摘要
目的:在天蓝色链霉菌Streptomyces coelicolor A3(2)中多效性调节因子AtrA(AtrA-c)可通过激活放线紫红素途径特异性的调节因子ActII-ORF4的转录来控制放线紫红素的产生。在灰色链霉菌Streptomyces griseus NBRC13350和阿维链霉菌Streptomyces avermitilis MA-4680中也发现了AtrA-c编码基因(atrA-c)的同源基因,分别影响链霉素和阿维菌素的生物合成。为探索球孢链霉菌C-1027(Streptomyces globisporus C-1027)中是否存在AtrA,克隆球孢链霉菌C-1027中atrA基因并进行生物信息学分析,为进一步确定其对力达霉素产生的调控作用及调控机制奠定基础。方法:采用在球孢链霉菌C-1027中异源表达AtrA-c,来确定AtrA-c对力达霉素产量的影响;通过Southern blot分析来判断在球孢链霉菌C-1027基因组中是否有atrA-c同源基因;PCR扩增方法获得球孢链霉菌C-1027 atrA基因(atrA-gl)并测序;通过多种生物信息学软件来分析atrA-gl及其与旁侧基因的组织结构、对已发现的AtrA蛋白进行同源性比对及亲缘关系分析。结果:在球孢链霉菌C-1027中异源表达天蓝色链霉菌AtrA-c蛋白,发现其对力达霉素的产量有影响。以atrA-c为探针,通过Southern blot分析显示球孢链霉菌C-1027基因组中存在atrA-c的同源基因。PCR扩增得到球孢链霉菌C-1027的atrA基因的全序列以及该基因上下游的旁侧序列(GenBank/EMBL/DDBJ登录号GU723707)。通过对球孢链霉菌C-1027、天蓝色链霉菌A3(2)、灰色链霉菌NBRC13350以及阿维链霉菌MA-4680 AtrA蛋白序列进行同源性分析发现,4种AtrA蛋白编码氨基酸序列一致性达到65%～87%,相似性高达70%～89%。并且,球孢链霉菌C-1027 atrA基因与相邻基因形成的组织结构与天蓝色链霉菌和灰色链霉菌完全一致。根据蛋白质同源性绘制进化树,发现球孢链霉菌AtrA蛋白与灰色链霉菌AtrA蛋白亲缘关系最近。结论:确定在球孢链霉菌C-1027中存在atrA同源基因并影响力达霉素的产�
Objective：AtrA （AtrA-c） was characterized as a transcriptional activator for actII-ORF4 gene which encoding a pathway-specific transcriptional activator of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3 （2）. Two atrA homologues in Streptomyces griseus NBRC13350 and Streptomyces avermitilis MA-4680 were identified to be regulators for the production of streptomycin and avermectin Bla respectively. The purpose is to identify and clone a new atrA homologue in Streptomyces globisporus C-1027 that can produce a chromoprotein antitumor antibiotic lidamycin. Methods： Heterologous expression of AtrA-c, Southern blotting, PCR, sequencing and bioinformatic analysis were used. Results：The heterologous expression of AtrA-c in S. globisporus C-1027 reduced the production of lidamycin production. Southern blotting analysis revealed that the S. globisporus C-1027 genome contains at least one copy of atrA-c homologue （atrA-gl）. atrA- gl and flanking gene fragments were successfully amplified with the primers designed by the alignment of amino acid sequences of AtrA from distinct Streptomyces （GenBank/EMBL/DDBJ accession number GU723707）. The alignments of amino acid sequences showed AtrA-gl shares highly conserved sequences with other homologous AtrA proteins, with a percentage of identity ranging from 65% to 87% and similarity ranging from 70% to 89%. The alignments of amino acid sequences of the flanking gene arrangement in their genomic context. Phylogenetic analysis AtrA-g have a relatively more closer relationship. Conclusion located outside the gene cluster in S. globisporus C-1027 for the stage for subsequent studies to elucidate the regulation fragments suggested four atrA genes have identical showed that S. globisporus AtrA-gl and S. griseus ： atrA-gl is the first cloned transcriptional regulator lidamycin production. The data presented here set of C-1027 biosynthesis, as well as for designing strategies for the construction of strains with enhanced C-1027 production.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2010年第8期52-59,共8页
China Biotechnology
基金
国家"十一五"计划"重大新药创制"科技重大专项(2009ZX09501-008)
国家自然科学基金(30973668
30572274)
国家"863"计划(2006AA02Z223)
教育部新世纪优秀人才支持计划(NCET-06-0157)
北京市自然科学基金(5102032)资助项目
