基于Ⅱ型毒素–抗毒素系统构建ε–聚赖氨酸合成酶的链霉菌稳定表达载体

Construction of ε-Poly-L-lysine synthetase expression vector of Streptomyces based on type Ⅱ toxin-antitoxin system

鉴于ε–聚赖氨酸収酵过程对抗生素压力条件的依赖,尝试基于Ⅱ型毒素–抗毒素系统(relBE2sca)构建无选择压力下稳定表达ε–聚赖氨酸的淀粉酶产色链霉菌表达系统:将抗毒素基因relB2sca与ε–聚赖氨酸合成酶基因pls克隆至表达载体,幵导入淀粉酶产色链霉菌pls缺失突变株,将毒素基因relE2sca整合至淀粉酶产色链霉菌的染色体(突变株YY3),获得包含ε–聚赖氨酸稳定表达的突变株YY1。经过多次传代,相比对照组,在不含抗生素压力条件下,突变株YY1依然能够稳定地合成ε–聚赖氨酸。毒素蛋白RelE2sca的表达会导致变铅青链霉菌、阿维链霉菌和链霉菌FR–008等常用链霉菌异源表达宿主的死亡,提示基于Ⅱ型毒素–抗毒素系统(relBE2sca)可作为一种通用的遗传标记。

Considering the dependence of ε-Poly-L-lysine fermentation on antibiotics, Streptomyces diastatochromogenes (S.diastatochromogenes) expression system, which could express the ε-Poly-L-lysine stably without the addition of antibiotics, was constructed based on the type Ⅱ toxin-antitoxin system relBE2sca. The antitoxin relB2sca and pls gene encoding the ε-PL synthetase were cloned into the expression vector, which was transformed into the S.diastatochromogenes without the pls gene, and the relE2sca was integrated into S. diastatochromogenes chromosome (mutant YY3), producing the mutant YY1, harboring the stable expression system of ε-Poly-L-lysine. Compared with the controls, the mutant YY1 still could synthesize the ε-Poly-L-lysine stably without the addition of antibiotic after several transmissions. Finally, the expression of RelE2sca was shown to be lethal to the common Streptomyces heterologous expression hosts, implying the potential of relBE2sca as a common genetic marker.