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Article

Research article

J. Microbiol. Biotechnol. 2019; 29(10): 1636-1643

Published online October 28, 2019 https://doi.org/10.4014/jmb.1907.07040

Copyright © The Korean Society for Microbiology and Biotechnology.

Biosynthesis of Two Hydroxybenzoic Acid-Amine Conjugates in Engineered Escherichia coli

Song-Yi Kim 1, Han Kim 1, Bong-Gyu Kim 2 and Joong-Hoon Ahn 1*

1Department of Integrative Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 05029, Republic of Korea, 2Department of Forest Resources, Gyeongnam National University of Science and Technology, Jinju 52725, Republic of Korea

Correspondence to:Joong-Hoon  Ahn
jhahn@konkuk.ac.kr

Received: July 17, 2019; Accepted: August 24, 2019

Abstract

Two hydroxybenzoyl amines, 4-hydroxybenzoyl tyramine (4-HBT) and N-2-hydroxybenzoyl tryptamine (2-HBT), were synthesized using Escherichia coli. While 4-HBT was reported to demonstrate anti-atherosclerotic activity, 2-HBT showed anticonvulsant and antinociceptive activities. We introduced genes chorismate pyruvate-lyase (ubiC), tyrosine decarboxylase (TyDC), isochorismate synthase (entC), isochorismate pyruvate lyase (pchB), and tryptophan decarboxylase (TDC) for each substrate, 4-hydroxybenzoic acid (4-HBA), tyramine, 2-hydroxybenzoic acid (2-HBA), and tryptamine, respectively, in E. coli. Genes for CoA ligase (hbad) and amide formation (CaSHT and OsHCT) were also introduced to form hydroxybenzoic acid and amine conjugates. In addition, we engineered E. coli to provide increased substrates. These approaches led to the yield of 259.3 mg/l 4-HBT and 227.2 mg/l 2-HBT and could be applied to synthesize diverse bioactive hydroxybenzoyl amine conjugates.

Keywords: Hydroxybenzoic acid, N-2-hydroxybenzoyl tryptamine, 4-hydroxybenzoyl tyramine, metabolic engineering

Introduction

Hydroxybenzoic acids are common chemicals found in plants. 2-HBA (2-hydroxybenzoic acid) serves as a signal molecule in plants [1] and 4-HBA (4-hydroxybenzoic acid) is a precursor for the synthesis of ubiquinol-6 in eukaryotes [2, 3]. These two compounds form amines with either tyramine or tryptamine; 4-Hydroxybenzoyl tyramine (4-HBT), found in Houttuynia cordata [4], has been reported to show anti-atherosclerotic activity [4]. N-2-hydroxybenzoyl tryptamine (2-HBT) has been studied as a benzoyl tryptamine analogue owing to its anticonvulsant [5, 6] and antinociceptive activities [7]. 2-HBT has been shown to relieve acute and chronic pain and inflammation [8].

Recently, biological synthesis of various chemicals, using Escherichia coli, has become prevalent. The biological pathway for the synthesis of target compounds is newly introduced into E. coli and engineered to increase the supply of substrates in E. coli. Precursors of 4-HBT and 2-HBT are available in E. coli; 4-HBA and 2-HBA are synthesized from chorismate [9, 10]. Tyramine and tryptamine are from tyrosine and tryptophan, respectively [11, 12]. Various BAHD (benzyl alcohol O-acetyltransferase (BEAT), anthocyanin O-hydroxycinnamoyl transferase (AHCT), anthranilate N-hydroxycinnamoyl/benzoyl trans-ferase (HCBT), and deacetyl vindoline 4-O-acetyltransferase (DAT)) acyltransferase families are known for amide formation between hydroxybenzoic acid and tyramine or tryptamine [13]. By expressing these genes in E. coli, amide formation between phenolic acid and various amines including tyramine, dopamine, tryptamine, and serotonin was conducted [14-16]. Combination of amine synthesis and hydroxybenzoic acid pathways in E. coli is considered to enable the synthesis of a new amide. Here, we report the synthesis of two new amides in E. coli, namely 4-HBT and 2-HBT. The route of synthesis for two hydroxybenzoic acids (4-HBA and 2-HBA) was introduced into E. coli. UbiC gene was overexpressed to provide a substrate for 4-HBT, and two genes, entC and pchB, were overexpressed to synthesize 2-HBA (Fig. 1). In addition, we engineered the shikimate pathway of E. coli to provide more substrates for the synthesis of 4-HBT and 2-HBT.

