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.