Cellular Biocatalysts Using Synthetic Genetic Circuits for Prolonged and Durable Enzymatic Activity

Cellular biocatalysts hold great promise for the synthesis of difficult to achieve compounds, such as complex active molecules. Whole-cell biocatalysts can be programmed through genetic circuits to be more efficient, but they suffer from low stability. The catalytic activity of whole cells decays under stressful conditions, such as prolonged incubation times or high temperatures. In nature, microbial communities cope with these conditions by forming biofilm structures. In this study, it is shown that the use of biofilm structures can enhance the stability of whole-cell biocatalysts. We employed two different strategies to increase the stability of whole-cell catalysts and decrease their susceptibility to high temperature. In the first approach, the formation of a biofilm structure is induced by controlling the expression of one of the curli component, CsgA. The alkaline phosphatase (ALP) enzyme was used to monitor the catalytic activity of cells in the biofilm structure. In the second approach, the ALP enzyme was fused to the CsgA curli fiber subunit to utilize the protective properties of the biofilm on enzyme biofilms. Furthermore, an AND logic gate is introduced between the expression of CsgA and ALP by toehold RNA switches and recombinases to enable logical programming of the whole-cell catalyst for biofilm formation and catalytic action with different tools. The study presents viable approaches to engineer a platform for biocatalysis processes.