Figure 1. Schematic pathway for the synthesis of 4-hydroxybenzoyl tyramine and N-2-hydroxybenzoyl tryptamine in E. coli. PpsA, phosphoenolpyruvate synthase; TktA, transketolase; AroG, phospho-2-dehydro-3-deoxyheptonate aldolase; AroL, shikimate kinase, EntC, isochorismate synthase; PchB, isochorismate pyruvate-lyase; Tdc, tryptophan decarboxylase; Hbad, hydroxybenzoate coenzyme A ligase; OsHCT, hydroxycinnamoyl transferase from O. sativa; TyDC, tyrosine decarboxylase; TyrA, prephenate dehydrogenase; TyrB, aromatic amino acid aminotransferase; UbiC, chorismate pyruvate lyase; CaSHT, serotonin N-hydroxycinnamoyl transferase from Capiscum annuum.

Materials and Methods

Constructs

PsTyDC and CaSHT were cloned previously [15]. PsTyDC was subcloned into BamHI/HindIII site of pETDuet-1 vector. The ubiC gene (CAA47181), encoding chorismate pyruvate-lyase from E. coli, was cloned by polymerase chain reaction (PCR) using primers 5’-aacatATGTCACACCCCGCGT-3’ and 5’-aaggtaccTTAGTAC AACGGTGACGCC-3’ (NdeI and KpnI sites are underlined), and then subcloned into NdeI/KpnI site of the pETDuet-1 containing PsTyDC. hbad gene (hydroxybenzoic acid-CoA ligase; U02033) was amplified with genomic DNA of Rhodopseudomonas palustris with primers 5’-aaggatccaGGATCCAATGCCGCTACGCGACTACA-3’ and 5’-aagcggccgcTCATCGTCCGTTGCCGG-3’ (BamHI and NotI sites are underlined) and then subcloned into BamHI/NotI site of pCDFDuet-1. CaSHT was subcloned into the BglII/KpnI site of pCDFDuet-1 harboring hbad (pC-hbad-CaSHT). BamHI site of tyrAf was deleted without any amino acid change and the resulting gene was subcloned into NdeI/KpnI of pACYCDuet-1 (pA-tyrAf). aroGf-ppsA-tktA and aroL-aroGf-ppsA-tktA, each containing a ribosome binding site (RBS), were subcloned into BamHI/NotI site of pA-tyrAf.

OsHCT (Os11g42370) was cloned using RT-PCR with primers, 5’-aagaattcaATGGAGATCACGAGCAGCG-3’ and 5’-aagcggccgcTTAAAGATGAGAGGGGATAGCATG-3’ and then subcloned into EcoRI/NotI site of pETDuet-1. BaTDC was subcloned into EcoRV/XhoI site of pETDuet-1 carrying OsHCT. Two genes, entC from E. coli and pchB from Pseudomonas fluorescens, were cloned into NdeI/KpnI site of pCDFDuet-1 carrying hbad.

The promoter of trp operon in E. coli was replaced with T7 to create the strain BT7P (Table 1). This was done by a two-step PCR: the first PCR was performed with FRT-PGK-gb2-neo-FRT DNA as a template, using 5’-tggtatatCTCCTTattaaagttaaacaaaattaCTA TAGTGAGTCGTATTAtaatacgactcactatagggctc-3’ (underlined capital letters show ribosome binding site (RBS), capital letters represent T7 promoter sequence, and underlined sequences are from FRT-pgk-gb2-neo-FRT cassette) as the forward primer and 5’-TGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGattaaccctcactaaagggcg-3’ (underlined capital letters are from trp operon and lower case letters are from FRT-pgk-gb2-neo-FRT cassette) as the reverse primer. The second PCR was performed with the template generated from the first PCR, using 5’-TAAGCGCCTTCGCAGGTTAGCTGTTCGAGAGTCGGTTTTTG TGTTTGCATtggtatatctccttattaaa-3’ (capital letters are from trp operon promoter region and underlined sequences are part of the first PCR product) as the forward primer and reverse primer from the 1st PCR. The second PCR product was used to replace the native trp promoter of E. coli with a T7 promoter using the Quick and Easy Conditional Knockout Kit (Gene Bridges, Germany). LB medium containing 50 μg/ml kanamycin was used to select the positive colonies. Positive clones were verified by colony PCR with two primers, 5’-CTGGCGTCAGGCAGCCATCG-3’ and 5’-CCAGCAGGGCTTCGCCGTTG-3’.

