Developing a Saccharomyces cerevisiae system for optimizing the expression of recombinant eukaryotic proteins. Two deletion mutants, which were hypersensitive to H2O2, were obtained by knocking out CTT1 and SOD2, respectively. The mutation rate of the mutants was up to over 4000 times of the spontaneous mutation rate when treated with H2O2. Endoglucanase Cel5A was used as a model enzyme to evaluate the system for improving the expression of the recombinant protein. Sixteen mutants of the RDKY3615 (ctt1a dagger) transformant and seven mutants of the RDKY3615 (sod2a dagger) transformant had significantly high Cel5A activity, while none mutants of the RDKY3615 transformant had significantly high enzyme activity. The combination of deletion mutagenesis and H2O2 treatment greatly accelerate the generation of genetic variants and will be a useful tool in improving the heterologous expression in S. cerevisiae.