De Novo Biosynthesis of p-Coumaric Acid in E. coli with a trans-Cinnamic Acid 4-Hydroxylase from the Amaryllidaceae Plant Lycoris aurea.

Yikui Li,Jie Li,Li Cheng,Binbin Qian,Ren Wang,Sheng Xu

doi:10.3390/molecules23123185

Abstract

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.

Highlights

Introduction pCoumaric 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 possesses potent anti-oxidant, antibacterial and anti-inflammatory properties, and serves as a conventional precursor for the production of flavors and fragrances used in edible and daily chemical products. p-Coumaric acid is a starting material for the preparation of environmentally degradable thermoplastics with liquid crystalline behavior [1]
Thereby, our observation suggested that the N-terminal 28 amino acids spanning the hydrophobic transmembrane region was responsible for leading LauC4H to localize in endoplasmic reticulum (ER) of plant cells
P-coumaric acid was de novo produced via the plant cytochrome P450 enzyme (CYP450)-involved biosynthetic route in Escherichia coli

Summary

Introduction

Introduction pCoumaric 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 possesses potent anti-oxidant, antibacterial and anti-inflammatory properties, and serves as a conventional precursor for the production of flavors and fragrances used in edible and daily chemical products. p-Coumaric acid is a starting material for the preparation of environmentally degradable thermoplastics with liquid crystalline behavior [1]. P-coumaric acid, as an upstream metabolite in the plant phenylpropanoid pathway, is a Molecules. 2 of 22 [7,8], common precursor numerous derivatives, such as other phenylpropanoids flavonoids [9,10], stilbenes [11] and anthocyanins [12]. P‐coumaric acid, as an upstream metabolite in the plant phenylpropanoid p-Coumaric acid has been biosynthesized mainly from tyrosine in engineered microbes. Pathway, is a common precursor for biosynthesizing numerous derivatives, such as other p-Coumaric acid, as a component of lignin, ubiquitously present in plants at a low concentration [13]. In plants,p‐Coumaric the biosynthesis of p-coumaric acid involves biochemical acid has been biosynthesized mainlytwo from tyrosine in processes: engineered phenylalanine microbes Phenylpropanoids [7,8], flavonoids [9,10],isstilbenes [11] and anthocyanins [12].

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