چكيده لاتين
Violacein is a bacterial pigment with diverse biological activities, including antimicrobial, antitumor, and antioxidant properties, making it a promising candidate for use as an antibiotic or anticancer agent. However, large-scale production of this compound faces several challenges, the most significant of which is the low yield in native producer strains. The aim of this study was to enhance violacein production in Janthinobacterium lividum through the application of abiotic stresses and bacterial adaptation to stress conditions. Initially, the effects of different carbon sources and the air: volume ratio on violacein production were investigated. Subsequently, the impact of hydrogen peroxide (H₂O₂), ampicillin (Amp), and acyl-homoserine lactone (AHL) on pigment synthesis was evaluated. In the next step, J. lividum was gradually adapted to increasing concentrations of ampicillin through serial passage. Finally, violacein production was assessed in the adapted strain in the presebce of optimal concentrations of H₂O₂, Amp, and AHL. Gene expression related to violacein biosynthesis was analyzed using real-time PCR (qPCR) and high-performance liquid chromatography (HPLC) was employed to measure changes in the production of L-tryptophan, the main precursor of violacein. Additionally, a genome-scale metabolic network for J. lividum was reconstructed using relevant databases. Flux Balance Analysis (FBA) was performed using the KBase platform, and after model validation, gene knockout simulations were conducted to identify targets for enhancing violacein biosynthesis. The highest violacein yield was obtained using glycerol as a carbon source and an air: volume ratio of 10% (in 100 mL flasks). Stresses caused by H₂O₂ (103 mg/L) and Amp (130 mg/L) significantly increased violacein production compared to the control (56 mg/L). In the presence of crude AHL, violacein production increased from 56 mg/L to 210 mg/L. The adapted strain produced approximately 1.3 g/L violacein in th presecen of stresses and AHL. qPCR results revealed that the expression of the AHL synthase gene (luxI) was downregulated under stress conditions and in the presence of glycerol. Expression of the vioA gene was upregulated in the presence of Amp, whereas H₂O₂ had no significant effect on its expression. HPLC analysis showed that the highest level of L-tryptophan production (1.64 mg/L) was observed under H₂O₂ stress, significantly higher than the control (0.55 mg/L). The genome-scale metabolic model, named iNB1336, consisted of 1336 reactions and 1309 metabolites. Simulations indicated that the knockout of genes encoding aromatic amino acid transaminase and tryptophan indole-lyase significantly increased flux of violacein production. Overall, this study demonstrated that microbial adaptation and abiotic stress application are effective, low-cost strategies for improving violacein production. Moreover, genome-scale metabolic modeling proved to be a powerful tool for predicting metabolic engineering strategies aimed at enhancing microbial metabolite biosynthesis.
