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