Abstract: The first important step toward a successful preparation of large and diverse promoter library with desired complexity, is to select a suitable mutagenesis strategy. To generate a promoter library of GAP promoter（pGAP）variants, mutations were introduced using error-prone PCR. After optimization of the conditions for EP-PCR random mutagenes, high mutation（error rate 1.1%）frequence was obtained using 1 ng/μL template and 10 mmol/L Mg²⁺, in combination with 25 thermal cycles. To increase mutational diversity and reach an appropriate error rate, three consecutive rounds of EP-PCR were carried out under the same conditions. After random sequencing of 10 clones from each round, an overall range of mutation rates from 1.1% to 4.0% was observed. Then, 250 clones containing pGAP variants were screened using the highthroughput screening approach in 48-deep-well plates. Among them, 5 mutants exhibited higher fluorescent intensity compared to the wild-type promoter.

Key words: Error-prone, PCR, Mutation frequency, Random mutagenesis, Pichia pastoris, GAP promoter