RESUMO
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.
Assuntos
Glucose , Redes e Vias Metabólicas , Glucose/metabolismo , Carbono/metabolismo , Bacillus subtilis/metabolismoRESUMO
Short-chain fatty acids (SCFA) such as propionate and butyrate are critical metabolites produced by the gut microbiota. Microbiome dysbiosis resulting in altered SCFA profiles is associated with certain diseases, including inflammatory bowel diseases (IBD), characterized by a reduction in butyrate concentration and active intestinal inflammation. There is an increasing interest in the use of engineered bacteria as diagnostic and therapeutic tools for gut diseases. In this study, we developed genetic circuits capable of sensing SCFA concentrations to build biosensors that express a response protein (superfolder green fluorescent protein [sfGFP]) in amounts inversely proportional to the SCFA concentration. We also built biotherapeutics expressing the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) using the same logic. The propionate biotherapeutic expressed larger amounts of mouse GM-CSF in the absence of propionate. The butyrate biotherapeutics presented the expected behavior only at the beginning of the kinetics and an accelerated response in the absence of butyrate. Overall, these genetic systems may function as complementary diagnostic tools for measuring SCFAs and as delivery vehicles for biotherapeutic molecules. IMPORTANCE Short-chain fatty acids are key molecules produced by the gut microbiome. Their concentrations are altered in certain diseases. Here, we created molecular biosensors that quantify the absence of propionate and butyrate, using logic "NOT" gates and bacterial promoters. Finally, we show that these genetic systems could be useful for the delivery of therapeutic molecules in the gut, in the absence of these acids.
RESUMO
Control of gene expression is crucial for several biotechnological applications, especially for implementing predictable and controllable genetic circuits. Such circuits are often implemented with a transcriptional regulator activated by a specific signal. These regulators should work independently of the host machinery, with low gratuitous induction or crosstalk with host components. Moreover, the signal should also be orthogonal, recognized only by the regulator with minimal interference with the host operation. In this context, transcriptional regulators activated by plant metabolites as flavonoids emerge as candidates to control gene expression in bacteria. However, engineering novel circuits requires the characterization of the genetic parts (e.g., genes, promoters, ribosome binding sites, and terminators) in the host of interest. Therefore, we decomposed the QdoR regulatory system of B. subtilis, responsive to the flavonoid quercetin, and reassembled its parts into genetic circuits programmed to have different levels of gene expression and noise dependent on the concentration of quercetin. We showed that only one of the promoters regulated by QdoR worked well in E. coli, enabling the construction of other circuits induced by quercetin. The QdoR expression was modulated with constitutive promoters of different transcriptional strengths, leading to low expression levels when QdoR was highly expressed and vice versa. E. coli strains expressing high and low levels of QdoR were mixed and induced with the same quercetin concentration, resulting in two stable populations expressing different levels of their gene reporters. Besides, we demonstrated that the level of QdoR repression generated different noise levels in gene expression dependent on the concentration of quercetin. The circuits presented here can be exploited in applications requiring adjustment of gene expression and noise using a highly available and natural inducer as quercetin.