Synthesis of chlorogenic acid and p ‐coumaroyl shikimate by expressing shikimate gene modules in Escherichia coli

Abstract

Aim: Chlorogenic acid and p-coumaroyl shikimate are hydroxycinnamic acid derivatives. These compounds are nutraceutical supplements due to their biological activities including prevention of cardiovascular disease and cancers. These two compounds were synthesized in Escherichia coli through two-culture system using two mutants, which are biochemically interdependent. The aim of this work was to improve the titres of their production in a single E. coli mutant in which all necessary genes were introduced. This was done by testing various shikimate gene combinations to determine the optimal gene combination for the synthesis of chlorogenic acid and p-coumaroyl shikimate. Methods and Results:A series of gene modules harbouring shikimate pathway genes were constructs. Six gene module constructs for chlorogenic acid synthesis and eight constructs for p-coumaric acid synthesis were tested in order to find the best one. Chlorogenic acid synthesis showed highest with the gene module construct containing ydiB, aroB, aroG f , ppsA and tktA. Using the E. coli strain, 109.7 mg L −1 chlorogenic acid was synthesized. The best gene module construct for the p-coumaroyl shikimate synthesis contained aroD and aroG f . In addition, we used two E. coli deletion mutant strains (ΔaroK and ΔaroL) to increase the final titre. The E. coli ΔaroK mutant harbouring this gene module construct synthesized 713.4 mg L −1 of p-coumaroyl shikimate. Conclusion:The chlorogenic acid synthesis using the current system was approximately 35.4% higher of the titre than titres obtained with an alternative method that depends on co-cultivation of two mutants. At the same time, production of pcoumaroyl shikimate increased 5.8 times. Significance and Impact of the Study:The current study's findings indicate that our selection of the shikimate gene module contributed to increases in the levels of the substrates and could be applied to synthesize other compounds whose synthesis requires intermediates of the shikimate pathway.

Yeast-Based Whole-Cell Factory for the Ecofriendly Synthesis of Vanillylamine as a Critical Step toward Capsaicin Production

“…Tyrosine is another precursor in the synthesis of vanillylamine that is directly converted into p-coumaric acid by tyrosine ammonia lyase (TAL). TAL from Rhodosporidium toruloides has been reported to possess a high capacity for synthesizing p-coumaric acid from tyrosine. , To increase the flux of phenylalanine and tyrosine, TAL , Aro9 , and PheA were introduced into the delta 1 site of the Z2 strain to generate the Z3 strain.…”

“…However, the amino acid negative effect caused by the upregulation of the shikimic acid pathway may limit the increase in vanillylamine production. 29 The synthesis of protocatechuic acid is likely another factor that interferes with the distribution of the precursor. The significant difference in the protocatechuic acid content observed between the Z2 and Z3 strains indicates the amount of 3-hydroxyshikimic acid converted into protocatechuic acid under the catalysis of PAL or TAL following the vanillylamine synthesis pathway.…”

“…As a common precursor of phenylalanine and tyrosine, prephenate is either converted into phenylpyruvate by prephenate dehydratase (Pha2) or into hydroxyphenylpyruvate by prephenate dehydrogenase (Tyr1), thereby entering the phenylalanine and tyrosine pathways via a transaminase (Aro9). 29,30 The bifunctional chorismate mutase−prephenate dehydratase (PheA) complex from E. coli can directly convert chorismate to phenylpyruvate, thereby replacing Aro7 and Pha2. 30 Tyrosine is another precursor in the synthesis of vanillylamine that is directly converted into p-coumaric acid by tyrosine ammonia lyase (TAL).…”

“…TAL from Rhodosporidium toruloides has been reported to possess a high capacity for synthesizing p-coumaric acid from tyrosine. 29,30 To increase the flux of phenylalanine and tyrosine, TAL, Aro9, and PheA were introduced into the delta 1 site of the Z2 strain to generate the Z3 strain.…”

Advances in Production of Hydroxycinnamoyl-Quinic Acids: From Natural Sources to Biotechnology

“…Recently, the synthesis of 5-CQA and p-coumaroyl shikimate by expressing shikimate gene modules in E. coli has been demonstrated. The overexpression of five genes of the shikimate pathway ( ppsA , tktA, aroGf, aroB, ydiB ) and heterologous genes TAL , HpaBC , 4CL , HST , and HQT resulted in the biosynthesis of 109.7 mg/L 5-CQA from glucose [ 146 ] ( Figure 6 ).…”