大肠杆菌乙酰-CoA羧化酶基因异源表达对变铅青链霉菌次级代谢的影响

Effect of heterologous expression of Escherichia coli acetyl-CoA carboxylase genes on the secondary metabolism of Streptomyces lividans

克隆组装了大肠杆菌的乙酰-CoA羧化酶基因acc,以研究其异源表达对变铅青链霉菌中2种"沉默"抗生素放线紫红素和十一烷基灵菌红素合成的影响。大肠杆菌ACC由4个基因编码,通过PCR扩增4个基因,并通过重叠延伸PCR加上ermE启动子,构建得到4个重组基因;再将重组基因依次组装得到异源表达质粒pHLZ11,接合转移到变铅青链霉菌中进行异源表达。结果显示:含有异源表达质粒的变铅青链霉菌接合转移子能合成放线紫红素而十一烷基灵菌红素产量提高。表明大肠杆菌acc基因异源表达能够激活变铅青链霉菌沉默抗生素的合成,并提高已有抗生素产量。

The Streptomyces lividans genome carries two gene clusters encoding the biosynthetic pathways of actinorhodin and undecylprodigiosin.However neither antibiotic is produced in S.lividans under the conditions of common laboratory growth.Malonyl-CoA as precursor was needed in the biosynthesis of both compounds catalyzed by the committed enzyme acetyl-CoA carboxylase（ACC）.This study was aimed to clone the acc genes from Escherichia coli and introduce them into S.lividans to investigate their effect on the production of actinorhodin and undecylprodigiosin.The E.coli ACC subunits are encoded by 4 acc genes.These acc genes were cloned with adding ermE promoter to each through overlapping extension PCR to gain 4 recombined genes.The recombinant genes were then cloned into pSET152 successively to gain a heterologous expression plasmid.The constructed plasmid was transferred into S.lividans by conjugation,and the antibiotic production in exconjugants was examined.The exconjugants could not only produce actinorhodin but also showed a prominent increase in the production of undecylprodigiosin.acc from E.coli can improve the production of pre-existing antibiotics and activate the expression of silent gene clusters as well in S.lividans.