(317c) Utilization of the Endogenous Toxin Gene for Selection of High Performing Microbial Cells for Bioproduction Enhancement

Biosynthetic behavior variation between individual cells inside a microbial population has been found to be an important factor determining the overall production performance. Such non-genetic variations can be addressed by implementing rationally designed population selection which uses exogenous antibiotics for removing low-performing cells from the population. Here, we develop a new engineering approach for continuous population selection without the need of using exogenous antibiotics. Specifically, an E. coli endogenous toxin gene whose product inhibits cell growth was engineered to express under the control of a metabolite-responsive biosensor. For the cells with low target product concentration, the toxin gene expression was upregulated, leading to growth arrest. For the cells with high product concentration, the expression of toxin gene was inhibited, which help these high-performers survive and get enriched during the selecting process. As a result, the overall biosynthetic performance of the entire culture could be improved. As a proof-of-concept, over-production of amino acid tryptophan and phenylalanine was achieved in metabolically engineered E. coli strains equipped with the designed toxin-based selection systems. Significant production improvement was achieved under all tested cultivation conditions, such as the use of different carbon substrates and cultivation temperatures. Moreover, we explored the use of other biosensors responsive to the target products for regulating the selection system, which also lead to considerable production increase. The findings of this work show the strong potential of population selection in microbial biosynthesis and pave the way for adapting toxin genes metabolic engineering applications.