AUTHOR=Huang Jingwen , Chen Jiuzhou , Wang Yu , Shi Tuo , Ni Xiaomeng , Pu Wei , Liu Jiao , Zhou Yingyu , Cai Ningyun , Han Shuangyan , Zheng Ping , Sun Jibin 

TITLE=Development of a Hyperosmotic Stress Inducible Gene Expression System by Engineering the MtrA/MtrB-Dependent NCgl1418 Promoter in Corynebacterium glutamicum

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.718511

DOI=10.3389/fmicb.2021.718511

ISSN=1664-302X

ABSTRACT=<p><italic>Corynebacterium glutamicum</italic> is an important workhorse for industrial production of diversiform bioproducts. Precise regulation of gene expression is crucial for metabolic balance and enhancing production of target molecules. Auto-inducible promoters, which can be activated without expensive inducers, are ideal regulatory tools for industrial-scale application. However, few auto-inducible promoters have been identified and applied in <italic>C. glutamicum</italic>. Here, a hyperosmotic stress inducible gene expression system was developed and used for metabolic engineering of <italic>C. glutamicum</italic>. The promoter of <italic>NCgl1418</italic> (P<italic><sub>NCgl1418</sub></italic>) that was activated by the two-component signal transduction system MtrA/MtrB was found to exhibit a high inducibility under hyperosmotic stress conditions. A synthetic promoter library was then constructed by randomizing the flanking and space regions of P<italic><sub>NCgl1418</sub></italic>, and mutant promoters exhibiting high strength were isolated <italic>via</italic> fluorescence activated cell sorting (FACS)-based high-throughput screening. The hyperosmotic stress inducible gene expression system was applied to regulate the expression of <italic>lysE</italic> encoding a lysine exporter and repress four genes involved in lysine biosynthesis (<italic>gltA</italic>, <italic>pck</italic>, <italic>pgi</italic>, and <italic>hom</italic>) by CRISPR interference, which increased the lysine titer by 64.7% (from 17.0 to 28.0 g/L) in bioreactors. The hyperosmotic stress inducible gene expression system developed here is a simple and effective tool for gene auto-regulation in <italic>C. glutamicum</italic> and holds promise for metabolic engineering of <italic>C. glutamicum</italic> to produce valuable chemicals and fuels.</p>