卷枝毛霉pyrG基因缺陷突变株的诱变筛选与鉴定

Isolation and Characterization of Mucor circinelloides pyr G Negative Mutant Strain

为制备新的遗传筛选标记用于构建高产番茄红素的工程菌株,实验运用化学诱变的手段,以N-甲基-N'-硝基-N-亚硝基胍为诱变剂,以番茄红素生产菌株卷枝毛霉MU616为出发菌株,诱变获得5株尿嘧啶缺陷型突变株,突变株在基本培养基中培养5天后仍不能生长。通过同源转化带有pyr G基因(编码乳清酸核苷-磷酸盐脱羧酶)的质粒p EPM1确定突变株Mt1、Mt4和Mt5为pyr G基因缺陷突变株。随之对pyr G突变株进行生长特性的研究和产番茄红素性能的检测,结果表明,其中突变株Mt4的生物量为(9.0±0.6)g/L,番茄红素产量在黑暗和光照条件下分别为(1 648±185)μg/g和(3 234±281)μg/g,均与出发菌株相似,适宜作为进行卷枝毛霉转化的带有遗传标记的受体菌。pyr G基因缺陷突变菌株的获得对构建高产番茄红素的卷枝毛霉工程菌具有重要的意义。

To generate genetic selective marker for constructing super-producing lycopene strains,chemical mutagenesis was applied in mutagenizing Mucor circinelloides MU616. Five uracil auxotroph strains of Mucor circcinelloides were isolated with nitrosoguanidine and selected by resistance to 5-fluoroorotic acid,and these mutants were unable to grow in the minimal medium even after 5 days＇ culture. Plasmid p EPM1 which harbors the pyr G gene encoding orotidine-5＇- monophosphate decarboxylase in Mucor circinelloides were used to transformed the representative uracil auxotroph strains,the result demonstrated that mutants Mt1,Mt4 and Mt5 were deficient in pyr G gene. However,the lycopene content analysis shown that only Mt4 maintained the capacity of accumulating lycopene [（ 1648 ± 185） μg / g and（ 3 234 ± 281） μg / g under dark and light conditions respectively],in addition,the biomass [（ 9. 0 ± 0. 6） g / L] and growth properties of Mt4 is also similar to the original strain MU616. It suggested that Mt4 could be used as the receipt strain for the subsequent molecular manipulation. The acquisition of the pyr G mutant strain is very important for construction of the super-producing lycopene strains by molecular genetic manipulation.