CAREER: Epigenetic Regulation of Gene Expression in Engineered Prokaryotes

The soil contains a complex web of microbes, dominated by bacteria species, and these microbes are essential for soil health, soil stability and crop production. Engineered varieties of soil bacteria may help protect our soils by detecting and removing pollutants, or reducing the nitrogen needed to fertilize farmlands. To engineer soil bacteria efficiently and with minimal risk to the environment, a better understanding of how important genes are turned on and off is needed.

This CAREER research focuses on epigenetic regulation – gene expression that is controlled by chemical modifications to the DNA-and its role in the survival and performance of native and engineered strains of soil bacteria. This work will be the source of projects for a research-oriented upper-level undergraduate course and for interactive educational resources that teach the principles of bioengineering to undergraduates.

Epigenetic regulation of gene expression via genome methylation in prokaryotes has been vastly understudied. Differences in genome methylation have been observed under changing growth conditions, such as between exponential and stationary phase growth, which suggests that the growth environment significantly influences epigenetic regulation. However, understanding of epigenetic regulation in synthetic biology chassis organisms beyond E. coli, and in real-world environmental contexts, is extremely limited.

Pseudomonas putida is a versatile Gram-negative organism that is native to soil, and is being developed as a chassis for numerous industrial and environmental applications. Yet, no comprehensive study of epigenetic regulation, nor any investigation of epigenetic effects on synthetic gene circuit function, has yet been reported in P. putida. This CAREER program will examine how genome methylation affects gene expression changes that are essential for the transition of P. putida from laboratory to soil conditions.

These epigenetic changes may similarly affect the function of integrated synthetic gene circuits. The overall objective of this proposal is to investigate the impact of genome methylation on gene expression and synthetic gene circuit function in P. putida within its native soil environment. To achieve the overall objective, two specific objectives will be pursued: 1) Determine the effect of genome methylation on endogenous gene expression of P. putida in soil. 2) Control synthetic gene circuit expression in P. putida by modulation of DNA methylation.

The proposed research will be complemented by educational activities that create new open-access higher education resources for teaching the core principles of synthetic biology. The research proposed in this CAREER program will uncover important epigenetic principles that permit engineered organisms to thrive within a complex environment. The knowledge will be broadly translatable to the optimization of genetically engineered microbes for a range of environmental and industrial applications.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.