控网络中位于sgcR1和sgcR2上游。实时定量RT-PCR结果显示,sgcR1和sgcR2的转录水平在S.globisporus R3KO中明显下降,而sgcR3的转录水平在S.globisporusR1R2KO中无明显变化,证明了sgcR3可调控sgcR1和sgcR2表达,这与交叉互补和SgcR3凝胶阻滞的实验结果一致。几个结构基因sgcA1、sgcC4、sgcD6和sgcE8在S.globisporus R3KO和S.globisporus R1R2KO中的表达量也都显著下降。本工作还构建了sgcR1和sgcR2的大肠杆菌表达质粒,并转入大肠杆菌BL21(DE3)进行了表达和纯化。为进一步研究sgcR1和sgcR2的功能奠定了基础。综上所述,本工作通过对球孢链霉菌C-1027力达霉素生物合成基因簇中sgcR3基因进行阻断、互补和过表达研究,确定了sgcR3的正调控功能。并对sgcR3的分子调控机制进行初步研究,确定了其靶基因为sgcR1R2,在途径特异性级联调控网络中位于sgcR1R2的上游,为阐明力达霉素生物合成调控网络并应用代谢工程手段构建力达霉素高产菌株奠定了基础。更多还原

【Abstract】 Lidamycin（C-1027）,produced by Streptomyces globisporus C-1027,is a novel enediyne antitumor antibiotic and has entered phaseⅡclinical trial in China recently.C-1027 biosynthetic gene cluster has been cloned,sequenced and characterized.Bioinformatic analysis and biochemical studies revealed a distinct iterative typeⅠenediyne polyketide synthase（PKSE） and provided a convergent biosynthetic strategy for C-1027 from four biosynthetic building blocks.However,almost nothing was known about the transcriptional regulation of enediyne antibiotic production prior to the present work.The biosynthetic gene cluster for 9-membered enediyne C-1027 contains at least three putative regulatory genes,i.e.,sgcR1,sgcR2 and sgcR3.The predicted gene products of sgcR1,sgcR2 and sgcR3 share sequence similarities to StrR,regulators of AraC/XylS family and TylR,respectively,sgcR1 and sgcR2 are two adjacent genes transcribed in the same direction with a gap of only 25 bp,suggesting that they might be transcriptionally coupled within an operon,sgcR2 and sgcR3 contain a TTA leucine codon respectively. Furthermore,the biosynthetic gene clusters for three 9-membered enediynes（C-1027, neocarzinostatin and maduropeptin） show high similarity in the organization of genes around these regulatory genes.Despite varying degrees of chromophore structural uniqueness,all homologues of three genes are located adjacent to the genes of PKSEs （sgcE,ncsE and mdpE） and the tailoring enzymes（E1 to E11）,which are responsible for the biosynthesis of enediyne core.This may represent a further indication for a regulatory role of the three genes in C-1027 biosynthesis.Disruption of sgcR3 abolished C-1027 production,suggesting that it is a positive regulator of C-1027 production.Complementation of the sgcR3 disrupted strain R3KO with intact sgcR3 gene restored C-1027 production and overexpression of sgcR3 in wild type strain resulted in a substantial increase in C-1027 production,confirming their positive regulatory role in C-1027 biosynthesis.SgcR3 was expressed and purified in E. coli BL21（DE3）.Purified N-terminal His₆-tagged SgcR3 showed specific DNA-binding activity for upstream sequence of sgcR1 and sgcR2 of C-1027 biosynthetic gene cluster.sgcR1,sgcR2 and sgcR1R2 with its own upstream sequence or the strong constitutive promoter ermE^*p were cloned into the vector pKC1139 and the recombinant plasmids were introduced into S.globisporus C-1027.Overexpression of sgcR1,sgcR2 and sgcR1R2 in wild type strain increased C-1027 production respectively,suggesting their positive role in C-1027 biosynthesis.The cross-complementation studies showed sgcR1R2 could functionally complement sgcR3 in trans,suggesting that sgcR1R2 occupies a lower rung than sgcR3 in the hierarchy of these genes.Consistent with this, the results from gene expression analysis via real time reverse transcriptase polymerase chain reaction（RT-PCR） showed that transcripts of sgcR1 and sgcR2 were significantly lower in R3KO mutant than in wild type strain,while sgcR3 transcripts were statistically at similar level in R1R2KO mutant as in wild type strain.A positive effect of sgcR1R2 and sgcR3 gene products on the transcription of biosynthetic structural genes such as sgcA1,sgcC4,sgcD6 and sgcE8 was also observed.To further investigate the functions of the sgcR1 and sgcR2,SgcR1 and SgcR2 were expressed and purified in E.coli BL21（DE3）.In this work,the results from disruption,complementation and overexpression analyses suggested a positive regulatory role of sgcR3 in C-1027 biosynthesis.Furthermore, purified SgcR3 showed specific DNA-binding activity for the upstream region of sgcR1R2.Consisting with this,cross-complementation experiments suggested that sgcR3 occupies a higher rung than sgcR1 and sgcR2 in the hierarchy of C-1027 regulatory genes. Additional evidence was observed through the study of the gene expression in mutants by using quantitative real time RT-PCR.The data presented in this work set the stage for subsequent studies to delineate the complexity of the regulation of C-1027 biosynthesis, as well as for designing strategies for the construction of strains with enhanced C-1027 production.更多还原