Table 1 . Plasmids and strains used in the present study..

Plasmids or E. coli strainsRelevant properties or genetic markerSource or reference
Plasmids
pACYCDuet-1P15A ori, CmrNovagen
pCDFDuet-1CloDE13 ori, StrrNovagen
pETDuet-1f1 ori, AmprNovagen
pC-hbad-CaSHTpCDFDuet-1 + hbad from Rhodopseudomonas palustris and CaSHT from Capiscum annuumThis study
pE-PsTyDC-ubiCpETDuet-1 + PsTyDC from Papaver somniferum and ubiC from Escherichia coliThis study
pA-aroGf-tyrAfpACYCDuet-1 + aroGf and tyrAf from E. coliThis study
pA-aroGf-ppsA-tktA-tyrAfpACYCDuet-1 + aroGf, ppsA, tktA, and tyrAf from E. coliThis study
pA-aroL-aroGf-ppsA-tktA-tyrAfpACYCDuet-1 + aroL, aroGf, ppsA, tktA, and tyrAf from E. coliThis study
pC-hbadpCDFDuet-1 + hbad from R. palustrisThis study
pC-hbad-entC-pchBpCDFDuet-1 + hbad from R. palustris and entC from E. coli and pchB from Pseudomonas fluorescensThis study
pE-OsHCTpETDuet-1 + HCT from Oryza sativaThis study
pE-OsHCT-BaTDCpETDuet-1 + HCT from Oryza sativa and TDC from Bacillus atrophaeusThis study
pA-aroL-aroGf-ppsA-tktApACYCDuet-1 + aroL, aroGf, ppsA, and tktA from E. coliThis study
Strains
BL21 (DE3)F- ompT hsdSB(rB- mB-) gal dcm lon (DE3)Novagen
B-tyrRBL21(DE3) ΔtyrRKim et al (2013)
B-tyrR/pheABL21(DE3) ΔtyrR/ΔpheAKim et al (2013)
B-trpEG/pheABL21(DE3) ΔtrpEG/ΔpheAThis study
BT7PBL21(DE3) trp promoter::T7 promoterThis study
B-4HBT-1BL21 (DE3) harboring pC-hbad-CaSHTThis study
B-4HBT-2BL21 (DE3) harboring pC-hbad-CaSHT and pE-PsTyDC-ubiCThis study
B-4HBT-3BL21 (DE3) harboring pACYCDuet-1, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-4HBT-4BL21 (DE3) harboring pA- aroGf-tyrAf, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-4HBT-5BL21 (DE3) harboring pA- aroGf-ppsA-tktA-tyrAf, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-4HBT-6BL21 (DE3) harboring pA-aroL-aroGf-ppsA-tktA-tyrAf, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-4HBT-7B-tyrR/pheA harboring pA-aroL-aroGf-ppsA-tktA-tyrAf, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-4HBT-8B-trpEG/pheA harboring pA-aroL-aroGf-ppsA-tktA-tyrAf, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-2-HBT-1BL21 (DE3) harboring pC-hbad and pE-OsHCTThis study
B-2-HBT-2BL21 (DE3) harboring pC-hbad and pE-OsHCT-BaTDCThis study
B-2-HBT-3BL21 (DE3) harboring pACYCDuet-1, pC-hbad-entC-pchB, and pE-OsHCT-BaTDCThis study
B-2-HBT-4B-tryR harboring pACYCDuet-1, pC-hbad-entC-pchB, and pE-OsHCT-BaTDCThis study
B-2-HBT-5BT7P harboring pACYCDuet-1, pC-hbad-entC-pchB, and pE-OsHCT-BaTDCThis study
B-2-HBT-6BT7P harboring pA-aroL-aroGf-ppsA-tktA, pC-hbad-entC-pchB, and pE-OsHCT-BaTDCThis study


