Agrochemical control of gene expression using evolved split RNA polymerase. II.

Agrochemical inducible gene expression system provides cost-effective and orthogonal control of energy and information flow in bacterial cells. However, the previous version of Mandipropamid inducible gene expression system (Mandi-T7) became constitutively active at room temperature. We moved the split site of the eRNAP from position LYS179 to position ILE109. This new eRNAP showed proximity dependence at 23 °C, but not at 37 °C. We built Mandi-T7-v2 system based on the new eRNAP and it worked in both Escherichia coli and Agrobacterium tumefaciens. We also induced GFP expression in Agrobacterium cells in a semi-in vivo system. The modified eRNAP when combined with the leucine zipper-based dimerization system, behaved as a cold inducible gene expression system. Our new system provides a means to broaden the application of agrochemicals for both research and agricultural application. Portions of this text were previously published as part of a preprint (https://www.biorxiv.org/content/10.1101/2024.04.02.587689v1).