热带假丝酵母诱变菌株筛选的两步浓缩与影印技术

Two-step enrichment and double-time replica plating technology for Candida tropicalis mutant selection

为提高热带假丝酵母转化烷烃生产长链二元酸的能力 ,建立了通过两步浓缩和双重影印技术筛选获得 α、ω-氧化增强、β-氧化减弱的热带假丝酵母诱变菌株的筛选体系。热带假丝酵母经过硫酸二乙酯和紫外诱变 ,其中的 β-氧化减弱或阻断的细胞在含有制霉菌素的 SEL1-1培养基中得到浓缩 (浓缩效率为 8倍 ) ;α、ω-氧化增强的突变株在含有氯丙嗪的 YPD培养基中被进一步浓缩 (浓缩效率为 3倍 )。此外 ,应用 SEL1影印技术 ,Alk-突变株可以被快速地分离出来。在 5× 10 8个诱变处理的细胞中应用上述体系筛选获得了 43株 β-氧化减弱或阻断的菌株。接着 ,通过 SEL2影印技术 ,获得了 10株 α。

This paper describes two step enrichment and double time replica plating technology to select mutants with enhanced α,ω oxidation and blocked β oxidation (Alk -). After C. tropicalis was mutated by DES and ultraviolet light, the Alk - mutants were enriched (first step enrichment had up to 8 fold in one round of enrichment) by treating with nystatin in medium SEL1 1. Then the mutagen treated cells were cultured in medium YPD containing chorpromazin for further enriching (the second step enrichment had up to 3 fold in one round of enrichment) the mutants with increasedα,ω oxidation capacity. The Alk - mutants were readily isolated by SEL 1 replica plating technology. A total of 43 Alk - mutants were isolated from 5×10 8 mutagen treated cells. In the following step, SEL2 replica plating technology was used for screening studied which showed that of the 43 Alk - mutants, 10 strains could rapidly produce DCA from alkane.