In this study, YlACL2 was inactivated by two methods: traditional approach based on homologous recombination and uracil marker and markerless system using CRISPR/Cas9. The efficiency of YlACL2 inactivation using traditional approach was 4% (one ΔYlacl2 strain out of 24 tested transformants) whereas knockout efficiency using CRISPR/Cas9 system was 75% (18 ΔYlacl2 strains out of 24 tested transformants). YlACL2 null mutant strains were not able to utilize citrate as a single carbon source. Growth kinetics was investigated in the media with glucose and acetate as a single carbon source. The fact that ΔYlacl2 is able to grow in the minimal medium with glucose as a single carbon source provides evidence that there is an alternative source of acetyl-CoA on carbohydrate substrates in Y. lipolytica.

在这项研究中，YlACL2通过两种方法失活：基于同源重组和尿嘧啶标记的传统方法以及使用CRISPR / Cas9的无标记系统。使用传统方法灭活Y1ACL2的效率为4％（24个测试的转化子中有一个ΔYlacl2菌株），而使用CRISPR / Cas9系统的敲除效率为75％（24个测试的转化子中有18个ΔYlacl2菌株）。Y1ACL2无效突变菌株不能利用柠檬酸盐作为单一碳源。在葡萄糖和乙酸盐为单一碳源的培养基中研究了生长动力学。ΔYlacl2的事实能够在以葡萄糖为单一碳源的基本培养基中生长，这提供了证据，表明Y的碳水化合物底物上还有乙酰辅酶A的替代来源。解脂脂。