定向进化 PobR 变构转录因子以生成 4-羟苯乙酸的生物传感器。

Directed evolution of the PobR allosteric transcription factor to generate a biosensor for 4-hydroxymandelic acid.

机构信息

School of Life Science, Northeast Forestry University, Harbin, 150040, Heilongjiang, People's Republic of China.

NEFU-China iGEM Team, Northeast Forestry University, Harbin, 150040, Heilongjiang, People's Republic of China.

出版信息

World J Microbiol Biotechnol. 2022 May 3;38(6):104. doi: 10.1007/s11274-022-03286-5.

DOI:10.1007/s11274-022-03286-5

PMID:35501522

Abstract

Hydroxy-mandelic acid (HMA) is widely applied in pharmaceuticals, food and cosmetics. In this study, we aimed to develop an allosteric transcription factors (aTFs) based biosensor for HMA. PobR, an aTF for HMA analog 4-hydroxybenzoic acid, was used to alter its selectivity and create novel aTFs responsive to HMA by directed evolution. We established a PobR mutant library with a capacity of 550,000 mutants using error-prone PCR and Megawhop PCR. Through our screening, two mutants were obtained with responsiveness to HMA. Analysis of each missense mutation indicating residues 122-126 were involved in its PobR ligand specificity. These results showed the effectiveness of directed evolution in switching the ligand specificity of a biosensor and improving HMA production.

摘要

羟基扁桃酸（HMA）广泛应用于医药、食品和化妆品。本研究旨在开发一种基于别构转录因子（aTFs）的 HMA 生物传感器。PobR 是 HMA 类似物 4-羟基苯甲酸的 aTF，通过定向进化改变其选择性并创建响应 HMA 的新型 aTFs。我们使用易错 PCR 和 Megawhop PCR 建立了一个容量为 55 万突变体的 PobR 突变体文库。通过筛选，获得了两个对 HMA 有响应的突变体。分析每个错义突变表明残基 122-126 参与了 PobR 配体特异性。这些结果表明，定向进化在改变生物传感器的配体特异性和提高 HMA 产量方面是有效的。