| Congresso Brasileiro de Microbiologia 2023 | Resumo: 948-1 | ||||
Resumo:Large-scale industrial production of compounds from engineered microorganisms imposes some challenges such as strain instability and the emergence of non-producing cells. One source of instability is the genetic heterogeneity intrinsic to cell populations, which hinders production due to low performers. Additionally, intense gene expression and target metabolite production arrest important cellular resources resulting in metabolic burden and reduced cell fitness. The problem escalates if the engineered strain accumulates toxic intermediates due to different enzyme efficiencies and unbalanced pathway gene expression. Compromised cell fitness is a driving force for the selection of non-producing mutant cells that naturally arise during cultivation. A proper balance of metabolic pathways is crucial for engineering microbial strains that can efficiently produce biochemicals at an industrial scale while maintaining cell fitness. 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 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 than 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. Palavras-chave: carbon catabolic repression, long-term fermentation, production escape, Bacillus subtilis, engineered bacteria Agência de fomento:Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) | |||||