Dynamic Regulation of Transporter Expression to Increase L-Threonine Production Using L-Threonine Biosensors

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, P_cysJ, P_cysD, and P_cysJH, 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.