(175e) Development of Toxr-like pH Regulator for the Optimization of Dahms Pathway in Engineered Escherichia coli

Bañares, A. B., Myongji University
Ramos, K. R. M., Myongji University
Lee, W. K., Myongji University
Nisola, G. M., Myongji University
Valdehuesa, K. N. G., Myongji University
Chung, W. J., Myongji University
Maintaining the pH of the medium at near physiological levels is crucial for the cellular processes, homeostasis, and growth of recombinant Escherichia coli strains designed to produce economically relevant compounds. One of the notorious problems in metabolic engineering is the production of acidic intermediates that could reduce the pH of the medium, creating an unsuitable extracellular environment for optimum growth and enzymatic function. Such condition elicits a stress response in the engineered strain leading to low yield and productivity. This problem is perennial to engineered E.coli utilizing the xylose oxidative pathway as the sole route for the synthesis of target compounds due to xylonate accumulation, most especially when xylose dehydrogenase (XDH) is overexpressed. Hence, in this study, a genetic switch is developed from a derivative of ToxR-like bacterial transmembrane one component regulator, which becomes activated at low extracellular pH. The designed-genetic switch was integrated into the Dahms pathway and was able to downregulate the expression of XDH at low pH. In effect, xylonate was produced at appropriate levels and rates. This limited the stress response and xylonate accumulation in the cell, leading to improved cell growth. Furthermore, the pH biosensor was utilized to increase the productivity and yield of ethylene glycol. To date, this is the first report on the use of a ToxR-like pH sensor in metabolic engineering. This technology is a promising tool for regulating the accumulation of acidic intermediates in other engineered metabolic pathways in E. coli.

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2018R1D1A1B07043993, No. 2018R1D1A1B07048107 and 22A20130012051(BK21Plus)) and by the Ministry of Science and ICT(2016R1C1B1013252).