Bacterial synthesis of N-hydroxycinnamoyl phenethylamines and tyramines
© Sim et al. 2015
Received: 14 August 2015
Accepted: 2 October 2015
Published: 13 October 2015
Hydroxycinnamic acids (HCAs) including cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid, are C6–C3 phenolic compounds that are synthesized via the phenylpropanoid pathway. HCAs serve as precursors for the synthesis of lignins, flavonoids, anthocyanins, stilbenes and other phenolic compounds. HCAs can also be conjugated with diverse compounds including quinic acid, hydroxyl acids, and amines. Hydroxycinnamoyl (HC) amine conjugates such as N-HC tyramines and N-HC phenethylamines have been considered as potential starting materials to develop antiviral and anticancer drugs.
We synthesized N-HC tyramines and N-HC phenethylamines using three different approaches in Escherichia coli. Five N-HC phenethylamines and eight N-HC tyramines were synthesized by feeding HCAs and phenethylamine or tyramine to E. coli harboring 4CL (encoding 4-coumarate CoA:ligase) and either SHT (encoding phenethylamine N-HC transferase) or THT (encoding tyramine N-HC transferase). Also, N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine were synthesized from p-coumaric acid using E. coli harboring an additional gene, PDC (encoding phenylalanine decarboxylase) or TDC (encoding tyrosine decarboxylase). Finally, we synthesized N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine from glucose by reconstructing the metabolic pathways for their synthesis in E. coli. Productivity was maximized by optimizing the cell concentration and incubation temperature.
We reconstructed the metabolic pathways for synthesis of N-HC tyramines and N-HC phenethylamines by expressing several genes including 4CL, TST or SHT, PDC or TDC, and TAL (encoding tyrosine ammonia lyase) and engineering the shikimate metabolic pathway to increase endogenous tyrosine concentration in E. coli. Approximately 101.9 mg/L N-(p-coumaroyl) phenethylamine and 495.4 mg/L N-(p-coumaroyl) tyramine were synthesized from p-coumaric acid. Furthermore, 152.5 mg/L N-(p-coumaroyl) phenethylamine and 94.7 mg/L N-(p-coumaroyl) tyramine were synthesized from glucose.
Plants synthesize phenolic compounds through the phenylpropanoid pathway. Hydroxycinnamic acids (HCAs) serve as precursors for the synthesis of lignins, flavonoids, anthocyanins, stilbenes and other phenolic compounds . Hydroxycinnamates can be conjugated with diverse compounds. In the formation of hydroxycinnamate conjugates, hydroxycinnamates such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid can serve as donors, while diverse chemicals including quinic acid, hydroxyl acids (maleic acid and tartaric acid), amino compounds (aromatic amino acids, choline, and anthranilic acids), and polysaccharides (glycerol, anthocyanin glycosides, flavonoid glycosides, and terpene glycosides) can be used as acceptors .
Hydroxycinnamoyl (HC) amides are an example of hydroxycinnamate conjugates, in which tyramine, phenethylamine, serotonin, anthranilate, tryptamine, or dopamine serve as HC acceptors. HC amides are formed in plants for defense against pathogens . For example, N-cinnamoyl phenylethylamine was reported to show antifungal activity . Accumulation of N-cinnamoyl tyramine and N-feruloyl tyramine in tomato (Lycopersicon eseulentum) was found to lead to resistance against Pseudomonas syringae . Also, some plants synthesize N-HC phenethylamine and N-HC tyramine. N-(p-Coumaroyl) tyramine and N-feruoyl tyramine are found in tomato , Solanum melongena , and Caspicum annuum . N-(p-Coumaoyl) phenethylamine has been isolated from Anomianthus dulcis .
Many plant metabolites have been used as starting molecules to develop new medicines [10, 11]. HC amides were originally known as antioxidants, like other plant phenolic compounds. However, recent studies have shown that HC amides could be developed into new candidate medicines. For example, hydroxycinnamate amide served as a backbone for the synthesis of antiviral compounds . Analogs of N-HC phenalkylamides are inhibitors of tyrosinase in human melanocytes, which has the potential to treat pigmentation-related disorders . N-HC tyramine showed anti-proliferative effects on cancer cells  and selectively induced the apoptosis of cancer cells .
