摘要
将依纽小单孢菌3′,4′-双脱羟基基因sisI组合到绛红小单孢菌G1008中,替代其中的同工异源酶基因genP,研究sisI基因异源表达的内在特征.以温敏型质粒pKC1139作为克隆载体,构建sisI替换genP的重组质粒pIP303,利用接合转移技术,将穿梭质粒导入绛红小单孢菌G1008基因组,影印筛选并通过PCR(聚合酶链式反应)鉴定,获得一株基因重组工程菌GIP95.借助TLC(薄层色谱)、HPLC(高效液相色谱)和MS(质谱)检测代谢产物组分及结构变化,结果表明突变株GIP95的代谢流未被阻断,仍能够合成庆大霉素C族组分.由此得出结论,外源基因依纽小单孢菌3′,4′-双脱羟基基因sisI参与了庆大霉素生物合成中绛红糖胺3′,4′-双脱羟基这一关键步骤,在绛红小单孢菌G1008中实现异源表达.
To research the heterologous expression of the inherent characteristics,sisI from Micromonospora inyoensis,which was presumed to be 3′,4′-double dehydroxylase gene,was assembled into Micromonospora purpurea G1008 and replaced the isoschizomer gene genP.Using the temperature-sensitive plasmid pKC1139 as cloning vector,the recombinant plasmid pIP303 for sisI-genPgene replacement was constructed and introduced into Micromonospora purpurea G1008 by conjugation.The combination mutant GIP95 was screened out by replica plating and PCR identification.Using TLC,HPLC and MS to analyze the components and structure changes of the metabolites.The results showed that the metabolic flow of the strain GIP95 was not blocked,which can still synthesize gentamicin C components.Gene sisIpresumed to be 3′,4′-double dehydroxylase gene from Micromonospora inyoensis was responsible for 3′,4′-double dehydroxylation of the magenta sugar amine in the biosynthesis of gentamicin,completing the heterologous expression in Micromonospora purpurea G1008.
出处
《延边大学学报（自然科学版）》
CAS
2016年第1期23-28,共6页
Journal of Yanbian University（Natural Science Edition）
