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

Chalcone synthase (CHS) catalyzes the rate-limiting step of (2)-naringenin (the essential flavonoid skeleton) biosynthesis. Improving the activity of the CHS by protein engineering enhances (2)-naringenin production by microbial fermentation and can facilitate the production of valuable flavonoids. A (2)-naringenin biosensor based on the TtgR operon was constructed in and its detection range was expanded by promoter optimization to 0-300 mg/L, the widest range for (2)-naringenin reported. The high-throughput screening scheme for CHS was established based on this biosensor. A mutant, CHS1 with a 2.34-fold increase in catalytic activity, was discovered by directed evolution and saturation mutagenesis. A pathway for biosynthesis of (2)-naringenin by CHS1 was constructed in , combined with CHS precursor pathway optimization, increasing the (2)-naringenin titer by 65.34% compared with the original strain. Fed-batch fermentation increased the titer of (2)-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.