Saccharomyces cerevisiae biofactory to produce naringenin using a systems biology approach and a bicistronic vector expression strategy in flavonoid production

Naringenin is the central flavonoid in the biosynthesis of several bioactive compounds and presents a growing demand for its nutraceutical properties. Naringenin extraction from plants is non-viable due to low yields, and microbial platforms could represent a controlled and sustained alternative to produce it using several metabolic engineering tools. This study shows the naringenin production in Saccharomyces cerevisiae from glucose through a combined approach of systems biology, enzyme criteria selection, and a molecular engineering strategy. In silico prediction using a mixed integer linear programming (MILP) algorithm showed that the phenylpropanoid pathway was the shortest and most viable metabolic pathway. Two biscistronic constructs were generated using the PTV-1 2A peptide sequence, and a naringenin biofactory was assembled with the phenylalanine ammonia-lyase/tyrosine ammonia-lyase genes encoding phenylalanine/tyrosine ammonia-lyase (Rhodobacter capsulatus), 4-coumaroyl (4 Cl) encoding a p-coumaroyl-CoA ligase (Solanum lycopersicum), CHS encoding chalcone synthase (Hypericum androsaemum), and CHI encoding a chalcone isomerase (Glycine max). Naringenin productivity in batch fermentation was about 40.67 +/- 3.47 mu g/Lh with a 6.10 +/- 0.52 mg/L titer (22.41 +/- 1.91 mu M) and a 3.26 +/- 1.36 mg/g yield (Y-P/S) with the detection of additional flavonoids. The obtained concentration is better than other related works in diverse engineered microorganisms. The results suggest a successful and optimizable alternative for the heterologous flavanone production in yeast combined with bicistronic expression mediated by a 2A peptide sequence for the first time. This strategy supports the production of extensive routes for other nutraceutical compounds.