For the synthesis of N-HC phenethylamines and N-HC tyramines, the two amino acids phenylalanine and tyrosine, respectively, serve as the key substrates. Phenethylamine and tyramine are synthesized from phenylalanine and tyrosine by phenylalanine decarboxylase (PDC) and tyrosine decarboxylase (TDC), respectively. PDC has been isolated and characterized from tomato  and TDC has been isolated from several plants including parsley, Papaver somniferum, Arabidopsis thaliana, and Catharanthus roseus [17–21]. PDC and TDC show catalytic activity for both phenylalanine and tyrosine as substrates, but have been named depending on their substrate preference. On the other hand, hydroxycinnamic acids are also synthesized from these two amino acids by phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) via the phenylpropanoid pathway . Therefore, the endogenous concentrations of tyrosine and phenylalanine are critical factors for the synthesis of N-HC tyramines and N-HC phenethylamines in Escherichia coli.
In this report, we synthesized N-HC tyramines and N-HC phenethylamines from tyrosine and phenylalanine using metabolically engineered E. coli. The shikimate pathway of E. coli which leads to the production of phenylalanine, tyrosine, and tryptophan, is well-characterized and strategies to increase the intracellular concentrations of tyrosine and phenylalanine are also known. . Using engineered E. coli strains, we were able to synthesize N-HC phenylethylamines and N-HC tyramines from glucose as well as from HCs.
Production of N-HC phenethylamines and N-HC tyramines in E. coli
Plasmids and strains used in the present study
Plasmids or E. coli strain
Relevant properties or genetic marker(s)
Source or references
P15A ori, Cmr
CloDE13 ori, Strr
f1 ori, Ampr
pACYCDuet carrying TAL from Saccharothrix espanaensis
pACYCDuet carrying TAL from S. espanaensis, aroG and tyrA from Escherichia coli
pACYCDuet carrying TAL from S. espanaensis, aroG fbr, and tyrA fbr from E. coli
pETDuet carrying PDC from Pseudomonas putida
pCDFDuet carrying 4CL from Oryza sativa and SHT from Capsicum annuum
pCDFDuet carrying 4CL from O. sativa and THT from C. annuum
pCDFDuet carrying TDC from Papaver somniferum, 4CL from O. sativa, and THT from C. annuum
F− ompT hsdS B (r B − m B − ) gal dcm lon (DE3)
BL21 harboring pC-Os4CL-SHT
BL21 harboring pC-Os4CL-SHT and pE-PDC
BL21 harboring pC-Os4CL-SHT, pA-SeTAL, and pE-PDC
BL21 harboring pC-Os4CL-SHT, pA-aroG-SeTAL-tyrA, and pE-PDC
BL21 harboring pC-Os4CL-SHT, pA-aroGfbr-SeTAL-tyrAfbr, and pE-PDC
BtyrR harboring pC-Os4CL-SHT, pA-aroGfbr-SeTAL-tyrAfbr, and pE-PDC
BtyrR-trpD harboring pC-Os4CL-SHT, pA-aroGfbr-SeTAL-tyrAfbr, and pE-PDC
BL21 harboring pC-Os4CL-THT
BL21 harboring pC-TDC-Os4CL-THT
BL21 harboring pC-TDC-Os4CL-THT and pA-aroG-tyrA
BL21 harboring pC-TDC-Os4CL-THT and pA-aroGfbr-tyrAfbr
BL21 harboring pC-TDC-Os4CL-THT and pA-SeTAL
BL21 harboring pC-TDC-Os4CL-THT and pA-aroG-SeTAL-tyrA
BL21 harboring pC-TDC-Os4CL-THT and pA-aroGfbr-SeTAL-tyrAfbr
BtyrR-pheA harboring pC-TDC-Os4CL-THT and pA-aroGfbr-SeTAL-tyrAfbr
BtyrR-trpD harboring pC-TDC-Os4CL-THT and pA-aroGfbr-SeTAL-tyrAfbr
Synthesis of N-(p-coumaroyl) phenethylamine in E. coli
