Abstract:[Background] Saccharomyces cerevisiae is widely used to produce industrial enzymes and pharmaceutical proteins. However, low protein production level and poor secretion efficiency are the major bottlenecks for its industrial applications. [Objective] To improve protein production by the recombinant yeast strains, and provide basis for development of robust yeast cell factory for heterologous protein secretion. [Methods] The cell wall protein encoding gene CWP2 was disrupted through CRISPR/cas9-based genome editing in the recombinant strain S. cerevisiae Y294-BGL, which can secret β-glucosidase. [Results] At 96 h of fermentation, the extracellular activity of β-glucosidase in the mutant Bcwp2△was improved by 53%, and intracellular activity was improved by 208%. No negative effect on cell growth was observed, and no significant change was observed in the tolerance to acetic acid and ethanol in the mutant yeast strain Bcwp2△. In addition, no difference in growth of the mutant in the presence of the endoplasmic reticulum stress inducers dithiothreitol and tunicamycin was observed. Further studies showed that less reactive oxygen species was accumulated inside the cells of the mutant. Disruption of CWP2 decreased transcription of genes associated with protein trafficking and secretion, as well as cell wall biosynthesis genes. [Conclusion] Disruption of the cell wall protein encoding gene CWP2 promotes extracellular β-glucosidase activity, and can be employed as a target in metabolic engineering of yeast protein production.
