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 (Delta aroK and Delta aroL) to increase the final titre. The E. coli Delta aroK 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 p-coumaroyl 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.