Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast
- Aymerick Eudes1, 2,
- Maxence Mouille1, 2,
- David S. Robinson3,
- Veronica T. Benites1, 2, 4,
- George Wang1, 2,
- Lucien Roux1, 2, 5,
- Yi-Lin Tsai1, 2,
- Edward E. K. Baidoo1, 2,
- Tsan-Yu Chiu1, 2,
- Joshua L. Heazlewood1, 2, 6,
- Henrik V. Scheller1, 2,
- Aindrila Mukhopadhyay1, 2,
- Jay D. Keasling1, 2, 7, 8,
- Samuel Deutsch3 and
- Dominique Loqué1, 2, 9Email authorView ORCID ID profile
© The Author(s) 2016
Received: 31 August 2016
Accepted: 6 November 2016
Published: 21 November 2016
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.
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.
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.
KeywordsYeast BAHD Antioxidant Therapeutics Flavors and fragrances CAPE
Chemical synthesis from non-renewable resources is the route most employed for production of chemicals used in both the pharmaceutical and flavor and fragrance industries. Several alternatives have emerged to limit the use of petroleum-based chemicals and to develop environmentally friendly methods through decreasing solvent utilization and reducing the carbon footprint of manufacturing processes. An example of such an alternative is biological synthesis via the use of engineered microbes for the production of fine and specialty chemicals. Because these processes consume renewable lignocellulosic biomass as a carbon source, they have reduced need for toxic chemicals and also offers consistent quality, scalability, simple extraction procedures and the potential for higher synthesis efficiency [1, 2]. In addition, biological synthesis could expand the chemical diversity of natural products, the structural complexity of which is sometimes challenging to achieve using multistep chemical synthesis . In this area, the industrial microorganism S. cerevisiae is a powerful host platform for the biosynthesis of plant secondary metabolites such as beta-carotene, amorphadiene, valencene, casbene, cubebol, linalool, patchoulol, resveratrol and vanillin. This is due to its food-grade status, its advantages in the expression of complex metabolic pathways, extensive knowledge regarding its use in large-scale production, the availability of genetic tools, and its biodiversity .
Hydroxycinnamic acids such as p-coumaric acid, caffeic acid, ferulic acid and sinapic acid possess antioxidant properties, antimicrobial activities and have been implicated in the prevention of cardiovascular diseases and cancer . Importantly, the pharmacokinetics and bioavailability of hydroxycinnamic acids, as well as their antimicrobial activity, are highly dependent on their chemical structures and the types of molecules to which they are conjugated [17, 18]. Moreover, certain benzoate conjugates are also used for their health benefits and therapeutic effects [19, 20], whereas benzoate alcohol esters are widely used as flavoring agents in the food and cosmetic industries [21, 22].
In this study, we investigated the potential of 10 members of the BAHD acyltransferase family for the production of diverse hydroxycinnamate and benzoate conjugates in S. cerevisiae after feeding with compatible acceptor molecules. These enzymes were selected based on their capacity to use hydroxycinnamoyl-CoAs and/or benzoyl-CoAs as donor molecules and their affinity for structurally divergent acceptors. We have previously reported on a two-gene strategy for co-expression in S. cerevisiae of Arabidopsis (Arabidopsis thaliana) 4-coumarate:CoA ligase 5 (4CL5) and HCBT for the synthesis of hydroxycinnamoyl anthranilates upon feeding with hydroxycinnamates and anthranilate . We also recently showed evidence that 4CL5 can accept benzoic acid as a substrate, which enabled the production of benzoyl anthranilates when co-expressed with HCBT . Here, taking advantage of 4CL5 substrate’s promiscuity, several BAHD acyltransferases were selected and co-expressed with 4CL5 in an attempt to synthesize in yeast novel hydroxycinnamate and benzoate esters and amides. Furthermore, although the yields of product formation were typically two to fivefold lower compared to those achieved with p-coumarate feedings, additional expression of tyrosine ammonia-lyase from Flavobacterium johnsoniae (FjTAL), which converts tyrosine into p-coumarate , enabled the synthesis of p-coumarate esters and amides directly from the endogenous tyrosine pool.
Results and discussion
Synthesis of rosmarinic acid and analogues
Synthesis of chlorogenic acid
Caffeoyl quinate (chlorogenic acid) and its derivatives have several beneficial properties for human health [6, 7, 26]. NtHQT, a BAHD enzyme from tobacco (Nicotiana tabacum), was selected for the synthesis of coumaroyl quinate and chlorogenic acid in S. cerevisiae because it uses both p-coumaroyl-CoA and caffeoyl-CoA as donors and quinate as its acceptor . We confirmed that feeding a strain expressing 4CL5 and NtHQT with quinate and p-coumarate or caffeate resulted in the production of the two target metabolites, namely coumaroyl quinate and chlorogenic acid (Additional file 1: Figure S2). The subsequent quantification of chlorogenic acid using an authentic standard showed that the culture medium contained 1.43 ± 0.21 µM (or 506 µg/L). To our knowledge, this is the first example of chlorogenic acid biosynthesis in yeast. Because of the conservation of the shikimate pathway between bacteria and yeast, engineering strategies for accumulation of quinate described in E. coli  could be easily transferred to our 4CL5-NtHQT yeast strain to achieve chlorogenic acid synthesis without feeding quinate.