Synthesis and Analysis of Reaction Products

To synthesize 4-HBT from 4-hydroxybenzoic acid and tyramine or 2-HBT from 2-hydroxybenzoic acid and tryptamine, E. coli strain B-4HBT-1 or B-2-HBT-1 was prepared [15] and 100 μM substrates were used. For the synthesis of 4-HBT and 2-HBT from glucose, E. coli strains were prepared as described before [17]. Briefly, transformants were grown in LB medium containing suitable antibiotics at 37°C for 18 h. The culture was inoculated into fresh medium containing suitable antibiotics at 37°C for 4 h. To synthesize 4-HBT from glucose, the cells were grown until OD600 = 1 and were harvested and resuspended in M9 containing 1% yeast extract, 2% glucose, 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and 50 μg/ml antibiotics. The cells were grown at 30°C with shaking for 24 h and extracted from ethyl acetate. To synthesize 2-HBT from glucose, the cells were grown until OD600 = 0.8 and IPTG was added to a final concentration of 1 mM and the culture was incubated at 18°C for 16 h. The cells were grown until OD600 = 3 and were harvested and resuspended in the same medium for the synthesis of 4-HBT. The cells were grown at 30°C with shaking for 24 h and extracted from ethyl acetate.

The reaction products were analyzed using high-performance liquid chromatography (HPLC; [15]) and nuclear magnetic resonance spectroscopy (NMR) [18]. These reaction products were purified from the culture filtrate with ethyl acetate followed by evaporation of the organic layer. The resulting sample was further purified using HPLC and the structure of purified compound was determined using NMR. The proton NMR data of the synthesized compounds were as follows; 4-Hydroxybenzoyl tyramine; 1H NMR (methanol-d4, 400MHz): δ 2.78 (2H, t, J = 7.5 Hz, H-α), 3.49 (2H, d, J = 7.5 Hz, H-β), 6.70 (4H, m, H-3/5, H-3'/5'), 7.05 (2H, d, J = 8.4 Hz, H-2/6), and 7.59 (2H, d, J = 8.7 Hz, H-2'/6'). N-2-Hydroxybenzoyl tryptamine; 1H NMR (acetone-d6, 400MHz): δ 3.07 (2H, t, J = 7.5 Hz, H-α), 3.72 (2H, t, J = 7.5 Hz, H-β), 6.68 (1H, dd, J = 7.9, 7.6 Hz, H-5'), 6.87 (1H, d, J = 8.4 Hz, H-3'), 7.01 (1H, dd, J = 7.9, 7.6 Hz, H-5), 7.09 (1H, dd, J = 8.2, 7.6 Hz, H-6), 7.21 (1H, s, H-2), 7.26 (1H, dd, J = 8.4, 7.6 Hz, H-4'), 7.37 (1H, d, J = 8.2 Hz, H-7), 7.65 (1H, d, J = 7.9 Hz, H-4), and 7.83 (1H, d, J = 7.9 Hz, H-6').

Results and Discussion

Synthesis of 4-Hydroxybenzoyl Tyramine

4-HBT is a conjugate of 4-HBA and tyramine. In order to form an amide bond, activation of 4-HBA by the attachment of CoA is a prerequisite. Conjugation of 4-hydroxybenzoyl-CoA and tyramine is further mediated by another enzyme. Hbad from R. palustris is known to attach CoA to 4-hydroxybenozic acid [19] and amide formation between tyramine and 4-hydroxybenzoyl-CoA was tested using benzyl alcohol O-acetyltransferase (BEAT). We tested the acetyltransferases from C. annuum (CaSHT). Two genes, hbad and CaSHT (pC-hbad-CaSHT), were transformed in E. coli. The resulting transformant (B-4HBT-1) was used to examine the synthesis of 4-hydroxybenzoyl tyramine by feeding 4-HBA and tyramine. HPLC analysis of the culture filtrate revealed a new peak, which had a different retention time from that of 4-HBA. Molecular mass of this compound was 257.11 Da, which is the predicted molecular mass of 4-HBT. Furthermore, the structure of this compound was confirmed using proton NMR (Materials and Methods).

