Fine-tuning of p-coumaric acid synthesis to increase (2S)-naringenin production in yeast

(2S)-Naringenin is a key precursor for biosynthesis of various highvalue flavonoids and possesses a variety of nutritional and pharmaceutical properties on human health. Systematic optimization approaches have been employed to improve (2S)naringenin production in different microbial hosts. However, very few studies have focused on the spatiotemporal distribution of (2S)naringenin and the related pathway intermediate p-coumaric acid, which is an important factor for efficient production. Here, we first optimized the (2S)-naringenin biosynthetic pathway by alleviating the bottleneck downstream of p-coumaric acid and increasing malonyl-CoA supply, which improved (2S)-naringenin production but significant accumulation of p-coumaric acid still existed extracellularly. We thus established a dual dynamic control system through combining a malonyl-CoA biosensor regulator and an RNAi strategy, to autonomously control the synthesis of p-coumaric acid with the supply of malonyl-CoA. Furthermore, screening potential transporters led to identification of Pdr12 for improved (2S)naringenin production and reduced accumulation of p-coumaric acid. Finally, a titer of 2.05 g/L (2S)-naringenin with negligible accumulation of p-coumaric acid was achieved in a fed batch fermentation. Our work highlights the importance of systematic control of pathway intermediates for efficient microbial production of plant natural products.