Efficient Biosynthesis of (2S)-Eriodictyol from (2S)-Naringenin in Saccharomyces cerevisiae through a Combination of Promoter Adjustment and Directed Evolution

The compound (2 S )-eriodictyol is an important flavonoid that can be derived from (2 S )-naringenin through flavonoid 3′-hydroxylase (F3′H) catalyzation. F3′H is a cytochrome P450 enzyme that requires a cytochrome P450 reductase (CPR) to function. However, P450s have limited applications in industrial scale biosynthesis, owing to their low activity. Here, an efficient Sm F3′H and a matched Sm CPR were identified from Silybum marianum . To improve the efficiency of Sm F3′H, we established a high-throughput detection method for (2 S )-eriodictyol, in which the promoter combination of Sm F3′H and Sm CPR were optimized in Saccharomyces cerevisiae . The results revealed that Sm F3′H/ Sm CPR should be expressed by using promoters with similar and strong expression levels. Furthermore, directed evolution was applied to further improve the efficiency of Sm F3′H/ Sm CPR. With the optimized promoter and mutated combinations Sm F3′H D285N / Sm CPR I453V, the (2 S )-eriodictyol titer was improved to 3.3 g/L, the highest titer in currently available reports. These results indicated that S. cerevisiae is an ideal platform for functional expression of flavonoid related P450 enzymes.