Preventing Production Escape Using an Engineered Glucose-Inducible Genetic Circuit.

A proper balance of metabolic pathways is crucial for engineering microbial strains that can efficiently produce biochemicals on an industrial scale while maintaining cell fitness. High production loads can negatively impact cell fitness and hinder industrial-scale production. To address this, fine-tuning gene expression using engineered promoters and genetic circuits can promote control over multiple targets in pathways and reduce the burden. We took advantage of the robust carbon catabolite repression system of Bacillus subtilis to engineer a glucose-inducible genetic circuit that supports growth and production. The circuit is resilient, enabling a quick switch in the production status when exposed to the correct carbon source. By performing serial cultivations for 61 generations under repressive conditions, we preserved the production capacity of the cells, which could be fully accessed by switching to glucose in the next cultivation step. Switching to glucose after 61 generations resulted in 34-fold activation and generated 70% higher production in comparison to standard cultivation in glucose. Conversely, serial cultivation under permanent induction resulted in 62% production loss after 67 generations alongside an increase in the culture growth rate. As a pathway-independent circuit activated by the preferred carbon source, our engineered glucose-inducible genetic circuit is broadly useful and imposes no additional cost to traditional production processes.

Engineering Microbial Living Therapeutics: The Synthetic Biology Toolbox.

Microbes can be engineered to act like living therapeutics designed to perform specific actions in the human body. From fighting and preventing infections to eliminating tumors and treating metabolic disorders, engineered living systems are the next generation of therapeutics. In recent years, synthetic biologists have greatly expanded the genetic toolbox for microbial living therapeutics, adding sensors, regulators, memory circuits, delivery devices, and kill switches. These advances have paved the way for successful engineering of fully functional living therapeutics, with sensing, production, and biocontainment devices. However, some important tools are still missing from the box. In this review, we cover the most recent biological parts and approaches developed and describe the missing tools needed to build robust living therapeutics.