大肠杆菌中吡咯喹啉醌合成途径的构建

Construction of Pyrroloquinoline Quinone Synthesis Pathway in Escherichia coli

摘要:

目前吡咯喹啉醌（Pyrroloquinoline quinone，PQQ）在大肠杆菌中的异源合成主要以质粒为表达载体进行，但是质粒载体难以进行合成途径多基因表达的系统优化，并且容易造成发酵不稳定。作者以大肠杆菌为底盘生物，利用CRISPR/Cas9基因编辑技术，在基因组水平系统优化PQQ的合成。将来源于Gluconobacter oxydans 621H的操纵子pqqABCDE引入底盘大肠杆菌，并进一步通过优化合成途径基因表达强度，敲除大肠杆菌自身抑制基因及强化PQQ的胞内需求与胞外转运等，获得了一株能够高效合成PQQ的工程菌，摇瓶发酵72 h时产量达到86.3 mg/L。以大肠杆菌为底盘构建PQQ高效合成途径的工作能够为后续以其他底盘生物生产PQQ及相关代谢产物提供借鉴。

Abstract:

Currently, the heterologous synthesis of pyrroloquinoline quinone (PQQ) in Escherichia coli is mainly achieved via plasmid vector, which is difficult to systematically optimize the synthesis pathway of multiple gene expression, and easy to cause fermentation instability. In this study, E. coli was used as the chassis organism to systematically optimize the PQQ synthesis at the genome-scale using CRISPR/Cas9 gene editing technology. The operon pqqABCDE from Gluconobacter oxydans 621H was introduced into the chassis E. coli. An engineered strain for highly-efficient synthesis of PQQ was obtained by further optimizing the gene expression strength in the synthesis pathway, knocking out the native inhibitor gene in E. coli, and enhancing the intracellular demand and extracellular transport of PQQ. A total of 86.3 mg/L PQQ was produced after 72 h fermentation in shake-flask. In this study, the construction of efficient PQQ synthesis pathway in the chassis E. coli could provide a reference for future production of PQQ or related metabolites in other chassis.