We also tested other hydroxybenzoic acids as substrates (benzoic acid, 2-HBA, 3-hydroxybenzoic acid [3-HBA], 4-HBA, and 3,4-dihydroxybenzoic acid [3,4-DHBA]), with either tryptamine or tyramine. Among 10 combinations, five benzoic acid derivatives with two amines (tryptamine and tyramine), and five conjugates (benzoyl tyramine, 4-hydroxybenozoyl tyramine, benzoyl tryptamine, 4-hydroxybenzoyl tryptamine, and 2-hydroxybenzoyl tryptamine) were synthesized. Since tyramine is an acyl group acceptor, 4-HBA was a better substrate than benzoic acid. On the other hand, with tryptamine as an acceptor, benzoic acid was the best donor followed by 4-HBA, 2-HBA, and 3-HBA.

We designed a pathway to synthesize 4-hydroxybenzoyl tyramine without feeding 4-HBA and tyramine. Tyramine was synthesized by decarboxylation of tyrosine using tyrosine decarboxylase from P. somniferum (PsTyDC). 4-HBA is synthesized by chorismate pyruvate-lyase (ubiC) from chorismate. Since E. coli contains ubiC gene, it can synthesize 4-HBA. However, in order to enhance the synthesis of 4-HBA, ubiC was overexpressed. Both PsTyDC and ubiC were transformed into B-4HBT-1 and the resulting transformant (B-4HBT-2) was tested for the synthesis of 4-hydroxybenzoyl tyramine. As shown in Fig. 2B, synthesis of 4-hydroxybenzoyl tyramine was indeed observed. We also observed unreacted 4-hyroxybenzoic acid, which indicated that the amount of tyramine was less than that of 4-HBA in E. coli. Synthesis of chorismate, the substrate of 4-HBA, did not undergo feedback inhibition, whereas synthesis of tyrosine, the substrate of tyramine, did [20]. In an attempt to synthesize more endogenous substrate, tyrosine, two genes, feedback-free versions of wild type, aroGf and tyrAf, were overexpressed. For chorismate synthesis, genes in the shikimate pathway were also overexpressed. Four constructs, having various combinations of genes of the shikimate pathway of E. coli, were prepared and tested for the production of 4-HBT. Overexpression of aroGf and tyrAf (strain B-4HBT-4) enhanced the yield of

Figure 2. Synthesis of 4-hydroxybenzoyl tyramine in E. coli strain B-4HBT-2. A, standard 4-hydroxybenzoic acid (S); B, the reaction products from the strain B-4HBT-2. S is likely to be 4-hydroxybenzic acid and P is 4- hydrobnezoyl tyramine.

4-HBT from 58.5 mg/l to 98.3 mg/l (Fig. 3A). Overexpression of two additional genes, tktA and ppsA (B-4HBT-5), both of which provide erythrose 4-phosphate and phosphoenolpy-ruvate, increased the synthesis of 4-HBT (113.2 mg/l) by probably providing more substrates for AroG. Furthermore, overexpression of aroL along with aroGf, tyrAf, tktA, and ppsA (strain B-4HBT-6) enhanced the synthesis of 4-hydroxybenzoyl tyramine (126.8 mg/l), which was a more than 2-fold increase compared to that in strain B-4HBT-3. Supplementation of chorismate by overexpressing shikimate pathway genes resulted in the increased synthesis of 4-HBT. This indicated that the downstream enzymes in this pathway (UbiC (chorismate pyruvate-lyase), PsTyDC (tyrosine decarboxylase), Hbad (hydroxybenzoate coenzyme A ligase), and CaSHT (Serotonin N-(hydroxycinnamoyl) transferase)) were not the bottleneck of the pathway.

Figure 3. Synthesis of 4-hydroxybenzoyl tyramine in E. coli expressing different shikimate pathway genes (A) and in E. coli mutants (B).