Among the several reaction products, we decided to synthesize N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine by feeding E. coli with only p-coumaric acid. For this, either a decarboxylase that can convert phenylalanine into phenethylamine or one that can convert tyrosine into tyramine was introduced into the E. coli strains HP-1 and HT-1. We engineered strain HP-2 to synthesize N-(p-coumaroyl) phenethylamine by transforming E. coli with PDC along with SHT and 4CL and then feeding them p-coumaric acid. Analysis of the cultures from strain HP-2 using HPLC showed a peak with the same retention time as that of N-(p-coumaroyl) phenethylamine and its structure as that compound was confirmed by MS and NMR (data not shown). Therefore, we decided to use the HP-2 strain for optimization of the production of N-(p-coumaroyl) phenethylamine. First, we tested the effect of initial cell density. The cell density of HP-2 was adjusted to an OD600 of 1, 3, 5, or 7. The production of N-(p-coumaroyl) phenethylamine increased from 22.8 mg/L at OD600 = 1, 55.7 mg/L at OD600 = 3, and 59.6 mg/L at OD600 = 5, but decreased to 51.6 mg/L at OD600 = 7. The optimal incubation temperature was determined at the optimized cell density of OD600 = 5. HP-2 grown at 25, 30, or 37 °C produced 43.1, 59.6, or 48.2 mg/L N-(p-coumaroyl) phenethylamine, respectively. The initial p-coumaric acid concentration was then optimized with the optimized cell density (OD600 = 5) and incubation temperature (30 °C). The strain HP-2 was fed with 0.2, 0.4, 0.6, 0.8, or 1.0 mM of p-coumaric acid and the production of N-(p-coumaroyl) phenethylamine was examined. HP-2 converted 81.8 % of 0.2 mM p-coumaric acid into N-(p-coumaroyl) phenethylamine, producing 43.7 mg/L N-(p-coumaroyl) phenethylamine. When the cells were fed with 0.4 mM p-coumaric acid, the conversion rate was 80.4 %, producing 85.9 mg/L N-(p-coumaroyl) phenethylamine. At higher p-coumaric acid concentrations, the conversion rate of p-coumaric acid was less than 50 % and the final yields of N-(p-coumaroyl) phenethylamine were 78.5, 74.0 and 59.6 mg/L, respectively, which were much lower than the yield at 0.4 mM p-coumaric acid. Using the optimized initial concentration of p-coumaric acid (0.4 mM), we monitored the production of N-(p-coumaroyl) phenethylamine and found that approximately 101.9 mg/L N-(p-coumaroyl) phenethylamine was produced after 12 h, after which the yield decreased until 24 h (data not shown). Approximately 95.3 % of p-coumaric acid was converted into N-(p-coumaroyl) phenethylamine.
Synthesis of N-(p-coumaroyl) tyramine in E. coli
In order to optimize the production of N-(p-coumaroyl) tyramine using the strain HT-9, the incubation temperature and initial cell density were optimized. The best temperature was determined to be 30 °C (57.1 mg/L) followed by 25 °C (52.5 mg/L), 37 °C (39.2 mg/L), and 18 °C (22.0 mg/L).
Using the strain HT-9, the incubation temperature and the initial cell density were optimized. HT-9 was grown at 18, 25, 30, or 37 °C. The highest production of p-coumaroyl tyramine occurred in cells grown at 30 °C (57.06 mg/L). The yields at 18, 25 and 37 °C were approximately 21.98, 52.47 and 39.16 mg/L, respectively. The optimal initial cell density was also examined. The production of N-(p-coumaroyl) tyramine increased from OD600 = 0.5 to OD600 = 2 but decreased at OD600 = 3.
