优化易错PCR条件以提高毕赤酵母GAP启动子文库突变效率

High-error-rate Random Mutagenesis of GAP Promoter in Pichia pastoris Using an Optimited Error Prone PCR

高效的随机突变策略对于构建含有丰富突变体的启动子文库至关重要。为了建立一个突变率适中并能获得较多有益突变子的易错PCR(error-prone PCR,EP-PCR)条件,实现毕赤酵母GAP启动子的高效突变,对EP-PCR反应条件进行了优化。考察了模板浓度、反应循环数和Mg2+浓度对EP-PCR的产物得率和突变率的影响后,确定了适于GAP启动子突变的EP-PCR条件:模板浓度、反应循环数和Mg2+浓度分别为1 ng/μL、25和10 mmol/L。优化EP-PCR条件后,GAP启动子突变率为1.1%,连续进行3轮EP-PCR后突变率可达到4.0%。利用优化后EP-PCR对GAP启动子进行随机突变,筛选了250个突变子,获得5个启动子强度高于野生型GAP启动子的突变体,有益突变达到了2%,可用于构建GAP启动子文库。

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.