Engineering bacterial hosts cells for robust growth at high external osmolarities

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Due to their relative simplicity and rapid growth rates bacteria have been engineered to produce a variety of bulk chemicals, from organic acids and diols to fuels such as ethanol and butanol. In line with their use as platforms for biotechnology there has been an increased interest in gaining control over bacterial growth rates. A very common, yet poorly understood, modulation of bacterial growth during bio production occurs in response to changes in external osmolarities. High-level excretion of metabolites and substrate feed during biotechnological applications constantly increases medium concentration and can result in limited cell growth and low volumetric productivity. Similarly, some of the less expensive media, used to decrease fabrication costs particularly in the areas where clean water is limited, are high in osmolarity.Increases in the external osmolarity were previously found to effect bacterial osmotic pressure. Based on these findings it was long thought that the change in osmotic pressure leads to decreased growth rates. We recently combined cutting-edge microscopy techniques for single cell studies to show that this is not the case. Upon an increase in external osmolarity osmotic pressure of Escherichia coli recovers to the initial pre-shock value, while the cells continue to grow slower immediately after (Biophys J, under review). Therefore the osmotic pressure does not explain the slowdown in growth. Instead, our preliminary measurements of cellular energetics indicate that the reduction in growth is due to the sustained energetic burden needed to maintain higher cytoplasmic concentration. The finding opens several novel possibilities of controlling cellular growth and production yields.In this project the student will be trained in a versatile set of interdisciplinary skills needed to explore a range of approaches to achieve more desirable responses to changes in external concentrations for biotechnology applications. The project has been structured to offer several approaches, and thus contingency options, with varying degrees of risk to ensure project success. The project aims are:(1) Determine the energetic costs of employing different osmoregulatory components during recovery and subsequent growth.The student will genetically engineer E.coli strains with a given combination of osmoregulatory components and use advanced microscopy techniques to measure the extent of changes in cellular energetics during subsequent growth. Our encouraging preliminary data indicated that this will generate simplified strains with a characteristic response to growth at higher external osmolarity.(2) Design synthetic pathways that enable E. coli to use accumulated osmolites as alternative sugar sources for production.The student will utilize increased osmolyte production during growth at higher external osmolarities, such as treahlose, to design synthetic circuit that allow E.coli to use accumulated treahlose for compound production.(3) Light power the proton motive force of E. coli to elevate available energy levels.The student will express a light-powered pump, proteorhodopsin, and test if artificially boosting cellular energetics with green light can enhance growth and production yields at high external osmolarity.(4) Apply the approaches from Aim 1-3 to INEOS proprietary bacteria.