Synthesis of glycerol hydroxycinnamates
Synthesis of polyamine hydroxycinnamates
Synthesis of monolignol hydroxycinnamates
Synthesis of malate hydroxycinnamates
Synthesis of alkyl hydroxycinnamates
Synthesis of cinnamoyl and benzoyl tropane analogues
Synthesis of benzoate alcohol esters and caffeate phenethyl ester (CAPE)
This study illustrates the potential of using BAHD acyltransferases from plants and S. cerevisiae as an expression host for the biological synthesis of valuable molecules that are usually produced from petrochemicals or found at very low concentration in plant extracts. In particular, expression of BAHD acyltransferases that use hydoxycinnamoyl-CoA and benzoyl-CoA allowed the synthesis of more than 30 different compounds which have applications across diverse sectors such as human health products and the flavor and fragrance industry. Exploring the substrate promiscuity of these BAHDs could considerably expand the number of molecules produced. Moreover, the use of BAHDs activity on alternate donors such as acetyl-CoA or malonyl-CoA should be explored for the biosynthesis of additional beneficial products.
The chemicals used for the S. cerevisiae feeding are listed in Additional file 1: Table S1. Rosmarinic acid was obtained from Spectrum Chemical Mfg. Corp. (New Brunswick, NJ), chlorogenic acid from Alfa Aesar (Haverhill, MA), ethyl benzoate, butyl benzoate, isopentyl benzoate and caffeate phenetyl ester from TCI America (Portland, OR), cinnamoyl 3β-tropine and phenethyl benzoate from Sigma-Aldrich (St. Louis, MO), and coniferyl ferulate from Biorbyt LLC (Berkeley, CA).
Strains, plasmids and media
Plasmids used in this study
URA3 selectable marker, arabidopsis 4CL5
P HXT7 -At4CL5-T CYC1 (URA3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, gateway cloning cassette
P HXT7 -At4CL5-T CYC1 -P PMA1 -attR1-Cm R -ccdB-attR2-T ADH1 (URA3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, DsRed dropout cassette
P HXT7 -At 4CL5-T CYC1 -P PMA1 -DsRed-T ADH1 (URA3, AmpR)
HIS3 selectable marker, Gateway cloning cassette
P TDH3 -attR1-Cm R -ccdB-attR2-T CYC1 (HIS3, AmpR)
HIS3 selectable marker, Flavobacterium johnsoniae TAL
P TDH3 -FjTAL-T CYC1 (HIS3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, Angelica sinensis FMT
P HXT7 -At4CL5-T CYC1 -P PMA1 -AsFMT-T ADH1 (URA3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, arabidopsis HHT3
P HXT7 -At4CL5-T CYC1 -P PMA1 -AtHHT3-T ADH1 (URA3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, arabidopsis SCT
P HXT7 -At4CL5-T CYC1 -P PMA1 -AtSCT-T ADH1 (URA3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, arabidopsis SDT
P HXT7 -At4CL5-T CYC1 -P PMA1 -AtSDT-T ADH1 (URA3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, Petunia x hybrida BPBT
P HXT7 -At4CL5-T CYC1 -P PMA1 -BPBT-T ADH1 (URA3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, Erythroxylum coca CS
P HXT7 -At4CL5-T CYC1 -P PMA1 -EcCS-T ADH1 (URA3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, Hordeum vulgare ACT
P HXT7 -At4CL5-T CYC1 -P PMA1 -HvACT-T ADH1 (URA3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, Nicotiana tabacum HQT
P HXT7 -At4CL5-T CYC1 -P PMA1 -NtHQT-T ADH1 (URA3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, Oryza sativa HCT4
P HXT7 -At4CL5-T CYC1 -P PMA1 -OsHCT4-T ADH1 (URA3, AmpR)
URA3 selectable marker, arabidopsis 4CL5, Trifolium pratense TpHCT2
P HXT7 -At4CL5-T CYC1 -P PMA1 -TpHCT2-T ADH1 (URA3, AmpR)
Construction of plasmids
A description of the plasmids used in this study is provided in Table 1. For the construction of the pDRf1-4CL5-DsRed vector, a red fluorescent protein (DsRed) dropout cassette sequence (kindly provided by Chris Anderson’s lab, UC Berkeley) was synthesized with flanking sequences (Genescript, Piscataway, NJ; Additional file 1: Data S1). It contains at the 5′-end the 18-bp sequence located upstream of the EcoRI site in pDRf1-4CL5-GW , followed by an EcoRI restriction site, the Gateway recombination site attB1 and a PmlI restriction site. At the 3′-end, it contains a PmlI restriction site, followed by the Gateway recombination site attB2, a PstI restriction site and the 15-bp sequence located upstream of the PstI site in pDRf1-4CL5-GW. This sequence was amplified by PCR using the oligonucleotides DsRed-dropout-fw and DsRed-dropout-rv (Additional file 1: Table S2) and inserted by In-Fusion cloning (Clonetech, Mountain View, CA) into a pDRf1-4CL5-GW vector digested with EcoRI and PstI to remove the Gateway cassette and yield the pDRf1-4CL5-DsRed vector. BAHD acyltransferase nucleotide sequences were codon-optimized for expression in S. cerevisiae using an empirically derived codon usage table. Codon optimization, including restriction site removal, and oligo design (150 mers) were performed using GeneDesign . Oligonucleotides were pooled for synthesis using acoustic deposition (Labcyte Echo 550), and synthesis was performed at the Joint Genome Institute using a two-step polymerase chain assembly (PCA) approach in 2 μL final volume as previously described . PCA products were purified by gel excision and cloned into a PmlI-digested pDRf1-4CL5-DsRed vector using Gibson assembly to generate the pDRf1-4CL5-BAHDs vectors. Plating and picking were performed using a QPix 400 system (Molecular Devices, Sunnyvale, CA). Eight colonies per construct were sequence-verified using PACBIO RSII system (Pacific Biosciences, Menlo Park, CA). Synthesized BAHD sequences and protein accession numbers are listed in Additional file 1: Data S1.
