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

BACKGROUND: BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals. RESULTS: For the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate. CONCLUSION: We demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates

MOESM1 of Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

Additional file 1: Figure S1. Synthesis of p-coumarate esters and amides from tyrosine in yeast. S. cerevisiae strains co-expressing tyrosine ammonia-lyase FjTAL and 4CL5 with LaAT1 (A), OsHCT4 (B), AtSCT (C), HvACT (D), TpHCT2 (E) or AtHHT3 (F) were fed with 4-hydroxyphenyllactate (A), nothing (B, C), agmatine (D), malate (E) or 1-dodecanol (F) for the synthesis of p-coumaroyl 4’-hydroxyphenyllactate (A), p-coumaroyl glycerol (B), N 1,N 8-disinapoyl spermidine (C), p-coumaroyl agmatine (D), p-coumaroyl malate (E) and dodecyl p-coumarate (F), respectively. Figure S2. Synthesis of quinate hydroxycinnamates in yeast. Representative LC-MS chromatograms obtained from analysis of the culture medium of a S. cerevisiae strain expressing 4CL5 and NtHQT are shown. The strain was fed with quinate and p-coumarate or caffeate for the synthesis of p-coumaroyl quinate (A) and chlorogenic acid (B), respectively. The LC-MS chromatogram of a solution of authentic chlorogenic acid is also shown (C). Table S1. List of acyl acceptors and donors used for the feedings of yeast strains expressing 4CL5 and BAHDs. Concentrations used in the culture medium are indicated. Table S2. Oligonucleotides used in this study. Data S1. Sequence of the red fluorescent protein dropout cassette and the codon-optimized sequences and accession numbers of BAHD acyltransferases used in this study