National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
ACS Synth Biol. 2020 Dec 18;9(12):3288-3297. doi: 10.1021/acssynbio.0c00346. Epub 2020 Nov 23.
The compound (2)-eriodictyol is an important flavonoid that can be derived from (2)-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 F3'H and a matched CPR were identified from . To improve the efficiency of F3'H, we established a high-throughput detection method for (2)-eriodictyol, in which the promoter combination of F3'H and CPR were optimized in . The results revealed that F3'H/CPR should be expressed by using promoters with similar and strong expression levels. Furthermore, directed evolution was applied to further improve the efficiency of F3'H/CPR. With the optimized promoter and mutated combinations F3'H/CPR, the (2)-eriodictyol titer was improved to 3.3 g/L, the highest titer in currently available reports. These results indicated that is an ideal platform for functional expression of flavonoid related P450 enzymes.
（2）-芹菜素是一种重要的类黄酮，可通过类黄酮 3'-羟化酶（F3'H）催化从（2）-柚皮苷中得到。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，由于其活性低，P450 在工业规模生物合成中的应用有限。在这里，从 中鉴定出一种有效的 F3'H 和匹配的 CPR。为了提高 F3'H 的效率，我们在 中建立了一种（2）-芹菜素的高通量检测方法，其中优化了 F3'H 和 CPR 的启动子组合。结果表明，F3'H/CPR 应该使用具有相似和强表达水平的启动子进行表达。此外，还应用定向进化进一步提高了 F3'H/CPR 的效率。使用优化的启动子和突变组合 F3'H/CPR，（2）-芹菜素的产量提高到 3.3 g/L，这是目前报道的最高产量。这些结果表明 是黄酮类相关 P450 酶功能表达的理想平台。
