Construction of a Chimeric Biosynthetic Pathway for the De Novo Biosynthesis of Rosmarinic Acid in Escherichia coli

Abstract

Hydroxycinnamic acid esters (HCEs) are widely-distributed phenylpropanoid-derived plant natural products. Rosmarinic acid (RA 7), the most well-known HCE, shows promise as a treatment for cancer and neurological disorders. In contrast to extraction from plant material or plant cell culture, a microbial production platform for HCEs could provide a sustainable, controlled means of production. Through the overexpression of a six-enzyme chimeric bacterial and plant pathway, we show the de novo biosynthesis of RA 7 and the related HCE isorinic acid (IA 8) in E. coli. Probing the pathway through precursor supplementation showed several potential pathway bottlenecks. We show HCE biosynthesis using three plant RAS orthologs exhibiting different levels of HCE biosynthesis, but the same ratio of IA 8 to RA 7 produced. This work serves as a proof of concept for a microbial production platform for HCEs using a modular biosynthetic approach to access diverse natural and non-natural HCEs.

Global Identification of the Full-Length Transcripts and Alternative Splicing Related to Phenolic Acid Biosynthetic Genes in Salvia miltiorrhiza

“…The main active components of S. miltiorrhiza are hydrophilic salvianolic acids (SAs), such as rosmarinic acid (RA) and lithospermic acid B (LAB; Wang et al, 2007 ), and lipophilic diterpenoid components, such as tanshinones I/IIA, dihydrotanshinone, and cryptotanshinone ( Lei et al, 2014 ). The availability of the nuclear and chloroplast genomes ( Qian et al, 2013 ) and transcriptome ( Hua et al, 2011 ; Luo et al, 2014 ), along with research related to the molecular regulation ( Zhang et al, 2013 , 2015 ; Tan et al, 2014 ; Li et al, 2015 ) and biosynthesis of its bioactive components ( Guo et al, 2013 , 2015 ; Bloch and Schmidt-Dannert, 2014 ), strongly favors S. miltiorrhiza as a potential model medicinal plant for TCM research.…”

Global dissection of the BAHD acyltransferase gene family in soybean: Expression profiling, metabolic functions, and evolution

“…Furthermore, several BAHD genes that catalyse the formation of diverse metabolites have been characterised in different plant species. For example, E. coli was used to express several BAHD enzymes, such as GmIMaT1, GmIMaT3 [2], GmMT7 [20], MtMaT1, 2, 3 [86], MaT4, 5, 6 [89] for the synthesis of malonylated flavones, isoflavones or anthocyanins, HQT for the synthesis of the antioxidant chlorogenic acid [33], hydroxycinnamoyl-CoA:hydroxyphenyllactate transferases for the production of rosmarinic acid [8], the hydroxycinnamoyl-CoA:glycerol transferase for the production of water-soluble antioxidants, hydroxycinnamate glycerol esters [34], and the hydroxycinnamoyl/benzoyl-CoA:anthranilate transferase (HCBT) for the synthesis of therapeutic benzoyl and hydroxycinnamoyl anthranilates [22]. The completion of genome sequencing projects of many plant species revealed the presence of BAHD family genes, such as the models Arabidopsis thaliana and Oryza sativa as well as orphan species, such as the cardoon (Cynara cardunculus, Asteraceae).…”

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

“…Such substrate flexibility has been explored for RA synthase from Coleus blumei and allowed biosynthesis of 13 RA analogues in E. coli [10]. Heterelogous pathways for the synthesis of the two acceptors, 4-hydroxyphenyllactate and 3,4-dihydroxyphenyllactate, from an inexpensive renewable carbon source has already been demonstrated in E. coli [8, 9] and could be implemented in yeast for sustainable and economical biosynthesis. We also demonstrated that expression of tyrosine ammonia-lyase (FjTAL) in addition to 4CL5 and LaAT1 results in the production of p -coumaroyl 4′-hydroxyphenyllactate when the FjTAL-4CL5-LaAT1 strain is fed with only 4-hydroxyphenyllactate (Additional file 1: Figure S1A).…”

“…Furthermore, several BAHD acyltransferases catalyze the formation of metabolites beneficial for human health, which has prompted their use as biocatalysts in engineered microbes. For example, engineered Escherichia coli strains have been developed to express BAHDs such as hydroxycinnamoyl-CoA:quinate transferases (HQT) for the synthesis of the antioxidant chlorogenic acid [6, 7]; hydroxycinnamoyl-CoA:hydroxyphenyllactate transferases for the production of rosmarinic acid [8–10]; hydroxycinnamoyl-CoA:glycerol transferase for the synthesis of the water-soluble antioxidants hydroxycinnamate glycerol esters [11] and hydroxycinnamoyl/benzoyl-CoA:anthranilate transferase (HCBT) for the production of therapeutic benzoyl and hydroxycinnamoyl anthranilates [12]. To our knowledge, the use of yeast strains engineered for the expression of BAHD acyltransferases has not been reported, except for the synthesis of hydroxycinnamoyl anthranilates using either HCBT or promiscuous hydroxycinnamoyl-CoA:shikimate transferases [13–16].…”