CRISPR/Cas9介导的果糖低消耗酿酒酵母工程菌的构建

CRISPR/Cas9-assisted Construction of Engineered Low-fructose-utilization Saccharomyces cerevisiae

摘要:

利用酿酒酵母为宿主，以果糖为原料合成D-阿洛酮糖具有食品方面的先天优势。为了减少酿酒酵母宿主本身对果糖的消耗，对酿酒酵母的己糖激酶同工酶2（Hexokinase isoenzyme 2，hxk2）基因进行编辑。本研究采用CRISPR/Cas9技术，构建了Cas9和gRNA共表达质粒pYES2-CG-Δhxk2，以酿酒酵母BY4741为出发菌株，URA3为筛选标记，采用高效电击转化法和胞内同源重组技术，获得hxk2基因缺陷株BY4741-Δhxk2。在此基础上，进行果糖发酵实验以评估突变菌株的果糖消耗速率。实验结果显示，发酵培养14 h时，缺陷株BY4741-Δhxk2果糖消耗速率为3.35 mg/h；与野生型菌株相比其下降了6.42%。此外，发酵培养22 h，BY4741-Δhxk2的OD600nm值为8.65，相比于野生型提高了6.40%。研究表明，己糖激酶hxk2基因的缺陷编辑可以一定程度降低酿酒酵母对果糖的利用，同时缺陷株较野生型表现出一定的生长优势，这为后续以酿酒酵母为宿主生产D-阿洛酮糖奠定了初步基础。

Abstract:

The production of D-psicose using D-fructose as the raw material and Saccharomyces cerevisiae as the host has inherent advantages in food applications. In order to reduce the consumption of fructose by the hostSaccharomyces cerevisiae itself, the hexokinase isoenzyme 2 (hxk2) gene was editedin Saccharomyces cerevisiae. In this study, CRISPR/Cas9 technology was used to construct Cas9 and gRNA co-expression plasmid pYES2-CG-?hxk2, and using Saccharomyces cerevisiae BY4741 as the starting strain and URA3 as the selection marker, hxk2-deficient strain BY4741-?hxk2 was obtained by high-efficiency electroporation-induced transformation and intracellular homologous recombination technology. On this basis, a fructose fermentation experiment was conducted to evaluate the consumption rate of D-fructose in the mutant strain BY4741-?hxk2. The experimental results showed that the fructose consumption rate in the BY4741-?hxk2 strain after 14 h of fermentation was 3.35 mg/h (which decreased by 6.42% compared with wild strain BY4741). In addition, the OD600 nm of BY4741-?hxk2 was 8.65 after 22h of fermentation (which increased by6.40% compared with BY4741). This research shows that deleting the hexokinase hxk2 gene can reduce the utilization of fructose by Saccharomyces cerevisiae to a certain extent, and the defective strain exhibited advantages in growth compared with the wild type. These findings lay a foundation for the subsequent production of D-psicose by Saccharomyces cerevisiae using BY4741-Δhxk2 as the host.