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(2S)-Naringenin, a (2S)-flavanone, is widely used in the food, chemical, and pharmaceutical industries because of its diverse physiological activities. The production of (2S)-naringenin in microorganisms provides an ideal source that reduces the cost of the flavonoid. To achieve efficient production of (2S)-naringenin in Saccharomyces cerevisiae (S. cerevisiae), we constructed a biosynthetic pathway from p-coumaric acid, a cost-effective and more efficient precursor. The (2S)-naringenin synthesis pathway genes were integrated into the yeast genome to obtain a (2S)-naringenin production strain. After gene dosage experiments, the genes negatively regulating the shikimate pathway and inefficient chalcone synthase activity were verified as factors limiting (2S)-naringenin biosynthesis. With fed-batch process optimization of the engineered strain, the titer of (2S)-naringenin reached 648.63 mg/L from 2.5 g/L p-coumaric acid. Our results indicate that the constitutive production of (2S)-naringenin from p-coumaric acid in S. cerevisiae is highly promising.

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The compound (2S)-eriodictyol is an important flavonoid that can be derived from (2S)-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 SmF3′H and a matched SmCPR were identified from Silybum marianum. To improve the efficiency of SmF3′H, we established a high-throughput detection method for (2S)-eriodictyol, in which the promoter combination of SmF3′H and SmCPR were optimized in Saccharomyces cerevisiae. The results revealed that SmF3′H/SmCPR should be expressed by using promoters with similar and strong expression levels. Furthermore, directed evolution was applied to further improve the efficiency of SmF3′H/SmCPR. With the optimized promoter and mutated combinations SmF3′H D285N /SmCPR I453V , the (2S)-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.

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