Next, we used E. coli mutant strains to supply more substrates, tyrosine and chorismate, thereby enhancing the synthesis of 4-hydroxybenzoyl tyramine. Two mutants were used for the purpose; TyrR/PheA (PheA competes with TyrA for prephenate to synthesize tyrosine) double mutant is already known to synthesize more tyrosine [20]. Since TrpEG converts chorismate into anthranilate for the synthesis of tryptophan, and PheA converts prephenate into phenylpyruvate for the synthesis of phenylalanine [21, 22], TrpEG/PheA double mutant would supply more substrates for 4-HBA and tyrosine. As expected, the synthesis of 4-hydroxybenzoyl tyramine was dramatically enhanced in these mutants (Fig. 3B); strain B-4HBT-6, -7, and -8 synthesized 124.0, 176.9, and 214.9 mg/l of 4-HBT, respectively. The TrpEG/PheA mutant (strain B-4HBT-8) produced approximately 70% more 4-HBT than the wild-type strain (strain B-4HBT-6). TrpEG/PheA seemed to have provided more of both substrates than the wild type. Using strain B-4HBT-8, we could synthesize approximately 259.3 mg/l of 4-hydroxybenzoyl tyramine after a 24 h reaction. 4-HBA was also monitored and its accumulation was observed when the synthesis of 4-HBT reached almost the maximum (Fig. 4).

Figure 4. Synthesis of 4-hydroxybenzoyl tyramine using E. coli strain B-4HBT-8.

Synthesis of N-2-Hydroxybenzoyl Tryptamine

2-HBT is a conjugate of 2-HBA and tryptamine. Two enzymatic reactions were necessary for its synthesis; attachment of CoA to 2-HBA and amide formation between 2-hydroxybenzoyl-CoA and tryptamine. Hydroxybenzoic acid-CoA ligase (hbad) is known to catalyze the first reaction [19]. To find a gene encoding amide formation, we used BAHD N-acyltransferases from rice [23]. One gene (OsHCT), along with hbad, was transformed into E. coli. 2-HBA and tryptamine were added to the culture medium of E. coli carrying OsHCT and hbad (E. coli B-2-HBT-1 in Table 1). An HPLC diagram showed that the E. coli B-2-HBT-1 synthesized a new product, which had the same molecular mass as 2-HBT. Proton NMR confirmed the synthesized compound as 2-HBT.

As an acyl group donor, benzoic acid, 2-HBA, 3-HBA, 4-HBA, and 3,4-DHBA were used. OsHCT was found to use four of the benzoic acids, except 3,4-DHBA, to synthesize the corresponding tryptamines. OsHCT could use tyramine as an acyl group acceptor with 3- HBA and benzoic acid as donors. However, it could not use 4-HBA as a donor (data not shown).

Next, we attempted to synthesize 2-HBT by feeding only 2-HBA. Since E. coli has endogenous tryptophan, we employed the tryptophan decarboxylase from Bacillus atrophaeus to synthesize tryptamine from tryptophan [16]. The resulting transformant (B-2-HBT-2) also synthesized 2-HBT when 2-HBA was supplied (data not shown).

Finally, to synthesize 2-HBT from glucose, the 2-HBA synthesis pathway was introduced into E. coli. Two genes, entC from E. coli and pchB from Pseudomonas fluorescens, converted chorismate to 2-HBA. E. coli strain B-2-HBT-2 was transformed with these two genes and the resulting transformant (E. coli B-2-HBT-3) was tested for the synthesis of 2-HBT. As expected, 2-HBT was indeed synthesized by E. coli B-2-HBT-3 (Fig. 5). 2-HBA was also observed, indicating the necessity of supply of tryptophan for synthesizing more 2-HBT. We tested two mutant strains; the first mutant had a deletion in the negative transcription regulator encoded by the tyrR gene, which was activated by tryptophan [24, 25]. The trp promoter of trp operon in the second mutant (E. coli strain BT7P in Table 1) was replaced by a stronger promoter T7. We prepared two strains (E. coli B-2-HBT-4 and E. coli B-2-HBT-5) and examined the synthesis of 2-HBT. E. coli B-2-HBT-5 synthesized the highest amount of 2-HBT (192.4 mg/l), followed by E. coli B-2-HBT-4 (183.9 mg/l) and E. coli B-2-HBT-3 (157.1 mg/l) (Fig. 6). The 2-HBA of these three strains was 55.2, 78.6, and 36.8 mg/l, respectively. E. coli BT7P supplied more tryptophan for the synthesis of 2-HBT. TyrR is a regulator of shikimate pathway and therefore, its deletion led to enhanced synthesis of chorismate, which could convert into both 2-HBA and tryptophan. Therefore, E. coli B-tyrR strain increased the synthesis of not only 2-HBT but also 2-HBA. We also introduced the construct pA-aroL-aroGf-ppsA-tktA, which was effective for the production of 4-HBT, into strain B-2-HBT-5. The resulting strain (B-2-HBT-6) produced a little more 2-HBT (227.8 mg/l) and even more 2-HBA (138.0 mg/l) than strain B-2-HBT-5. This result showed that a balanced supply of both substrates is critical for the final yield; by overexpressing four genes of the shikimate pathway, sufficient amounts of 2-HBA were provided. But, the final yield of 2-HBT was not increased due to the tryptophan. Therefore, the bottleneck of 2-HBT synthesis was the synthesis of tryptophan.

