Development and Analysis of Autonomous Metabolite Valves

The ability of microbes to convert organic materials (feedstock) into a vast array of chemical products makes them ideally suited as chemical factories. Advances in this technology require redirecting natural and engineered metabolic pathways (i.e. the pathways of chemical reactions in the cell) towards the production of the desired chemical. One means of redirecting microbial metabolism is to manipulate the pathways so that the desired amount of metabolites flows toward the desired pathway at the appropriate time. This is akin to opening a valve to permit the flow of chemicals into a factory at the appropriate time and in the appropriate amount. In this study a set of bioengineering tools will be developed for detecting accumulation in a microbe of a signalling molecule and for detecting the depletion of a nutrient in the growth medium. When the targeted concentrations of the signal molecule or nutrient are reached the expression of specific metabolic pathways will be altered appropriately to increase production of the desired chemical. This toolkit will be made broadly available to synthetic biology and metabolic engineering researchers. Workforce training and development will be provided to young career (undergraduate and graduate students) scientists through an interdisciplinary research experience.

There has been great interest in developing strategies to control the flux of metabolites at the appropriate time and in the appropriate amounts to optimize yields of microbially synthesized chemicals. To achieve high product yields requires balancing the need for increasing cellular biomass with the diversion of metabolites towards the synthesis of the desired product. An ideal system would allow dynamic control of metabolite flow towards product synthesis without the need for any user-provided inputs. This project aims to develop and analyze the use of autonomous metabolite valves to control the appropriate flux of metabolites through a heterologous pathway at the appropriate time. In contrast to other systems that rely on exogenous amendments to control metabolite flux this system will rely on the intracellular accumulation of microbially synthesized signals or the depletion of a specific nutrient in the growth medium. At targeted concentrations of the signal molecule or nutrient, gene expression will altered to increase the flux of metabolites toward synthesis of the desired product. The objectives of this study will be to: 1) develop and analyze quorum sensing-based metabolite valves; 2) develop and analyze metabolite valves based on the natural phosphate starvation response; and 3) integrate the quorum sensing and phosphate starvation valves into a single strain to assess system compatibility. While a specific metabolic pathway will be investigated for valve performance as proof of concept, it is also envisioned that these devices will be useful for more general physiological studies such as the impact of metabolic shifts on cellular behavior.