Five N-HC phenethylamines and eight N-HC tyramines from 12 HCAs and corresponding amines (phenethylamine and tyramine) were synthesized. Two enzymatic reactions are required to synthesize N-HC phenethylamines or N-HC tyramines in E. coli. The first step is the activation of HC by 4CL which results in the formation of HC-CoA. The second step is the conjugation of HC-CoA to phenethylamine or tyramine, which is catalyzed by either SHT or THT. Based on the results of HT-1 biotransformation, Os4CL used in this study could use at least eight HCAs as substrates (p-coumaric acid, m-coumaric acid, o-coumaric acid, caffeic acid, ferulic acid, cinnamic acid, 3-methoxycinnamic acid, and 4-methoxycinnamic acid). However, HCAs having two methoxy groups did not yield any reaction products. It is not clear whether this result is due to the failure of Os4CL to convert HCA into HC-CoA or due to the formation of conjugates between HC-CoA and tyramine or phenethylamine by THT or SHT. These results also suggested that THT, which was used for the formation of N-HC tyramines, had broader acyl donor specificity than did SHT, which was used for the synthesis of N-HC phenethylamines.
It is not clear whether the different rates of N-HC phenethylamines and N-HC tyramines formation observed can be attributed to the first step or the second step. It is, however, clear that Os4CL used p-coumaric acid or caffeic acid more efficiently than it used ferulic acid . In the formation of N-HC tyramine, N-feruloyl tyramine was synthesized more effectively than N-(p-coumaroyl) tyramine or N-caffeoyl tyramine. These observations suggest that the second reaction catalyzed by THT determines the rate of product formation.
We synthesized N-(p-coumaroyl) phenethylamines and N-(p-coumaroyl) tyramines using three different approaches. Firstly, HC and either phenethylamine or tyramine were used as substrates. Two genes, 4CL and either SHT for N-(p-coumaroyl) phenethylamine biosynthesis or THT for N-(p-coumaroyl) tyramine biosynthesis, were transformed into E. coli and various HC-amines were successfully synthesized. Secondly, HC and either phenylalanine or tyrosine were used. For this, an additional gene, either PDC or TDC was introduced to convert phenylalanine or tyrosine into phenethylamine or tyramine, respectively. Thirdly, we synthesized N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine from amino acids (phenylalanine and tyrosine, respectively) by introducing TAL into E. coli. In these approaches, the yield of the major product was much higher than that of byproduct(s). It is likely that it is related to the specificity of the enzymes employed in this study. During screening of HCTs using biotransformation, we observed that both SHT and THT used both phenethylamine and tyramine (Fig. 2), although SHT showed a preference for phenethylamine and THT showed a preference for tyramine. Previous in vitro kinetic data have also shown that SHT has a higher preference for phenethylamine than for tyramine . This is one reason that only a small amount of byproduct is observed. Moreover, the amino acid decarboxylases (PDC and TDC) used in this study showed high substrate specificities. PDC from Pseudomonas putida showed higher substrate specificity for phenylalanine than for tyrosine  and TDC from P. somniferum showed activity for tyrosine but not for phenylalanine . Furthermore, the TAL we used was previously found to show higher substrate specificity for tyrosine  than for phenylalanine. Therefore, byproducts were rarely synthesized. Taken together, using specific enzymes for each reaction step contributed to the synthesis of a specific product with only the formation of a small amount of byproduct(s).
HC-amine conjugates such as N-HC tyramines and N-HC phenethylamines have been considered as potential starting materials for development of antiviral and anticancer drus. We synthesized N-HC tyramines and N-HC phenethylamines using E. coli harboring 4CL and either THT or SHT. Approximately 101.9 mg/L N-(p-coumaroyl) phenethylamine and 495.4 mg/L N-(p-coumaroyl) tyramine were synthesized from p-coumaric acid using E. coli harboring an additional gene either PDC or TDC. Finally, we synthesized 152.5 mg/L N-(p-coumaroyl) phenethylamine and 94.7 mg/L N-(p-coumaroyl) tyramine from glucose by engineering the shikimate pathway of E. coli and reconstructing the pathway toward synthesis of N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine. Productivity was further improved by optimizing the cell concentration and incubation temperature.