Figure 5. Synthesis of N-2-hydroxybenzoyl tryptamine from glucose using E. coli strain B-2-HBT-3. A, standard 2-hydroxybenzoic acid (S); B, reaction products from B-2-HBT-3. S is likely to be 2-hydroxybenzoic acid and P is N-2-hydroxybenzoyl tryptamine.
Figure 6. Synthesis of N-2-hydroxybenzoyl tyramine in different E. coli strains.

Using B-2-HBT-6, the synthesis of 2-HBT was monitored for 24 h (Fig. 7). 2-HBT yield continued to increase until 15 h (227.2 mg/l) and then remained almost unchanged. However, 2-HBA started to accumulate rapidly from 15 h, indicating that tryptophan was the limiting factor.

Figure 7. Synthesis of N-2-hydroxybenzoyl tryptamine using E. coli strain B-2-HBT-6.

We synthesized two hydroxybenzoic acid-amine conjugates. To maximize the synthesis of both products, we overexpressed four genes (aroGf, tktA, ppsA, and aroL) to ensure the supply of increased substrates. In addition, mutant strains were used during the synthesis of 4-HBT to increase two substrates, chorismate and tyrosine, more effectively. More tryptophan was supplied by replacing the trp operon promoter. Fine tuning of substrates is, therefore, a critical issue in increasing the final yield.

Two HCTs, CaSHT, and OsHCT, used diverse substrates. CaSHT used diverse hydroxycinnamic acid derivatives (o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, etc.) and various amines including tryptamine, serotonin, and tyramine [15, 16]. It was shown that CaSHT expanded its substrates to the benzoic acid derivatives. OsHCT not only used tryptamine but also tyramine. Promiscuity of CaSHT and OsHCT was useful in synthesizing a variety of diverse amine conjugates, thereby enabling novel biological activities of the synthesized compounds to be explored.

Acknowledgments

This work was funded by a grant from the Next-Generation BioGreen 21 Program (PJ01326001), Rural Development Administration, Republic of Korea, and a grant from Konkuk University’s research support program for its faculty on sabbatical leave in 2018.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Schematic pathway for the synthesis of 4-hydroxybenzoyl tyramine and N-2-hydroxybenzoyl tryptamine in E. coli. PpsA, phosphoenolpyruvate synthase; TktA, transketolase; AroG, phospho-2-dehydro-3-deoxyheptonate aldolase; AroL, shikimate kinase, EntC, isochorismate synthase; PchB, isochorismate pyruvate-lyase; Tdc, tryptophan decarboxylase; Hbad, hydroxybenzoate coenzyme A ligase; OsHCT, hydroxycinnamoyl transferase from O. sativa; TyDC, tyrosine decarboxylase; TyrA, prephenate dehydrogenase; TyrB, aromatic amino acid aminotransferase; UbiC, chorismate pyruvate lyase; CaSHT, serotonin N-hydroxycinnamoyl transferase from Capiscum annuum.
Journal of Microbiology and Biotechnology 2019; 29: 1636-1643https://doi.org/10.4014/jmb.1907.07040

Fig 2.

