Abstract:[Objective] In this study, the effect of the mutant srp4039 gene on isobutanol tolerance of Saccharomyces cerevisiae was studied.[Methods] Firstly, the wild-type SRP40 gene and the mutant srp4039 gene were respectively cloned from the chromosome DNAs of the wild-type strain W303-1A and the mutant EMS39 of S. cerevisiae. Then, SRP40 and srp4039 were respectively ligated to YCplac22 to construct the recombinant plasmids YCplac22-SRP40 and YCplac22-srp4039. The obtained recombinant plasmids and the YCplac22 empty plasmid were respectively transformed into the wild-type strain W303-1A to construct the engineering strains W303-1A-SRP40, W303-1A-srp4039, and W303-1A-control. The three engineering strains were then fermented in the complete media containing 1.0% isobutanol, 1.3% isobutanol, 8.0% ethanol, and 0.5% isoamyl alcohol, respectively. The cell density (OD600) was measured, and the specific growth rate in 2-10 h was calculated. The three engineering strains were heated at 55℃ for 4 min and then diluted for observation of cell growth on the plate. Finally, the amino acid sequences of SRP40 and srp4039 were analyzed by bioinformatics tools.[Results] In the medium without isobutanol (0% isobutanol), the three strains showed no significant difference. After fermentation for 24 h in the complete media containing 1.0% isobutanol and 1.3% isobutanol, W303-1A-srp4039 showed the cell density 1.12 and 1.06 times that of W303-1A-SRP40, and 1.10 and 1.10 times that of W303-1A-control, respectively; the specific growth rate of W303-1A-srp4039 was 1.07 and 1.10 times as high as that of W303-1A-SRP40, and 1.10 and 1.10 times as high as that of W303-1A-control, respectively. After fermentation for 24 h in the complete media containing 8.0% ethanol and 0.5% isoamyl alcohol, W303-1A-srp4039 showed the cell density 1.12 and 1.01 times that of W303-1A-SRP40, and 1.17 and 1.07 times that of W303-1A-control, respectively; W303-1A-srp4039 showed the specific growth rate 1.37 and 1.07 times as high as that of W303-1A-SRP40, and 1.31 and 1.09 times as high as that of W303-1A-control, respectively. Moreover, W303-1A-srp4039 still grew better than the other two strains after heat shock. The bioinformatics analysis showed that Srp4039 protein had a similar structure to the pleuralin-1 of diatom.[Conclusion] We found that the srp4039 mutant gene can enhance the tolerance of S. cerevisiae to isobutanol, and plays a role in enhancing the tolerance to ethanol, isoamyl alcohol, and heat. Srp4039 protein has a similar structure with the pleuralin-1 of diatom, which indicates that Srp4039 protein may have the function of pleuralin-1 and play a role in cell wall maintenance. These findings will provide new ideas for improving the tolerance of S. cerevisiae to ethanol, isobutanol, isoamyl alcohol, and high temperature.
