De Novo Biosynthesis of p-Coumaric Acid in E-coli with a trans-Cinnamic Acid 4-Hydroxylase from the Amaryllidaceae Plant Lycoris aurea
De Novo Biosynthesis of p-Coumaric Acid in E-coli with a trans-Cinnamic Acid 4-Hydroxylase from the Amaryllidaceae Plant Lycoris aurea
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使用来自石蒜科植物石蒜的反式肉桂酸 4-羟化酶在大肠杆菌中从头生物合成对香豆酸
DOI：
10.3390/molecules23123185
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发表时间：
2018-12-01
期刊：
影响因子：
4.6
通讯作者：
Wang, Ren
中科院分区：
文献类型：
--
作者：
Li, Yikui;Li, Jie;Wang, Ren
对香豆酸是一种可商购的酚羧酸，在营养品、制药、材料和化学工业中具有大量重要应用。对香豆酸已经在一些工程菌中生物合成，但尚未研究大肠杆菌中植物CYP 450参与的生物合成途径的潜力。本研究从黄花石蒜（Lycoris aurea（L' Hér.）通过快速扩增cDNA末端。然后，N-末端28个氨基酸的LauC 4 H的特征在于，在拟南芥原生质体的内质网膜的亚细胞定位。在大肠coli中，不含N端膜锚区的LauC 4 H与A.拟南芥细胞色素P450酶（CYP 450），并通过全细胞生物转化、高效液相色谱检测和液质联用分析验证其催化反式肉桂酸转化为对香豆酸。进一步用A. thaliana中，对香豆酸在重组E.大肠杆菌细胞。通过调节细胞内NADPH的水平，对香豆酸的产量显着提高了9.18倍，并在摇瓶中达到156.09 μM的滴度。具有功能性LauC 4 H的重组细胞为通过植物CYP 450参与的途径生物生产p-香豆酸甚至其他衍生物提供了有前途的底盘。
p-Coumaric acid is a commercially available phenolcarboxylic acid with a great number of important applications in the nutraceutical, pharmaceutical, material and chemical industries. p-Coumaric acid has been biosynthesized in some engineered microbes, but the potential of the plant CYP450-involved biosynthetic route has not investigated in Escherichia coli. In the present study, a novel trans-cinnamic acid 4-hydroxylase (C4H) encoding the LauC4H gene was isolated from Lycoris aurea (L’ Hér.) Herb via rapid amplification of cDNA ends. Then, N-terminal 28 amino acids of LauC4H were characterized, for the subcellular localization, at the endoplasmic reticulum membrane in protoplasts of Arabidopsis thaliana. In E. coli, LauC4H without the N-terminal membrane anchor region was functionally expressed when fused with the redox partner of A. thaliana cytochrome P450 enzyme (CYP450), and was verified to catalyze the trans-cinnamic acid to p-coumaric acid transformation by whole-cell bioconversion, HPLC detection and LC-MS analysis as well. Further, with phenylalanine ammonia-lyase 1 of A. thaliana, p-coumaric acid was de novo biosynthesized from glucose as the sole carbon source via the phenylalanine route in the recombinant E. coli cells. By regulating the level of intracellular NADPH, the production of p-coumaric acid was dramatically improved by 9.18-fold, and achieved with a titer of 156.09 μM in shake flasks. The recombinant cells harboring functional LauC4H afforded a promising chassis for biological production of p-coumaric acid, even other derivatives, via a plant CYP450-involved pathway.