For the construct of the pRS423-GW vector, two DNA fragments covering the full backbone including P TDH3 and T CYC1 sequences while excluding XKS1 encoding sequence of the pRS423.XI vector were amplified by PCR using the oligonucleotide pairs pRS423-fw1/pRS423-rv1 and pRS423-fw2/pRS423-rv2 (Additional file 1: Table S2) and the pRS423.XI vector as a template . A third fragment corresponding to the Gateway cassette was amplified using the oligonucleotides GW-fw/GW-rv (Additional file 1: Table S2) and the pDRf1-4CL5-GW vector as a template . The pRS423-GW vector was constructed by assembling the three PCR fragments via In-Fusion cloning (Clonetech, Mountain View, CA). For the construct of the pRS423-FjTAL vector, a nucleotide sequence encoding FjTAL (GenBank accession number AKE50827.1) flanked with the attB1 (5′-end) and attB2 (3′-end) Gateway recombination sites was synthesized for expression in S. cerevisiae (Genescript, Piscataway, NJ) and cloned into the Gateway pDONR221 entry vector by BP recombination (Life technologies, Foster City, CA). An entry clone was LR recombined with the pRS423-GW vector to generate the pRS423-FjTAL construct. Plasmids are available upon request through the JBEI-ICE registry (http://public-registry.jbei.org) .
Production of hydroxycinnamate and benzoate conjugates
Overnight cultures from single colonies of recombinant S. cerevisiae harboring the pDRf1-4CL5-BAHDs and pDRf1-4CL5  constructs were grown using 2X YNB medium without amino acids, supplemented with 6% glucose and 2X CSM-ura. These cultures were used to inoculated 4 mL of fresh minimal medium at an OD600 = 0.15 and shaken at 200 rpm at 30 °C. Precursors were added 5 h post inoculation at the concentrations indicated in Additional file 1: Table S1. These concentrations were selected to be below toxicity levels and avoid growth inhibition. The cultures were shaken at 200 rpm at 30 °C for 24 h in the presence of the precursors for the production of hydrocinnamate and benzoate conjugates. Yeast colonies harboring the pDRf1-4CL5 control vector were grown under similar conditions. Yeast colonies co-transformed with the pRS423-FjTAL plasmid and the pDRf1-4CL5-LaAT1, pDRf1-4CL5-OsHCT4, pDRf1-4CL5-AtSCT, pDRf1-4CL5-HvACT, pDRf1-4CL5-TpHCT2, or pDRf1-4CL5-AtHHT3 vector were grown under similar conditions except that the medium was supplemented with 2X CSM-ura-his. For the detection of metabolites, an aliquot of the culture medium was collected and cleared by centrifugation (21,000×g for 5 min at 4 °C), mixed with an equal volume of cold methanol:water (1:1, v/v), and filtered using Amicon Ultra centrifugal filters (3000 Da MW cutoff regenerated cellulose membrane; Millipore, Billerica, MA) prior to analysis using high-performance liquid chromatography (HPLC), electrospray ionization (ESI), and time-of-flight (TOF) mass spectrometry (MS). Alternatively, for the detection of dodecyl hydroxycinnamates, caffeate phenethyl ester and benzoate alcohol esters, the culture medium was cleared by centrifugation and mixed with 2 mL of hexane for extraction with vortexing. The hexane phase was collected after centrifugation and concentrated under a nitrogen stream prior to HPLC-TOF-MS analysis. For the detection of coniferyl ferulate, yeast cells were harvested by centrifugation, washed and resuspended with 1 mL of HPLC grade water, and mixed with 2 mL of hexane for extraction with vortexing. The hexane phase was collected after centrifugation and concentrated under a nitrogen stream prior to HPLC-ESI-TOF-MS analysis.
LC–MS analysis of hydroxycinnamate and benzoate conjugates
Standard solutions of chlorogenic acid, rosmarinic acid, coniferyl ferulate and cinnamoyl 3β-tropine were prepared in methanol and water (1:1, v/v) and those of caffeate phenethyl ester and benzoate alcohol esters were prepared in hexane. All metabolites were separated via an Agilent 1200 Series Rapid Resolution high performance liquid chromatography (HPLC) system. The systems autosampler tray was set to 6 °C. The HPLC system was coupled to an Agilent Technologies 6210 LC/TOF mass spectrometer (MS) via either electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), which was used for MS detection and metabolite identification. Drying gas temperature, drying gas pressure, nebulizing gas pressure and capillary voltage were set to 330 °C, 10 or 11 L/min, 25 lb/in2, and 3500 V (in positive or negative ion modes), respectively, unless stated otherwise. For APCI, the vaporizer and corona were set to 350 °C and 4 µA, respectively, unless stated otherwise.
