Department of Integrative Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul, 05029, Republic of Korea.
Department of Forest Resources, Gyeongnam National University of Science and Technology, 33 Dongjin-ro, Jinju-si, Gyeongsangman-do, 52725, South Korea.
Microb Cell Fact. 2018 Mar 22;17(1):46. doi: 10.1186/s12934-018-0896-9.
Hydroxycinnamoyl anthranilates, also known as avenanthramides (avns), are a group of phenolic alkaloids with anti-inflammatory, antioxidant, anti-itch, anti-irritant, and antiatherogenic activities. Some avenanthramides (avn A-H and avn K) are conjugates of hydroxycinnamic acids (HC), including p-coumaric acid, caffeic acid, and ferulic acid, and anthranilate derivatives, including anthranilate, 4-hydroxyanthranilate, and 5-hydroxyanthranilate. Avns are primarily found in oat grain, in which they were originally designated as phytoalexins. Knowledge of the avns biosynthesis pathway has now made it possible to synthesize avns through a genetic engineering strategy, which would help to further elucidate their properties and exploit their beneficial biological activities. The aim of the present study was to synthesize natural avns in Escherichia coli to serve as a valuable resource.
We synthesized nine avns in E. coli. We first synthesized avn D from glucose in E. coli harboring tyrosine ammonia lyase (TAL), 4-coumarate:coenzyme A ligase (4CL), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and anthranilate synthase (trpEG). A trpD deletion mutant was used to increase the amount of anthranilate in E. coli. After optimizing the incubation temperature and cell density, approximately 317.2 mg/L of avn D was synthesized. Avn E and avn F were then synthesized from avn D, using either E. coli harboring HpaBC and SOMT9 or E. coli harboring HapBC alone, respectively. Avn A and avn G were synthesized by feeding 5-hydroxyanthranilate or 4-hydroxyanthranilate to E. coli harboring TAL, 4CL, and HCBT. Avn B, avn C, avn H, and avn K were synthesized from avn A or avn G, using the same approach employed for the synthesis of avn E and avn F from avn D.
Using different HCs, nine avns were synthesized, three of which (avn D, avn E, and avn F) were synthesized from glucose in E. coli. These diverse avns provide a strategy to synthesize both natural and unnatural avns, setting a foundation for exploring the biological activities of diverse avns.
羟基肉桂酰基邻氨基苯甲酸酯，也称为燕麦蒽酰胺（avns），是一组具有抗炎、抗氧化、抗瘙痒、抗刺激和抗动脉粥样硬化活性的酚类生物碱。一些燕麦蒽酰胺（avn A-H 和 avn K）是羟基肉桂酸（HC）的共轭物，包括对香豆酸、咖啡酸和阿魏酸，以及邻氨基苯甲酸衍生物，包括邻氨基苯甲酸、4-羟基邻氨基苯甲酸和 5-羟基邻氨基苯甲酸。avns 主要存在于燕麦谷物中，最初被指定为植物抗毒素。avns 生物合成途径的知识现在使得通过遗传工程策略合成 avns 成为可能，这将有助于进一步阐明它们的性质并利用它们有益的生物学活性。本研究的目的是在大肠杆菌中合成天然 avns，作为一种有价值的资源。
我们在大肠杆菌中合成了 9 种 avns。我们首先在含有酪氨酸氨裂解酶（TAL）、4-香豆酸：辅酶 A 连接酶（4CL）、邻氨基苯甲酸 N-羟基肉桂酰/苯甲酰基转移酶（HCBT）和邻氨基苯甲酸合酶（trpEG）的大肠杆菌中从葡萄糖合成 avn D。使用 trpD 缺失突变体增加大肠杆菌中邻氨基苯甲酸的含量。优化孵育温度和细胞密度后，约合成 317.2 mg/L 的 avn D。然后，分别使用含有 HpaBC 和 SOMT9 的大肠杆菌或仅含有 HapBC 的大肠杆菌，从 avn D 合成 avn E 和 avn F。通过向含有 TAL、4CL 和 HCBT 的大肠杆菌中添加 5-羟基邻氨基苯甲酸或 4-羟基邻氨基苯甲酸，合成 avn A 和 avn G。使用与从 avn D 合成 avn E 和 avn F 相同的方法，从 avn A 或 avn G 合成 avn B、avn C、avn H 和 avn K。
使用不同的 HCs，在大肠杆菌中合成了 9 种 avns，其中 3 种（avn D、avn E 和 avn F）可从葡萄糖中合成。这些不同的 avns 提供了一种合成天然和非天然 avns 的策略，为探索不同 avns 的生物学活性奠定了基础。
