Construction of an engineered Saccharomyces cerevisiae for lignin peroxidase production

Abstract:

[Objective] To obtain engineered Saccharomyces cerevisiae with high-yield of lignin peroxidase. [Methods] We cloned a constitutive promoter PGK, an exogenous protein secretion signal peptide α-factor, lignin peroxidases (LiP) genes and a terminator CYC1. We constructed complete expression box (PαLC) by overlap extension PCR method. Then, we established expression vector of lignin peroxidase S. cerevisiae via rDNA integration method, to achieve multi-copy expression of lignin peroxidase in S. cerevisiae. Then, we identified the copy number via droplet digital PCR technology, to explore the relationship between the copy number and protein expression amount. [Results] We got the engineered strain of S. cerevisiae to produce lignin peroxidase with the copy number of 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 via rDNA integration method, through enzymatic activity determination, it shows that the enzymatic activity is as highest as 367 U/L when the copy number is 7. [Conclusion] In this study, lignin peroxidase was expressed in S. cerevisiae, and the relationship between gene copy number and enzyme activity was studied, which is of great significance to the development of lignin degradation technology.