A HPLC-ESI-TOF-MS method previously described in  was used for the separation and detection of rosmarinic acid and its derivatives, coumaroyl quinate and malate hydroxycinnamate esters. The same method was used for coniferyl ferulate except that the drying gas temperature was set at 200 °C. A similar method was used for glycerol hydroxycinnamate esters with the following modifications: The mobile phase was composed of water (solvent A) and methanol (solvent B), and metabolites were separated via gradient elution under the following mobile phase compositions: % B was linearly increased from 30% B to 98% B in 12 min, held at 98% B for 0.6 min, decreased from 98% B to 30% B in 0.2 min and held at 30% B for a further 2.8 min. The total run time was 15.6 min. A drying gas temperature of 200 °C was used throughout. A HPLC-ESI-TOF-MS method previously described in  was used for the separation and detection of polyamine hydroxycinnamates amides and tropane alkaloids.
Separation of benzoate alcohol esters was conducted on a Phenomenex Kinetex XB-C18 column (100 mm length, 2.1 mm internal diameter, and 2.6 µm particle size; Phenomenex, Torrance, CA). The mobile phase was composed of water (solvent A) and methanol (solvent B). The elution gradient was as follows: % B was linearly increased from 60% B to 100% B in 5.0 min, held at 100% B for 2.0 min, decreased from 100% B to 60% B in 0.2 min and held at 60% B for a further 4.5 min. A flow rate of 0.3 mL/min was used until 7.0 min, increased from 0.3 to 0.45 mL/min in 0.2 min and held at 0.45 mL/min for 4.5 min. The total run time was 11.7 min. The column compartment was set to 55 °C. A sample injection volume of 2 µL was used throughout. APCI was conducted using in the positive ion mode for the detection of [M + H]+ ions. The same HPLC column and column compartment temperature were used for the separation of CAPE. The mobile phase was composed of water (solvent A) and acetonitrile (solvent B) and the elution gradient was as follows: % B was increased linearly from 25% B to 100% B in 3.0 min, held at 100% B for 1.0 min, decreased from 100% B to 25% B in 0.2 min and held at 25% B for a further 4.0 min. A flow rate of 0.3 mL/min was used until 4.0 min, increased from 0.3 to 0.45 mL/min in 0.2 min and held at 0.45 mL/min for the remaining 4.0 min. The total run time was 8.2 min. A sample injection volume of 0.5 µL was used throughout. ESI was conducted in the negative ion mode for the detection of [M–H]− ions.
Separation of dodecyl hydroxycinnamates was conducted on a Phenomenex Kinetex XB-C18 column (100 mm length, 3.0 mm internal diameter and 2.6 μm particle size). A sample injection volume of 3 μL was used throughout. The column compartment was set to 55 °C. The mobile phase was composed of water (solvent A) and methanol (solvent B). The elution gradient was as follows: % B was increased linearly from 65% B to 98% B in 1.47 min, held at 98% B for 9.2 min, decreased from 98% B to 65% B in 0.33 min and held at 65% B for a further 2.0 min. A flow rate of 0.42 mL/min was used until 10.67 min, increased from 0.42 to 0.65 mL/min in 0.33 min and held at 0.65 mL/min for 2.0 min. The total run time was 13.0 min. APCI was conducted in the positive ion mode for the detection of [M + H]+ ions. The corona and capillary voltage were set to 15 µA and 3000 V, respectively. A nebulizing gas pressure of 30 lb/in2 and a drying gas temperature of 200 °C were used throughout.
A HPLC-ESI TOF-MS method was used to detect chlorogenic acid. The chromatography was done using a Phenomenex Kinetex XB-C18 column (100 mm length, 3 mm internal diameter and 2.6 µm particle size) using the aforementioned HPLC system. A sample injection volume of 5 μL was used throughout. The mobile phase was composed of 0.1% formic acid in water (solvent A) and in methanol (solvent B). The elution gradient was as follows: % B was increased linearly from 25 to 77.5% B in 3.0 min, then increased from 77.5 to 97.1% B in 0.3 min, held at 97.1% B for 1.0 min, decreased from 97.1 to 25% B in 0.4 min and held at 25% B for a further 2 min. A flow rate of 0.42 mL/min was used until 4.3 min, then increased from 0.42 to 0.65 mL/min in 0.4 min and held at 0.65 mL/min for 2 min. The total run time was 6.7 min. The column compartment and sample tray were set to 50 °C and 6 °C, respectively. A sample injection volume of 3 µL was used throughout. ESI was conducted in the negative ion mode for the detection of [M–H]− ions. The nebulizing gas pressure was set to 30 lb/in2.
Data acquisition and processing were performed by the MassHunter software package (Agilent Technologies Inc., Santa Clara, CA). For each compound detected, the measured masses agreed with the expected theoretical masses within less than 5 ppm mass error.
Angelica sinensis feruloyl-CoA:monolignol transferase
Arabidopsis thaliana caffeoyl-CoA:fatty alcohol transferase
Arabidopsis thaliana p-coumaroyl-CoA:spermidine transferase
Arabidopsis thaliana sinapoyl-CoA:spermidine transferase
Arabidopsis thaliana 4-coumarate:CoA ligase 5
BEAT (benzoyl alcohol O-acetyltransferase), AHCT (anthocyanin O-hydroxycinnamoyl transferase), HCBT (anthranilate N-hydroxycinnamoyl/benzoyl transferase), and DAT (deacetyl vindoline 4-O-acetyltransferase)
benzoyl-CoA:benzyl alcohol/phenylethanol benzoyltransferase
caffeate phenethyl ester
Erythroxylum coca cocaine synthase
Flavobacterium johnsoniae tyrosine ammonia-lyase
Hordeum vulgare p-coumaroyl-CoA:agmatine transferase
liquid chromatography-mass spectrometry
Nicotiana tabacum hydroxycinnamoyl-CoA:quinate transferase
Oryza sativa hydroxycinnamoyl-CoA:shikimate transferase 4
Trifolium pratense hydroxycinnamoyl-CoA:shikimate transferase 2
AE wrote the manuscript. DL edited the document. AE, LR, MM, VTB and Y-LT performed the experiments. T-YC generated the p423-GW yeast vector. EEKB and GW conducted the LC–MS analyses. DR and SD synthesized the genes and assembled them in the yeast expression vector. AE, AM, HVS, JDK, SD and DL supervised the research. AM, DL, HVS, JDK, JLH and SD provided chemicals and financial support. All authors read and approved the final manuscript.
