Abstract
Hydroxycinnamic acids (HCs) are natural compounds that form conjugates with diverse compounds in nature. Ethyl caffeate (EC) is a conjugate of caffeic acid (an HC) and ethanol. It has been found in several plants, including Prunus yedoensis, Polygonum amplexicaule, and Ligularia fischeri. Although it exhibits anticancer, anti-inflammatory, and antifibrotic activities, its biosynthetic pathway in plants still remains unknown. This study aimed to design an EC synthesis pathway and clone genes relevant to the same. Genes involved in the caffeic acid synthesis pathway (tyrosine ammonia-lyase (TAL) and p-coumaric acid hydroxylase (HpaBC)) were introduced into Escherichia coli along with 4-coumaroyl CoA ligase (4CL) and acyltransferases (AtCAT) cloned from Arabidopsis thaliana. In presence of ethanol, E. coli harboring the above genes successfully synthesized EC. Providing more tyrosine through the overexpression of shikimate-pathway gene-module construct and using E. coli mutant enhanced EC yield; approximately 116.7 mg/L EC could be synthesized in the process. Synthesis of four more alkyl caffeates was confirmed in this study; these might potentially possess novel biological properties, which would require further investigation.
Highlights
Hydroxycinnamic acids (HCs) are abundant in nature. They are synthesized via phenylpropanoid pathway in plants and serve as the building blocks for other phenolic compounds, such as flavonoids, coumarin, lignin, proanthocyanidins, cutin, and suberin [1]
We reported the successful synthesis of Ethyl caffeate (EC) in E. coli
The results indicated that AtCAT1 could conjugate caffeoyl-coenzyme A (CoA) with ethanol to make EC (Fig. 2)
Summary
Hydroxycinnamic acids (HCs) are abundant in nature. They are synthesized via phenylpropanoid pathway in plants and serve as the building blocks for other phenolic compounds, such as flavonoids, coumarin, lignin, proanthocyanidins, cutin, and suberin [1]. High-molecular-weight alcohols, such as C18, C20, and C22 alkan-1-ols, have been reported to be synthesized from long-chain fatty acids in plants [12], how the ethyl group is provided during the synthesis of EC still remains a mystery. Among the variety of phytochemicals, phenolic compounds are mostly synthesized in microbial systems, since the biological synthetic genes have been characterized in many plants and hosts are engineered to provide more substrates [17]. The final titers of caffeic acid, reported in various publications, are quite different; one report used tyrosine-overproducing E. coli by introducing tyrAfbr, ppsA, tktA, and aroGfbr while deleting pheLA and tyrA genes, and overexpressing TAL. By introducing genes for the conjugation of caffeic acid and ethanol into an engineered E. coli strain, and providing ethanol, we could successfully synthesize EC
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