Constructs and E. coli strains
Os4CL was previously cloned in our lab  and was subcloned into the EcoRI/NotI sites of pCDFDuet1 (pC-Os4CL). HC-CoA: serotonin N-(HC) transferase (CaSHT) from Capsicum annuum (GenBank accession number AF329463.1) for the synthesis of N-(p-coumaroyl) phenethylamine was cloned using reverse transcription-polymerase chain reaction (RT-PCR). cDNA was isolated from the leave of C. annuum. PCR was carried out using primers, 5′-ATGATATCGATGGCTTCTGCTCCTCAACCACCAA-3′ (EcoRV site is underlined.) as a forward primer and 5′-ATCTCGAGCTAACAGCTTCCTGCACCATTTTTCT-3′ (XhoI site is underlined.) as a reverse primer. The resulting PCR product was subcloned into the EcoRV/XhoI sites of pC-Os4CL and named pC-Os4CL-SHT.
PDC from P. putida (GenBank accession number BK006920) was amplified using PCR using genomic DNA as a template using 5′-ATCATATGGTGACCCCCGAACAATTCCGCC-3′ (NdeI site is underlined.) as a forward primer and 5′-AACTCGAGTCAGCCCTTGATCACGTCCTGC-3′ (XhoI site is underlined.) as a reverse primer. The resulting PCR product was subcloned into the NdeI/XhoI sites of pET-Duet1 (Novagene, Madison, WI, USA) and the construct was named pE-PDC. TDC from Papaver somniferum was synthesized after codon optimization for E. coli based on the published sequence (GenBank U08598.1) and subcloned into BamHI/HindIII sites of pCDF. The construct formed was named pC-TDC. Os4CL was amplified with a forward primer containing T7 promoter sequence and HindIII site and a reverse primer containing a NotI site. The resulting PCR product was subcloned into the HindIII/NotI sites of pC-TDC and the construct was named pC-TDC-Os4CL. Tyramine N-HC transferase (THT) from C. annuum (GenBank accession number AY819700.1) was cloned using RT-PCR. The resulting PCR product was subcloned into the NdeI/EcoRV sites of pC-TDC-Os4CL and the construct was named pC-TDC-Os4CL-THT.
pA-SeTAL, pA-aroG-SeTAL-tyrA, and pA-aroGfbr-SeTAL-tyrAfbr were cloned previously .
Synthesis of N-HC phenethylamines and N-HC tyramines
HCAs including p-coumaric acid, m-coumaric acid, o-coumaric acid, caffeic acid, ferulic acid, cinnamic acid, 3-methoxycinnamic acid, 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, 2,4-dimethoxycinnamic acid, sinapic acid, and 3,4,5-trimethoxycinnamic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Preference of HCA for the synthesis of N-HC phenethylamines or N-HC tyramines was tested as follows. E. coli strains HP-1 and (Table 1) for the synthesis of N-HC phenethylamines and N-HC tyramines, respectively were grown at 37 °C for 18 h in LB medium containing 50 μg/mL spectinomycin. The culture was inoculated into a fresh new LB medium containing 50 μg/mL spectinomycin. Cells were grown until OD600 = 0.8, at which point 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added, and continued to grow at 18 °C for a further 24 h. The cells were harvested and resuspended in M9 medium containing 2 % (w/v) glucose, 1 % (w/v) yeast extract, either 500 μM HCA (for the synthesis of N-HC phenethylamines) or 1 mM HCA (for the synthesis of HC-tyramine), 100 μg/ml antibiotic(s), and 1 mM IPTG. Fourteen HC derivatives (p-coumaric acid, m-coumaric acid, o-coumaric acid, caffeic acid, ferulic acid, cinnamic acid, 3-methoxycinnamic acid, 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, 2,4-dimethoxycinnamic acid, sinapic acid, and 3,4,5-trimethoxycinnamic acid) were used, individually. The cultures were incubated at 30 °C for 3 h and then extracted with ethylacetate. After drying the aqueous layer and dissolving it in dimethylsulfoxide (DMSO), the product was analyzed by HPLC. The relative N-HC phenethylamine or N-HC tyramine formation rates were calculated relative to that of the best substrate, which was denoted as 100 %.