We thank Sabin Russell for editing this manuscript.
JDK has financial conflicts of interest in Amyris and Lygos. DL and HVS have financial conflicts of interest in Afingen Inc.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files. Plasmids are available upon request through the JBEI-ICE registry (http://public-registry.jbei.org).
This work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research and the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes.
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- Keasling JD. Manufacturing molecules through metabolic engineering. Science. 2010;330:1355–8.View ArticleGoogle Scholar
- Nielsen J, Keasling JD. Engineering cellular metabolism. Cell. 2016;164:1185–97.View ArticleGoogle Scholar
- Sun H, Liu Z, Zhao H, Ang EL. Recent advances in combinatorial biosynthesis for drug discovery. Drug Des Devel Ther. 2015;12:823–33.Google Scholar
- Hong KK, Nielsen J. Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries. Cell Mol Life Sci. 2012;69:2671–90.View ArticleGoogle Scholar
- D’Auria JC. Acyltransferases in plants: a good time to be BAHD. Curr Opin Plant Biol. 2006;9:331–40.View ArticleGoogle Scholar
- Kim BG, Jung WD, Mok H, Ahn JH. Production of hydroxycinnamoyl-shikimates and chlorogenic acid in Escherichia coli: production of hydroxycinnamic acid conjugates. Microb Cell Fact. 2013;12:15.View ArticleGoogle Scholar
- Cha MN, Kim HJ, Kim BG, Ahn JH. Synthesis of chlorogenic acid and p-coumaroyl shikimates from glucose using engineered Escherichia coli. J Microbiol Biotechnol. 2014;24:1109–17.View ArticleGoogle Scholar
- Bloch SE, Schmidt-Dannert C. Construction of a chimeric biosynthetic pathway for the de novo biosynthesis of rosmarinic acid in Escherichia coli. ChemBioChem. 2014;15:2393–401.View ArticleGoogle Scholar
- Jiang J, Bi H, Zhuang Y, Liu S, Liu T, Ma Y. Engineered synthesis of rosmarinic acid in Escherichia coli resulting production of a new intermediate, caffeoyl-phenyllactate. Biotechnol Lett. 2016;38:81–8.View ArticleGoogle Scholar
- Zhuang Y, Jiang J, Bi H, Yin H, Liu S, Liu T. Synthesis of rosmarinic acid analogues in Escherichia coli. Biotechnol Lett. 2016;38:619–27.View ArticleGoogle Scholar
- Kim IA, Kim BG, Kim M, Ahn JH. Characterization of hydroxycinnamoyltransferase from rice and its application for biological synthesis of hydroxycinnamoyl glycerols. Phytochemistry. 2012;76:25–31.View ArticleGoogle Scholar
- Eudes A, Juminaga D, Baidoo EE, Collins FW, Keasling JD, Loqué D. Production of hydroxycinnamoyl anthranilates from glucose in Escherichia coli. Microb Cell Fact. 2013;12:62.View ArticleGoogle Scholar
- Eudes A, Baidoo EE, Yang F, Burd H, Hadi MZ, Collins FW, Keasling JD, Loqué D. Production of tranilast [N-(3′,4′-dimethoxycinnamoyl)-anthranilic acid] and its analogs in yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2011;89:989–1000.View ArticleGoogle Scholar
- Eudes A, Teixeira Benites V, Wang G, Baidoo EE, Lee TS, Keasling JD, Loqué D. Precursor-directed combinatorial biosynthesis of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates in Saccharomyces cerevisiae. PLoS ONE. 2015;10:e0138972.View ArticleGoogle Scholar
- Eudes A, Pereira JH, Yogiswara S, Wang G, Teixeira Benites V, Baidoo EE, Lee TS, Adams PD, Keasling JD, Loqué D. Exploiting the substrate promiscuity of hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase to reduce lignin. Plant Cell Physiol. 2016;57:568–79.View ArticleGoogle Scholar
- Moglia A, Goitre L, Gianoglio S, Baldini E, Trapani E, Genre A, Scattina A, Dondo G, Trabalzini L, Beekwilder J, Retta SF. Evaluation of the bioactive properties of avenanthramide analogs produced in recombinant yeast. BioFactors. 2015;41:15–27.View ArticleGoogle Scholar
- El-Seedi HR, El-Said AM, Khalifa SA, Göransson U, Bohlin L, Borg-Karlson AK, Verpoorte R. Biosynthesis, natural sources, dietary intake, pharmacokinetic properties, and biological activities of hydroxycinnamic acids. J Agric Food Chem. 2012;60:10877–95.View ArticleGoogle Scholar
- Guzman JD. Natural cinnamic acids, synthetic derivatives and hybrids with antimicrobial activity. Molecules. 2014;19:19292–349.View ArticleGoogle Scholar
- Mo H, Tatman D, Jung M, Elson CE. Farnesyl anthranilate suppresses the growth, in vitro and in vivo, of murine B16 melanomas. Cancer Lett. 2000;157:145–53.View ArticleGoogle Scholar
