文献类型：期刊文献

英文题名：Rational design and characterization of enhanced alcohol-inducible synthetic promoters in Pichia pastoris

作者：Liu, Qi[1];Li, Yun-hao[1];Tao, Liu-fei[1];Yang, Jia-yi[1];Zhang, Yi-lun[1];Cai, Meng-hao[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg, Shanghai, Peoples R China

年份：2025

卷号：91

期号：1

外文期刊名：APPLIED AND ENVIRONMENTAL MICROBIOLOGY

收录：;EI(收录号：20250617835140);WOS:【SCI-EXPANDED(收录号:WOS:001381007100001)】；

基金：This study was supported by Young Scientist Fund of National Natural Science Foundation of China (32201206), National Key Research and Development Program of China (2022YFC2805102), and China Postdoctoral Science Foundation (2022M711146).

语种：英文

外文关键词：Pichia pastoris; methanol; ethanol; synthetic promoter

摘要：The C1 and C2 alcohols hold great promise as substrates for biomanufacturing due to their low cost and rich resources. Pichia pastoris is considered a preferred host for methanol and ethanol bioconversion due to its natural utilization of methanol and ethanol. However, the scarcity of strong and tightly regulated alcohol-inducible promoters limits its extended use. This study aimed to develop enhanced methanol- and ethanol-inducible promoters capable of improving gene expression in P. pastoris. Rational design strategies were employed to rewire the upstream regulatory sequence of the methanol-inducible P-AOX1, generating several high-strength methanol-inducible promoters with a stringent regulatory pattern. Eleven strong promoters were identified from 36 endogenous ethanol-inducible candidates recognized from transcriptome analysis. Core promoter regions, the crucial element influencing transcriptional strength, were also characterized. Five high-activity core promoters were then combined with four upstream regulatory sequences of high-strength promoters, resulting in four groups of synthetic promoters. Ultimately, the highly active methanol-inducible P-A13 and ethanol-inducible P-0688 and PsynIV-5 were selected for the expression of an alpha-amylase and yielded enzyme activity 1.6, 2.6, and 4.5 times higher as compared to that of P-AOX1. This work expands the genetic toolkit available for P. pastoris, providing more precise and efficient options for regulating gene expression. It benefits the use of P. pastoris as an efficient platform for the C1 and C2 alcohol-based biotransformation in industrial biotechnology.