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Molecules 2018, 23(12), 3185; https://doi.org/10.3390/molecules23123185

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

1,2,†
,
1,†
,
1
,
1
,
1,2
and
1,2,*
1
Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China
2
The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing 210014, China
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Received: 30 September 2018 / Revised: 18 November 2018 / Accepted: 29 November 2018 / Published: 3 December 2018
(This article belongs to the Special Issue Natural Product Enzymes in Biosynthesis and Biocatalysis)
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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. View Full-Text
Keywords: p-coumaric acid; trans-cinnamic acid 4-hydroxylase; Lycoris aurea; Escherichia coli; synthetic biology p-coumaric acid; trans-cinnamic acid 4-hydroxylase; Lycoris aurea; Escherichia coli; synthetic biology
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Li, Y.; Li, J.; Qian, B.; Cheng, L.; Xu, S.; Wang, R. De Novo Biosynthesis of p-Coumaric Acid in E. coli with a trans-Cinnamic Acid 4-Hydroxylase from the Amaryllidaceae Plant Lycoris aurea. Molecules 2018, 23, 3185.

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