Dynamic Regulation of Transporter Expression to Increase L-Threonine Production Using L-Threonine Biosensors
Sumeng Wang, Ruxin Hao, Xin Jin, Xiaomeng Li, Qingsheng Qi, Quanfeng Liang
Abstract
The cytotoxicity of overexpressed transporters limits their application in biochemical production. To overcome this problem, we developed a feedback circuit for L-threonine production that uses a biosensor to regulate transporter expression. First, we used IPTG-induced rhtA regulation, L-threonine exporter, to simulate dynamic regulation for improving L-threonine production, and the results show that it had significant advantages compared with the constitutive overexpression of rhtA. To further construct a feedback circuit for rhtA auto-regulation, three L-threonine sensing promoters, PcysJ, PcysD, and PcysJH, were characterized with gradually decreasing strength. The dynamic expression of rhtA with a threonine-activated promoter considerably increased L-threonine production (21.19 g/L) beyond that attainable by the constitutive expression of rhtA (8.55 g/L). Finally, the autoregulation method was used in regulating rhtB and rhtC to improve L-threonine production and achieve a high titer of 26.78 g/L (a 161.01% increase), a yield of 0.627 g/g glucose, and a productivity of 0.743 g/L/h in shake-flask fermentation. This study analyzed in detail the influence of dynamic regulation and the constitutive expression of transporters on L-threonine production. For the first time, we confirmed that dynamically regulating transporter levels can efficiently promote L-threonine production by using the end-product biosensor.