依纽小单胞菌2-脱氧蟹肌醇合成酶基因的克隆与表达

Cloning and Expression of 2-deoxy-scyllo-inosose Synthase Gene sisB from Micromonospora inyoensis

从伊纽小单孢菌(Micromonospora inyoensis)扩增参与紫苏霉素生物合成的2-脱氧蟹肌醇合成酶基因sisB,并分别将其克隆到筛选载体pUC18和表达载体pET-30a上,其开放阅读框长1 185 bp,编码含394个氨基酸残基(41.94 ku)的多肽链,将pET-sisB转化大肠杆菌E.coliBL21(DE3),使sisB基因实现表达.基因sisB的碱基序列与棘孢小单孢菌(M.echinospora)的基因gntB的碱基序列的同源性高达93%.预测的SisB蛋白序列与GntB蛋白序列的同源性为95.2%.基因sisB是继紫苏霉素抗性基因srm1(AY661430)和参与紫苏霉素生物合成的糖基转移酶基因sisD(DQ250992)和sisZ(DQ250994)后,从依纽小单孢菌克隆到的又一新基因.该基因在GenBank的接受号为DQ250993.

The gene sisB,a novel 2-deoxy-scyUo-inosose synthase gene, was cloned. The DNA fragment containing s/sB gene （the key gene to take part in sisomicin biosynthesis） was isolated from Micromonospora inyoensis and cloned into pUC18 vector. Its ORF is 1 185 bp coding 394 AA （41.94 ku）. Sequence analysis verified that s/sB gene and its amino acids sequence showed 93% and 95.2% identity with gntB （gentamicin biosynthesis gene from M. echinospora）, respectively. The prediction of structure of sisB suggested it is also a 2-dcoxy-scyllo-inosose synthase protein. The sisB expression vector pET-sisB was constructed by fusion of the flanking sisB ORF in the pET-30a plasrnid. SisB protein was expressed by IPTG induction in E. coli BL21（DE3）. sisB is another gene cloned from M. inyoensis after sisomicin resistance gene srm1, glycosyltransferase gene sisD and sisZ.