Fine-tuning of coumaric acid synthesis to increase naringenin production in yeast

(2 S )-Naringenin is a key precursor for biosynthesis of various high-value flavonoids and possesses a variety of nutritional and pharmaceutical properties on human health. Systematic optimization approaches have been employed to improve (2 S )-naringenin production in different microbial hosts. However, very few studies have focused on the spatiotemporal distribution of (2 S )-naringenin and related pathway intermediate p -coumaric acid, which is an important factor for efficient production. Here, we show that fine-turning of p -coumaric acid synthesis enables alleviated cell burden and improved (2 S )-naringenin production in yeast. First, we systematically optimized the (2 S )-naringenin biosynthetic pathway by alleviating the bottleneck downstream of p -coumaric acid and increasing malonyl-CoA supply, which improved (2 S )-naringenin production but significant amounts of p -coumaric acid still accumulated outside the cell. We further established a dual dynamic control system through combing a malonyl-CoA biosensor regulator and an RNAi strategy, to autonomously control the synthesis of p -coumaric acid and downregulate a pathway competing for malonyl-CoA. The optimized strains remarkably decreased extracellular accumulation of p -coumaric acid and simultaneously improved (2 S )- naringenin production. Finally, production of 933 mg/L of (2 S )-naringenin could be achieved by using minimal medium with negligible accumulation of p -coumaric acid. Our work highlights the importance of systematic control of pathway intermediates for efficient microbial production of plant natural products.