- Singh S. Chemistry, design, and structure-activity relationship of cocaine antagonists. Chem Rev. 2000;100:925–1024.View ArticleGoogle Scholar
- Adams TB, Cohen SM, Doull J, Feron VJ, Goodman JI, Marnett LJ, Munro IC, Portoghese PS, Smith RL, Waddell WJ, Wagner BM. Expert panel of the flavor and extract manufacturers association: the FEMA GRAS assessment of benzyl derivatives used as flavor ingredients. Food Chem Toxicol. 2005;43:1207–40.View ArticleGoogle Scholar
- Del Olmo A, Calzada J, Nuñez M. Benzoic acid and its derivatives as naturally occurring compounds in foods and as additives: uses, exposure and controversy. Crit Rev Food Sci Nutr. 2015. doi:https://doi.org/10.1080/10408398.2015.1087964.Google Scholar
- Jendresen CB, Stahlhut SG, Li M, Gaspar P, Siedler S, Förster J, Maury J, Borodina I, Nielsen AT. Highly active and specific tyrosine ammonia-lyases from diverse origins enable enhanced production of aromatic compounds in bacteria and saccharomyces cerevisiae. Appl Environ Microbiol. 2015;81:4458–76.View ArticleGoogle Scholar
- Kim GD, Park YS, Jin YH, Park CS. Production and applications of rosmarinic acid and structurally related compounds. Appl Microbiol Biotechnol. 2015;99:2083–92.View ArticleGoogle Scholar
- Landmann C, Hücherig S, Fink B, Hoffmann T, Dittlein D, Coiner HA, Schwab W. Substrate promiscuity of a rosmarinic acid synthase from lavender (Lavandula angustifolia L.). Planta. 2011;234:305–20.View ArticleGoogle Scholar
- Ma CM, Kawahata T, Hattori M, Otake T, Wang L, Daneshtalab M. Synthesis, anti-HIV and anti-oxidant activities of caffeoyl 5,6-anhydroquinic acid derivatives. Bioorg Med Chem. 2010;18:863–9.View ArticleGoogle Scholar
- Niggeweg R, Michael AJ, Martin C. Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat Biotechnol. 2004;22:746–54.View ArticleGoogle Scholar
- Juminaga D, Baidoo EE, Redding-Johanson AM, Batth TS, Burd H, Mukhopadhyay A, Petzold CJ, Keasling JD. Modular engineering of l-tyrosine production in Escherichia coli. Appl Environ Microbiol. 2012;78:89–98.View ArticleGoogle Scholar
- Kikugawa M, Tsutsuki H, Ida T, Nakajima H, Ihara H, Sakamoto T. Water-soluble ferulic acid derivatives improve amyloid-β-induced neuronal cell death and dysmnesia through inhibition of amyloid-β aggregation. Biosci Biotechnol Biochem. 2016;80:547–53.View ArticleGoogle Scholar
- Evans KO, Compton DL, Laszlo JA, Appell M. Feruloyl glycerol and 1,3-diferuloyl glycerol antioxidant behavior in phospholipid vesicles. Chem Phys Lipids. 2016;195:1–11.View ArticleGoogle Scholar
- Ohta S, Fujimaki T, Uy MM, Yanai M, Yukiyoshi A, Hirata T. Antioxidant hydroxycinnamic acid derivatives isolated from Brazilian bee pollen. Nat Prod Res. 2007;21:726–32.View ArticleGoogle Scholar
- Hadjipavlou-Litina D, Garnelis T, Athanassopoulos CM, Papaioannou D. Kukoamine A analogs with lipoxygenase inhibitory activity. J Enzyme Inhib Med Chem. 2009;24:1188–93.View ArticleGoogle Scholar
- Yingyongnarongkul BE, Apiratikul N, Aroonrerk N, Suksamrarn A. Synthesis of bis, tris and tetra(dihydrocaffeoyl) polyamine conjugates as antibacterial agents against VRSA. Arch Pharm Res. 2008;31:698–704.View ArticleGoogle Scholar
- Ma CM, Nakamura N, Hattori M. Inhibitory effects on HIV-1 protease of tri-p-coumaroylspermidine from Artemisia caruifolia and related amides. Chem Pharm Bull. 2001;49:915–7.View ArticleGoogle Scholar
- Fixon-Owoo S, Levasseur F, Williams K, Sabado TN, Lowe M, Klose M, Joffre Mercier A, Fields P, Atkinson J. Preparation and biological assessment of hydroxycinnamic acid amides of polyamines. Phytochemistry. 2003;63:315–34.View ArticleGoogle Scholar
- Li Z, Fennie MW, Ganem B, Hancock MT, Kobaslija M, Rattendi D, Bacchi CJ, O’Sullivan MC. Polyamines with N-(3-phenylpropyl) substituents are effective competitive inhibitors of trypanothione reductase and trypanocidal agents. Bioorg Med Chem Lett. 2001;11:251–4.View ArticleGoogle Scholar
- Yamamoto A, Nakamura K, Furukawa K, Konishi Y, Ogino T, Higashiura K, Yago H, Okamoto K, Otsuka M. A new nonpeptide tachykinin NK1 receptor antagonist isolated from the plants of Compositae. Chem Pharm Bull. 2002;50:47–52.View ArticleGoogle Scholar
- Zhao G, Qin GW, Gai Y, Guo LH. Structural identification of a new tri-p-coumaroylspermidine with serotonin transporter inhibition from safflower. Chem Pharm Bull. 2010;58:950–2.View ArticleGoogle Scholar
