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

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Efficient Biosynthesis of (2S)-Eriodictyol from (2S)-Naringenin in Saccharomyces cerevisiae through a Combination of Promoter Adjustment and Directed Evolution
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-11-23 , DOI: 10.1021/acssynbio.0c00346
Song Gao _{1,

2} , Xiaoyu Xu ₂ , Weizhu Zeng ₂ , Sha Xu _{1,

2} , Yunbin Lyv _{1,

2} , Yue Feng ₃ , Guoyin Kai ₃ , Jingwen Zhou _{1,

2,

4,

5} , Jian Chen _{1,

2,

4,

5}

Affiliation

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.

中文翻译：

通过启动子调整和定向进化相结合，在酿酒酵母中从 (2S)-柚皮素高效生物合成 (2S)-圣草酚

( 2S )-圣草酚是一种重要的黄酮类化合物，可通过类黄酮3'-羟化酶(F3'H)催化从( 2S )-柚皮素衍生而来。 F3'H 是一种细胞色素 P450 酶，需要细胞色素 P450 还原酶 (CPR) 才能发挥作用。然而，由于 P450 活性较低，其在工业规模生物合成中的应用受到限制。在此，从水飞蓟中鉴定出高效的Sm F3'H 和匹配的Sm CPR。为了提高Sm F3'H的效率，我们在酿酒酵母中优化了Sm F3'H和Sm CPR的启动子组合，建立了( 2S )-圣草酚的高通量检测方法。结果表明， Sm F3'H/ Sm CPR 应该使用具有相似且强表达水平的启动子来表达。此外，应用定向进化进一步提高Sm F3'H/ Sm CPR的效率。通过优化的启动子和突变组合Sm F3′H ^D285N / Sm CPR ^I453V ，( 2S )-圣草酚滴度提高至3.3 g/L，为目前报道中的最高滴度。这些结果表明酿酒酵母是类黄酮相关 P450 酶功能表达的理想平台。

更新日期：2020-12-18

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