Project Outline

There is a growing trend for the development of biological processes for production of organic chemicals such as biofuels, platform chemicals, and precursors for synthesis of a range of products (Yang21). These processes are greener than the traditional chemical synthesis routes, typically requiring less energy and fewer solvents, and producing less hazardous wastes. For example, E. coli is currently used commercially for the production of 1,3-propanediol and 1,4-butanediol. However, many of the products of these processes damage the membranes of E. coli and are therefore toxic, severely limiting yields.

Most cells have the ability to pump out toxic molecules. Gram-negative bacteria have multiple mechanisms for efflux of noxious chemicals, the best-studied being the RND efflux pumps. These are tripartite pumps which span the inner and outer membrane and use proton motive force to drive export of a wide range of endogenous and exogenous chemicals, including antibiotics and organic solvents. The major E. coli RND efflux pump AcrAB-TolC confers organic solvent tolerance (OST) to a range of chemicals, as well as mediating antibiotic resistance.

We have recently discovered a fundamental link between the AcrAB-TolC efflux pump and bacterial membrane potential (Whittle23). Inactivation of AcrAB-TolC results in an increase in the membrane potential of the cell – this results in alterations in global gene regulation, later entry into stationary phase, and a change in microbial physiology.

This link between efflux and membrane potential has major implications for industrial processes. The proton motive force (which makes up part of the membrane potential) is generated via NADH by energy metabolism and is used to drive ATP synthesis as well as other cellular processes such as efflux. NADH and ATP are also required for production of many organic chemical products. Therefore, cellular resources need to be balanced between the requirements of growth, product generation, and efflux (therefore solvent tolerance). Improvements to process yield can only be realised by balancing these needs.

In this project, we will explore these relationships, determining how membrane potential, efflux, and organic solvent tolerance are balanced. We will develop new ways to measure organic solvent tolerance and other aspects of physiology using single cell analysis. We will also seek to develop bacterial strains that are more solvent tolerant and also more productive, balancing the different needs of the cell.

References

1. Yang D, Prabowo CPS, Eun H, Park SY, Cho IJ, Jiao S, Lee SY. Escherichia coli as a platform microbial host for systems metabolic engineering. Essays Biochem. 2021 Jul 26;65(2):225-246. doi: 10.1042/EBC20200172. PMID: 33956149.
2. Emily E Whittle, Oluwatosin Orababa, Alexander Osgerby, Sarah J Element, Jessica MA Blair, Tim W Overton. bioRxiv 2023.04.03.535035; doi: https://doi.org/10.1101/2023.04.03.535035

Techniques

Basic microbiology and culture; bioreactor growth of bacteria; molecular biology (cloning, Gibson assembly); efflux assays; flow cytometry; FACS; analysis of physical characteristics of cells (eg MATH assay, zeta potential, contact angle); microscopy (fluorescent / confocal / EM); microensapsulation assays and sorting of droplets.