- Luo J, Fuell C, Parr A, Hill L, Bailey P, Elliott K, Fairhurst SA, Martin C, Michael AJ. A novel polyamine acyltransferase responsible for the accumulation of spermidine conjugates in Arabidopsis seed. Plant Cell. 2009;21:318–33.View ArticleGoogle Scholar
- Burhenne K, Kristensen BK, Rasmussen SK. A new class of N-hydroxycinnamoyltransferases. Purification, cloning, and expression of a barley agmatine coumaroyltransferase (EC 220.127.116.11). J Biol Chem. 2003;278:13919–27.View ArticleGoogle Scholar
- Kim SK, Jin YS, Choi IG, Park YC, Seo JH. Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocellulose-derived inhibitors through modulation of spermidine contents. Metab Eng. 2015;29:46–55.View ArticleGoogle Scholar
- Chou SC, Everngam MC, Sturtz G, Beck JJ. Antibacterial activity of components from Lomatium californicum. Phytother Res. 2006;20:153–6.View ArticleGoogle Scholar
- Ho CC, Kumaran A, Hwang LS. Bio-assay guided isolation and identification of anti-Alzheimer active compounds from the root of Angelica sinensis. Food Chem. 2009;114:246–52.View ArticleGoogle Scholar
- Chen C, Wu C, Lu X, Yan Z, Gao J, Zhao H, Li S. Coniferyl ferulate, a strong inhibitor of glutathione S-transferase isolated from radix Angelicae sinensis, reverses multidrug resistance and downregulates P-glycoprotein. Evid Based Complement Alternat Med. 2013;2013:639083.Google Scholar
- Wilkerson CG, Mansfield SD, Lu F, Withers S, Park JY, Karlen SD, Gonzales-Vigil E, Padmakshan D, Unda F, Rencoret J, Ralph J. Monolignol ferulate transferase introduces chemically labile linkages into the lignin backbone. Science. 2014;344:90–3.View ArticleGoogle Scholar
- Jansen F, Gillessen B, Mueller F, Commandeur U, Fischer R, Kreuzaler F. Metabolic engineering for p-coumaryl alcohol production in Escherichia coli by introducing an artificial phenylpropanoid pathway. Biotechnol Appl Biochem. 2014;61:646–54.View ArticleGoogle Scholar
- van Summeren-Wesenhagen PV, Voges R, Dennig A, Sokolowsky S, Noack S, Schwaneberg U, Marienhagen J. Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli. Microb Cell Fact. 2015;14:79.View ArticleGoogle Scholar
- Kimura Y, Okuda H, Okuda T, Hatano T, Arichi S. Studies on the activities of tannins and related compounds, X. Effects of caffeetannins and related compounds on arachidonate metabolism in human polymorphonuclear leukocytes. J Nat Prod. 1987;50:392–9.View ArticleGoogle Scholar
- Sullivan M. A novel red clover hydroxycinnamoyl transferase has enzymatic activities consistent with a role in phaselic acid biosynthesis. Plant Physiol. 2009;150:1866–79.View ArticleGoogle Scholar
- Liu P, Jarboe LR. Metabolic engineering of biocatalysts for carboxylic acids production. Comput Struct Biotechnol J. 2012;3:e201210011.View ArticleGoogle Scholar
- Anselmi C, Centini M, Andreassi M, Buonocore A, La Rosa C, Facino RM, Sega A, Tsuno F. Conformational analysis: a tool for the elucidation of the antioxidant properties of ferulic acid derivatives in membrane models. J Pharm Biomed Anal. 2004;35:1241–9.View ArticleGoogle Scholar
- Jayaprakasam B, Vanisree M, Zhang Y, Dewitt DL, Nair MG. Impact of alkyl esters of caffeic and ferulic acids on tumor cell proliferation, cyclooxygenase enzyme, and lipid peroxidation. J Agric Food Chem. 2006;54:5375–81.View ArticleGoogle Scholar
- Kosma DK, Molina I, Ohlrogge JB, Pollard M. Identification of an Arabidopsis fatty alcohol:caffeoyl-Coenzyme A acyltransferase required for the synthesis of alkyl hydroxycinnamates in root waxes. Plant Physiol. 2012;160:237–48.View ArticleGoogle Scholar
- Runguphan W, Keasling JD. Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Metab Eng. 2014;21:103–13.View ArticleGoogle Scholar
- Sheng J, Stevens J, Feng X. Pathway compartmentalization in peroxisome of Saccharomyces cerevisiae to produce versatile medium chain fatty alcohols. Sci Rep. 2016;6:26884.View ArticleGoogle Scholar
- Fodor G, Dharanipragada R. Tropane alkaloids. Nat Prod Rep. 1994;11:443–50.View ArticleGoogle Scholar
- Grynkiewicz G, Gadzikowska M. Tropane alkaloids as medicinally useful natural products and their synthetic derivatives as new drugs. Pharmacol Rep. 2008;60:439–63.Google Scholar
