Controlled Protein Degradation for Development of Metabolite Valves

The expression of heterologous pathways in microbial hosts offers interesting opportunities for production of both commodity chemicals and specialty compounds.  Gene knockouts are often employed to redirect flux into these pathways, but in cases where the competing enzymes are part of central metabolism of the host, this may result in poor cell health and slow growth on the desired substrate.  We would like to develop strategies for dynamically modulating the abundance of native enzymes within the host cell, allowing for switching between growth and production modes.  One system that could potentially benefit from implementation of such a “metabolite valve” strategy is the pathway for production of glucaric acid in E. coli previously developed in our lab.  The initial substrate for the glucaric acid pathway, glucose-6-phosphate, is utilized by the cell in both glycolysis and the pentose phosphate pathway, and static knockout strategies involving these central metabolic pathways can be detrimental to growth and recombinant protein expression. 

To develop the metabolite valve concept for this system, we have implemented a strategy based on controlled degradation of a key glycolytic enzyme, phosphofructokinase.  Control of enzyme abundance at the post-translational level through degradation allows for rapid response time and can help overcome growth-mediated buffering effects seen with transcriptional control.  Combining this with tuning of gene expression levels, we have been able to develop an E. coli strain with a “growth mode” very close to wild type and a “production mode” with decreased glycolytic flux.  Ongoing work focuses on induction of the system in response to culture conditions to allow for autonomous switching.