sb3c00134_si_005.xlsx (92.49 kB)
Preventing Production Escape Using an Engineered Glucose-Inducible Genetic Circuit
datasetposted on 2023-09-29, 09:17 authored by Leonardo F. Tavares, Nathan V. Ribeiro, Vitória F. B. Zocca, Graciely G. Corrêa, Laura A. S. Amorim, Milca R. C. R. Lins, Danielle B. Pedrolli
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.
preferred carbon sourcepermanent induction resultedmaintaining cell fitnessengineering microbial strainsefficiently produce biochemicalscorrect carbon source67 generations alongsideperforming serial cultivationsculture growth ratenext cultivation stepindependent circuit activated61 generations resultedtraditional production processeshigh production loadsinducible genetic circuit61 generationsserial cultivationsupports growthgenetic circuitsstandard cultivationscale productionproduction statusproduction lossproduction capacityhigher productiontook advantagerepressive conditionsquick switchproper balancepromote controlmultiple targetsindustrial scalehinder industrialgenerated 70fully accessedfold activationbroadly usefulbacillus subtilisadditional cost