Применение CRISPR/Cas9-системы редактирования генома в дрожжах Yarrowia lipolytica для инактивации гена YIACL2, кодирующего АТФ-зависимую цитратлиазу

Abstract

In this study, Y1ACL2 was inactivated by two methods: traditional approach based on homologous recombination and uracil marker and markerless system using CRISPR/Cas9. The efficiency of YIACL2 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). YIACL2 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. Yarrowia lipolytica, CRISPR/Cas9 system, ATP citrate lyase, YIACL2. The work was carried out using the equipment of the Unique Scientific Facility of BRC VKPM with technical support of the Centre for Collective Use of NRC «Kurchatov Institute» -GosNIIgenetika and with financial support of Russian Federation (state task No. 595-00003-19 PR) as well as partially supported by grant No. MK-2241.2019.7.