Longping Branch Graduate School, Hunan University, Changsha 410082, China; Hunan Agriculture Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China; Hunan Key Lab of Fruits & Vegetables Storage, Processing, Quality and Safety, Hunan Agricultural Products Processing Institute, Changsha 410125, China.
Longping Branch Graduate School, Hunan University, Changsha 410082, China; Hunan Agriculture Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China; Hunan Key Lab of Fruits & Vegetables Storage, Processing, Quality and Safety, Hunan Agricultural Products Processing Institute, Changsha 410125, China.
J Biotechnol. 2022 Mar 10;347:67-76. doi: 10.1016/j.jbiotec.2022.02.008. Epub 2022 Feb 19.
Hesperetin, a methoxylated flavanone, has numerous biological activities. Access to this compound is currently restricted by its low abundance in plants, which limits its practical applicability. To provide an alternative, eco-friendly production source, we developed a biosynthetic pathway of hesperetin in an engineered Escherichia coli consortium, which was fed with naringenin as a precursor and demonstrated good hesperetin production. The biosynthetic pathway was divided into two modules. The first recombinant host harbored the pathway genes from two different species: a flavonoid 3'-hydroxylase (F3'H) gene from Gentiana triflora and a cytochrome P450 reductase (CPR) gene from Arabidopsis thaliana. The second strain heterologously expressed a gene encoding a flavonoid 4'-O-methyltransferase (MpOMT) from Mentha × piperita, which was N-terminally fused to a Sumo tag. A construct expressing a 29 aa N-terminally truncated F3'H and CPR was the most effective combination for the conversion of naringenin. The strain expressing the Sumo-tagged MpOMT protein exhibited an increase in the final hesperetin titer, reaching 5.9 mg/L. Simultaneous overexpression of metK (coding for the endogenous S-adenosyl-l-methionine [SAM] synthase) further improved the hesperetin titer by 25.1%. Finally, the designed E. coli consortium harboring the two modules efficiently converted naringenin to hesperetin (37.1 mg/L). This work reports the construction of a multi-step in vivo cascade biocatalyst for the biotransformation of naringenin to hesperetin. It also illustrates the potential of the E. coli consortium system for producing other O-methylated flavonoids.
橙皮素是一种甲氧基黄酮类化合物，具有多种生物学活性。由于其在植物中的含量较低，限制了其实际应用，目前获取该化合物的途径有限。为了提供一种替代方法，我们开发了一种在工程大肠杆菌联合体中合成橙皮素的生物合成途径，该途径以柚皮苷为前体，具有良好的橙皮素生产能力。该生物合成途径分为两个模块。第一个重组宿主携带来自两个不同物种的途径基因：来自三叶龙胆的黄酮 3'-羟化酶（F3'H）基因和来自拟南芥的细胞色素 P450 还原酶（CPR）基因。第二个菌株异源表达了一个编码薄荷 4'-O-甲基转移酶（MpOMT）的基因，该基因来自薄荷，N 端融合了 Sumo 标签。表达 29 个氨基酸 N 端截断 F3'H 和 CPR 的构建体是将柚皮苷转化为橙皮素最有效的组合。表达 Sumo 标记的 MpOMT 蛋白的菌株最终橙皮素产量增加，达到 5.9mg/L。同时过表达 metK（编码内源性 S-腺苷甲硫氨酸[SAM]合酶）可使橙皮素产量进一步提高 25.1%。最后，含有两个模块的设计大肠杆菌联合体有效地将柚皮苷转化为橙皮素（37.1mg/L）。本工作报道了构建一种多步体内级联生物催化剂，用于将柚皮苷生物转化为橙皮素。它还说明了大肠杆菌联合体系统生产其他 O-甲基化黄酮类化合物的潜力。