- Schmidt GW, Jirschitzka J, Porta T, Reichelt M, Luck K, Torre JC, Dolke F, Varesio E, Hopfgartner G, Gershenzon J, D’Auria JC. The last step in cocaine biosynthesis is catalyzed by a BAHD acyltransferase. Plant Physiol. 2015;167:89–101.View ArticleGoogle Scholar
- Qualley AV, Widhalm JR, Adebesin F, Kish CM, Dudareva N. Completion of the core β-oxidative pathway of benzoic acid biosynthesis in plants. Proc Natl Acad Sci USA. 2012;109:16383–8.View ArticleGoogle Scholar
- Palazón J, Navarro-Ocaña A, Hernandez-Vazquez L, Mirjalili MH. Application of metabolic engineering to the production of scopolamine. Molecules. 2008;13:1722–42.View ArticleGoogle Scholar
- Boatright J, Negre F, Chen X, Kish CM, Wood B, Peel G, Orlova I, Gang D, Rhodes D, Dudareva N. Understanding in vivo benzenoid metabolism in petunia petal tissue. Plant Physiol. 2004;135:1993–2011.View ArticleGoogle Scholar
- Tolba MF, Azab SS, Khalifa AE, Abdel-Rahman SZ, Abdel-Naim AB. Caffeic acid phenethyl ester, a promising component of propolis with a plethora of biological activities: a review on its anti-inflammatory, neuroprotective, hepatoprotective, and cardioprotective effects. IUBMB Life. 2013;65:699–709.View ArticleGoogle Scholar
- Murtaza G, Karim S, Akram MR, Khan SA, Azhar S, Mumtaz A, Bin Asad MH. Caffeic acid phenethyl ester and therapeutic potentials. Biomed Res Int. 2014;2014:145342.Google Scholar
- Zhang P, Tang Y, Li NG, Zhu Y, Duan JA. Bioactivity and chemical synthesis of caffeic acid phenethyl ester and its derivatives. Molecules. 2014;19:16458–76.View ArticleGoogle Scholar
- Dudareva N, D’Auria JC, Nam KH, Raguso RA, Pichersky E. Acetyl-CoA:benzylalcohol acetyltransferase–an enzyme involved in floral scent production in Clarkia breweri. Plant J. 2002;14:297–304.View ArticleGoogle Scholar
- D’Auria JC, Chen F, Pichersky E. Characterization of an acyltransferase capable of synthesizing benzylbenzoate and other volatile esters in flowers and damaged leaves of Clarkia breweri. Plant Physiol. 2002;130:466–76.View ArticleGoogle Scholar
- Sun J, Alper HS. Metabolic engineering of strains: from industrial-scale to lab-scale chemical production. J Ind Microbiol Biotechnol. 2015;42:423–36.View ArticleGoogle Scholar
- Kim B, Cho BR, Hahn JS. Metabolic engineering of Saccharomyces cerevisiae for the production of 2-phenylethanol via Ehrlich pathway. Biotechnol Bioeng. 2014;111:115–24.View ArticleGoogle Scholar
- Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM, Connelly C, Davis K, Dietrich F, Dow SW, El Bakkoury M, Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, Jones T, Laub M, Liao H, Liebundguth N, Lockhart DJ, Lucau-Danila A, Lussier M, M’Rabet N, Menard P, Mittmann M, Pai C, Rebischung C, Revuelta JL, Riles L, Roberts CJ, Ross-MacDonald P, Scherens B, Snyder M, Sookhai-Mahadeo S, Storms RK, Véronneau S, Voet M, Volckaert G, Ward TR, Wysocki R, Yen GS, Yu K, Zimmermann K, Philippsen P, Johnston M, Davis RW. Functionnal characterization of the S. cerevisiae genome by deletion and parallel analysis. Science. 1999;285:901–6.View ArticleGoogle Scholar
- Richardson SM, Nunley PW, Yarrington RM, Boeke JD, Bader JS. GeneDesign 3.0 is an updated synthetic biology toolkit. Nucleic Acids Res. 2010;38:2603–6.View ArticleGoogle Scholar
- Heins RA, Cheng X, Nath S, Deng K, Bowen BP, Chivian DC, Datta S, Friedland GD, D’Haeseleer P, Wu D, Tran-Gyamfi M, Scullin CS, Singh S, Shi W, Hamilton MG, Bendall ML, Sczyrba A, Thompson J, Feldman T, Guenther JM, Gladden JM, Cheng JF, Adams PD, Rubin EM, Simmons BA, Sale KL, Northen TR, Deutsch S. Phylogenomically guided identification of industrially relevant GH1 β-glucosidases through DNA synthesis and nanostructure-initiator mass spectrometry. ACS Chem Biol. 2014;9:2082–91.View ArticleGoogle Scholar
- Reider Apel A, Ouellet M, Szmidt-Middleton H, Keasling JD, Mukhopadhyay A. Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae. Sci Rep. 2016;6:19512.View ArticleGoogle Scholar
- Ham TS, Dmytriv Z, Plahar H, Chen J, Hillson NJ, Keasling JD. Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools. Nucleic Acids Res. 2012;40:e141.View ArticleGoogle Scholar
- Bokinsky G, Baidoo EE, Akella S, Burd H, Weaver D, Alonso-Gutierrez J, Garcia-Martin H, Lee TS, Keasling JD. HipA-triggered growth arrest and b-lactam tolerance in Escherichia coli are mediated by RelA-dependent ppGpp synthesis. J Bacteriol. 2003;195:3173–82.View ArticleGoogle Scholar