Improvement of Chalcone Synthase Activity and High-Efficiency Fermentative Production of (2 S )-Naringenin via In Vivo Biosensor-Guided Directed Evolution.

Chalcone synthase (CHS) catalyzes the rate-limiting step of (2 S )-naringenin (the essential flavonoid skeleton) biosynthesis. Improving the activity of the CHS by protein engineering enhances (2 S )-naringenin production by microbial fermentation and can facilitate the production of valuable flavonoids. A (2 S )-naringenin biosensor based on the TtgR operon was constructed in Escherichia coli and its detection range was expanded by promoter optimization to 0-300 mg/L, the widest range for (2 S )-naringenin reported. The high-throughput screening scheme for CHS was established based on this biosensor. A mutant, Sj CHS1S208N with a 2.34-fold increase in catalytic activity, was discovered by directed evolution and saturation mutagenesis. A pathway for de novo biosynthesis of (2 S )-naringenin by Sj CHS1S208N was constructed in Saccharomyces cerevisiae , combined with CHS precursor pathway optimization, increasing the (2 S )-naringenin titer by 65.34% compared with the original strain. Fed-batch fermentation increased the titer of (2 S )-naringenin to 2513 ± 105 mg/L, the highest reported so far. These findings will facilitate efficient flavonoid biosynthesis and further modification of the CHS in the future.