title: Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: Aymerick
authors.initials: A
authors.lastname: Eudes
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: Maxence
authors.initials: M
authors.lastname: Mouille
authors.affiliation: Joint Genome Institute, Walnut Creek, CA, 94598, USA.
authors.forename: David S
authors.initials: DS
authors.lastname: Robinson
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.|Graduate Program, San Francisco State University, San Francisco, CA, 94132, USA.
authors.forename: Veronica T
authors.initials: VT
authors.lastname: Benites
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: George
authors.initials: G
authors.lastname: Wang
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.|Master Program, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland.
authors.forename: Lucien
authors.initials: L
authors.lastname: Roux
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: Yi-Lin
authors.initials: YL
authors.lastname: Tsai
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: Edward E K
authors.initials: EE
authors.lastname: Baidoo
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: Tsan-Yu
authors.initials: TY
authors.lastname: Chiu
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.|School of BioSciences, The University of Melbourne, Melbourne, VIC, 3010, Australia.
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Heazlewood
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: Henrik V
authors.initials: HV
authors.lastname: Scheller
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.
authors.forename: Aindrila
authors.initials: A
authors.lastname: Mukhopadhyay
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA.|Department of Chemical & Biomolecular Engineering and Department of Bioengineering, University of California, Berkeley, CA, 94720, USA.|Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Alle´, 2970, Hørsholm, Denmark.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Joint Genome Institute, Walnut Creek, CA, 94598, USA.
authors.forename: Samuel
authors.initials: S
authors.lastname: Deutsch
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St., 4th Floor, Emeryville, CA, 94608, USA. dloque@lbl.gov.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720, USA. dloque@lbl.gov.|CNRS, UMR5240, Microbiologie, Adaptation et Pathogénie, Université Claude Bernard Lyon 1, INSA de Lyon, 10 rue Raphaël Dubois, 69622, Villeurbanne, France. dloque@lbl.gov.
authors.forename: Dominique
authors.identifier: http://orcid.org/0000-0002-2060-6611
authors.initials: D
authors.lastname: Loqué
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D001565:Benzoates / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism*; D006801:Humans; D015003:Yeasts / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D001565:Benzoates; D003373:Coumaric Acids; D000217:Acyltransferases
keywords: Antioxidant; BAHD; CAPE; Flavors and fragrances; Therapeutics; Yeast