The metabolites were analyzed using an UltiMate 3000 HPLC (Thermo Scientific, Waltham, MA, USA) equipped with a photo diode array (PDA) detector and a C18 reversed-phase column (4.60 × 250 mm, 3.5 μm particle size, Varian, Palo Alto, CA, USA). The mobile phase consisted of 0.1 % (v/v) formic acid in water and acetonitrile. The program was as follows: 20 % (v/v) acetonitrile at 0 min, 60 % at 8 min, 90 % at 12 min, 90 % at 15 min, 20 % at 15.1 min, 20 % at 20 min. The flow rate was 1 ml/min and UV absorbance was monitored at 278 and 310 nm.
Mass spectrometry (MS) was carried out as described previously . The structures of N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine were determined using nuclear NMR . The NMR data were as follows; N-(p-coumaroyl) phenethylamine: 1H NMR (400 MHz, DMSO-d 6 ) δ 7.39 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 15.7 Hz, 1H), 7.29 (m, 2H), 7.22 (d, J = 7.97, 2H), 7.20 (dd, J = 16.4, 7.5 Hz, 1H), 6.80 (d, J = 8.6 Hz, 2H), 6.42 (d, J = 15.7 Hz, 1H), 3.41 (m, 2H), 2.78 (t, J = 3.5 Hz, 2H); 13C NMR (100 MHz, DMSO-d 6 ) δ 165.3, 158.8, 139.5, 138.6, 129.2, 128.6, 128.3, 126.0, 125.9, 118.6, 115.7, 40.3, 35.2.
N-(p-Coumaroyl) tyramine: 1H NMR (400 MHz, DMSO-d 6 ) δ 8.00 (s, NH), 7.38 (m, 2H), 7.31 (d, J = 15.7 Hz, 1H), 7.00 (m, 2H), 6.79 (m, 2H), 6.68 (m, 2H), 6.40 (d, J = 15.7 Hz, 1H), 3.32 (dd, J = 7.4, 6.3 Hz, 2H), 2.64 (t, J = 7.4 Hz, 2H); 13C NMR (100 MHz, DMSO-d 6 ) δ 165.3, 158.8, 155.6, 138.5, 129.5, 129.4, 129.1, 125.9, 118.7, 115.7, 115.1, 40.6, 34.4.
N-(p-Coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine were collected and used as standards for the calculation of the production of other N-HC phenethylamines and N-HC tyramines, respectively.
Total synthesis of N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine
Overnight cultures of E. coli strains, HTs or HPs (Table 1) were inoculated into 15 ml of fresh LB medium containing appropriate antibiotics and were cultured to OD600 = 1. Cells were harvested by centrifugation and cell density was adjusted to OD600 = 2 with 10 mL of M9 medium containing 2 % (w/v) glucose, 1 % (w/v) yeast extract, 50 μg/mL antibiotics, and 1 mM IPTG in a 100 mL flask. The cells were grown at 30 °C with shaking at 180 rpm for 24 h. To analyze product formation, cell growth was monitored by determining the absorbance at 600 nm. Culture supernatants were collected, extracted twice with an equal volume of ethyl acetate, and then dried under vacuum. The dried samples were dissolved in DMSO and analyzed using HPLC.
The mean and the standard deviation (SD) were calculated from triplicate experiments. Analysis of variance (ANOVA) was carried out using Tukey’s method, with significance at a P value of 0.01, using Microsoft Excel.
JHA initiated and coordinated the project. GYS, SMY, BGK, and JHA performed experiments and analyzed data. GYS, SMY, and JHA wrote the paper. All authors read and approved the final manuscript.
This work was supported by a grant from the Next-Generation BioGreen 21 Program (PJ00948301), Rural Development Administration, and Priority Research Centers Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2009-0093824). We thank Professor Jonathan Walton, Michigan State University, for editing the manuscript.
The authors declare that they have no competing interests.
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