Random Mutagenesis by Insertion of Error-Prone PCR Products to the Chromosome of Bacillus subtilis.
Random Mutagenesis by Insertion of Error-Prone PCR Products to the Chromosome of Bacillus subtilis.
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通过将易错 PCR 产物插入枯草芽孢杆菌染色体进行随机诱变。
DOI：
10.3389/fmicb.2020.570280
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发表时间：
2020
影响因子：
5.2
通讯作者：
Yan X
中科院分区：
文献类型：
--
作者：
Ye B;Li Y;Tao Q;Yao X;Cheng M;Yan X
枯草芽孢杆菌是酶定向进化的理想宿主，其底物不能跨细胞膜运输。然而，在枯草杆菌中产生突变文库存在文库小、质粒不稳定和杂合性等问题。在此，通过在枯草杆菌染色体上插入容易出错的聚合酶链式反应(EpPCR)产物，建立了一个庞大的随机突变体文库。具体地说，利用基于聚合酶链式反应的多聚体方法将epPCR产物与侧翼区域和抗生素耐药标记融合，产生插入构建体。将插入载体转化到枯草芽孢杆菌SCK6菌株的超级细胞中，通过同源重组将epPCR产物整合到染色体上。通过共表达同源重组促进蛋白NgAgo，增加感受态细胞数，增加侧翼区域长度，提高了插入载体的转化效率。使用每个μg插入构建的方法构建了一个包含5.31x105个随机突变体的文库，这足以进行定向进化。文库生成过程在1天内完成。通过提高枯草杆菌甲基对硫磷水解酶(MPH)对毒死蜱的活性和提高MPH的分泌水平，证实了该方法的有效性。综上所述，本工作提供了一种快速有效的方法将epPCR产物整合到枯草杆菌的染色体中，促进了目标蛋白的定向进化和表达优化。
Bacillus subtilis is an attractive host for the directed evolution of the enzymes whose substrates cannot be transported across cell membrane. However, the generation of a mutant library in B. subtilis suffers problems of small library size, plasmid instability, and heterozygosity. Here, a large library of random mutant was created by inserting error-prone PCR (epPCR) products to the chromosome of B. subtilis. Specifically, the epPCR product was fused with flanking regions and antibiotic resistant marker using a PCR-based multimerization method, generating insertion construct. The epPCR product was integrated into the chromosome via homologous recombination after the insertion construct was transformed into the supercompetent cells of B. subtilis strain SCK6. The transformation efficiency of the insertion construct was improved through co-expressing homologous recombination-promoting protein NgAgo, raising the number of competent cells, and increasing the length of flanking regions. A library containing 5.31 × 105 random mutants was constructed using per μg insertion construct, which is sufficient for directed evolution. The library generation process was accomplished within 1 day. The effectiveness of this method was confirmed by improving the activity of Methyl Parathion Hydrolase (MPH) toward chlorpyrifos and by enhancing the secretion level of MPH in B. subtilis. Taken together, the present work provides a fast and efficient method to integrate epPCR products into the chromosome of B. subtilis, facilitating directed evolution and expression optimization of target proteins.
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原核 Argonaute 蛋白增强细菌中的同源序列定向重组。
影响因子：
14.9
作者：
Fu, Lei;Xie, Caiyun;Zhang, Anding
通讯作者：
Zhang, Anding
影响因子：
2.7
作者：
Shafikhani, S;Siegel, RA;Schellenberger, V
通讯作者：
Schellenberger, V
影响因子：
14.9
作者：
Melnikov, A;Youngman, PJ
通讯作者：
Youngman, PJ
影响因子：
6.8
作者：
Goldsmith, Moshe;Tawfik, Dan S.
通讯作者：
Tawfik, Dan S.
DOI：
10.1007/bf00273591
发表时间：
1991-04-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
BRON, S;HOLSAPPEL, S;PEETERS, BPH
通讯作者：
PEETERS, BPH