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

Area of Science:

• Synthetic biology
• Bacterial gene regulation

Background:

• Agrochemical inducible gene expression systems offer cost-effective control in bacteria.
• Previous Mandipropamid (Mandi-T7) system showed constitutive activity at room temperature.

Purpose of the Study:

• To engineer a more robust and controllable Mandipropamid inducible gene expression system.
• To enhance the application scope of agrochemical-based gene regulation.

Main Methods:

• Modified the split site of the engineered RNA polymerase (eRNAP) from LYS179 to ILE109.
• Developed the Mandi-T7-v2 system using the modified eRNAP.
• Tested the system in *Escherichia coli* and *Agrobacterium tumefaciens*.
• Demonstrated GFP expression induction in *Agrobacterium* semi-in vivo.

Main Results:

• The new eRNAP exhibited proximity dependence at 23 °C but not at 37 °C.
• Mandi-T7-v2 system functioned effectively in both *E. coli* and *A. tumefaciens*.
• The modified eRNAP, with a leucine zipper system, acted as a cold-inducible system.

Conclusions:

• The improved Mandi-T7-v2 system provides orthogonal and controllable gene expression.
• This advancement broadens the utility of agrochemicals in research and agriculture.
• The cold-inducible nature expands potential applications for bacterial systems.