Intellectual merit: This research project was centered on the development of experimental tools to control the flow of metabolites for engineered biosynthetic pathways.   It continued work funded previously by the National Science Foundation, in which we created ?metabolite valves? that enable the diversion of carbon-containing substrates from biomass synthesis towards product formation in a controllable manner.  Such valves are considered to be particularly useful in applications and scenarios where competing reactions that result in low yield of target product on substrate cannot be eliminated through gene knock-outs.  The resulting system is analogous to the use of inducible promoters to decouple growth and product formation for recombinant protein synthesis, in order to produce an operational mode in which a ?biomass-generating? phase can initially be favored to boost the biocatalytic potential, but then be subsequently down-regulated in favor of a ?product-generating? phase. Our primary achievement for the current project was to demonstrate that this regulation could be designed and implemented using autonomous systems that eliminated the need for both the addition of components to the culture after inoculation and the human intervention required for any such additions.  To do so, we first employed quorum-sensing systems that would repress the expression of an essential endogenous gene as a function of culture density.  Applying the control system to a pathway for myo-inositol synthesis resulted in improvements in product titers that ranged from 20% to 5.5-fold greater than the wild-type control.  Further extension of the pathway to produce glucaric acid revealed that the valve strains were able to produce significant amounts of product while the controls only generated acetate in shake flask cultures.  We also showed that the improvements resulting from the valves were consistent across multiple production scales.  We next integrated this system with a second layer of control, in which the valve was combined with metabolite-driven regulation of the expression of a pathway enzyme.  When integrated, these two methods were roughly twice as effective as either applied in isolation.  Lastly, we have begun to add multiple layers of dynamic gene control at multiple points in a given pathway. This involves building additional quorum-sensing systems to enable autonomous modulation of gene expression, and then combining multiple tools together in the same strain to allow synergistic boosts in pathway productivity.  Our preliminary results from this last extension of the work are quite exciting and should be published within the next year.  Overall, this work significantly expanded the area of ?dynamic metabolic engineering,? as we increased the scope of products targeted as well as the mechanisms to achieve regulation.

Broader impacts:  This project partially supported two graduate students, the first of whom received his doctoral degree in the summer of 2017 and is currently employed full-time in the biotech industry.  The second student continues to matriculate in the doctoral program at MIT.  An undergraduate student also worked on the project for one year.  The main work arising from this project was published in two high-profile journals:  Nature Biotechnology (2017) and Proceedings of the National Academy of Sciences USA (2018).  The former manuscript has been cited 79 times in 2 years, reflecting the impact of the work.  We have also completed several material transfer agreements distributing strains arising from this work to other research laboratories.  Finally, this work has been presented at several university seminars and conferences over the past 3 years, being well-received each time.  Collectively, this indicates that the funded research has had a significant impact on the broader science and engineering community.

Last Modified: 02/14/2019
Modified by: Kristala L Prather