Figure 2.Synthesis of 4-hydroxybenzoyl tyramine in E. coli strain B-4HBT-2. A, standard 4-hydroxybenzoic acid (S); B, the reaction products from the strain B-4HBT-2. S is likely to be 4-hydroxybenzic acid and P is 4- hydrobnezoyl tyramine.
Journal of Microbiology and Biotechnology 2019; 29: 1636-1643https://doi.org/10.4014/jmb.1907.07040

Fig 3.

Figure 3.Synthesis of 4-hydroxybenzoyl tyramine in E. coli expressing different shikimate pathway genes (A) and in E. coli mutants (B).
Journal of Microbiology and Biotechnology 2019; 29: 1636-1643https://doi.org/10.4014/jmb.1907.07040

Fig 4.

Figure 4.Synthesis of 4-hydroxybenzoyl tyramine using E. coli strain B-4HBT-8.
Journal of Microbiology and Biotechnology 2019; 29: 1636-1643https://doi.org/10.4014/jmb.1907.07040

Fig 5.

Figure 5.Synthesis of N-2-hydroxybenzoyl tryptamine from glucose using E. coli strain B-2-HBT-3. A, standard 2-hydroxybenzoic acid (S); B, reaction products from B-2-HBT-3. S is likely to be 2-hydroxybenzoic acid and P is N-2-hydroxybenzoyl tryptamine.
Journal of Microbiology and Biotechnology 2019; 29: 1636-1643https://doi.org/10.4014/jmb.1907.07040

Fig 6.

Figure 6.Synthesis of N-2-hydroxybenzoyl tyramine in different E. coli strains.
Journal of Microbiology and Biotechnology 2019; 29: 1636-1643https://doi.org/10.4014/jmb.1907.07040

Fig 7.

Figure 7.Synthesis of N-2-hydroxybenzoyl tryptamine using E. coli strain B-2-HBT-6.
Journal of Microbiology and Biotechnology 2019; 29: 1636-1643https://doi.org/10.4014/jmb.1907.07040

Table 1 . Plasmids and strains used in the present study..

Plasmids or E. coli strainsRelevant properties or genetic markerSource or reference
Plasmids
pACYCDuet-1P15A ori, CmrNovagen
pCDFDuet-1CloDE13 ori, StrrNovagen
pETDuet-1f1 ori, AmprNovagen
pC-hbad-CaSHTpCDFDuet-1 + hbad from Rhodopseudomonas palustris and CaSHT from Capiscum annuumThis study
pE-PsTyDC-ubiCpETDuet-1 + PsTyDC from Papaver somniferum and ubiC from Escherichia coliThis study
pA-aroGf-tyrAfpACYCDuet-1 + aroGf and tyrAf from E. coliThis study
pA-aroGf-ppsA-tktA-tyrAfpACYCDuet-1 + aroGf, ppsA, tktA, and tyrAf from E. coliThis study
pA-aroL-aroGf-ppsA-tktA-tyrAfpACYCDuet-1 + aroL, aroGf, ppsA, tktA, and tyrAf from E. coliThis study
pC-hbadpCDFDuet-1 + hbad from R. palustrisThis study
pC-hbad-entC-pchBpCDFDuet-1 + hbad from R. palustris and entC from E. coli and pchB from Pseudomonas fluorescensThis study
pE-OsHCTpETDuet-1 + HCT from Oryza sativaThis study
pE-OsHCT-BaTDCpETDuet-1 + HCT from Oryza sativa and TDC from Bacillus atrophaeusThis study
pA-aroL-aroGf-ppsA-tktApACYCDuet-1 + aroL, aroGf, ppsA, and tktA from E. coliThis study
Strains
BL21 (DE3)F- ompT hsdSB(rB- mB-) gal dcm lon (DE3)Novagen
B-tyrRBL21(DE3) ΔtyrRKim et al (2013)
B-tyrR/pheABL21(DE3) ΔtyrR/ΔpheAKim et al (2013)
B-trpEG/pheABL21(DE3) ΔtrpEG/ΔpheAThis study
BT7PBL21(DE3) trp promoter::T7 promoterThis study
B-4HBT-1BL21 (DE3) harboring pC-hbad-CaSHTThis study
B-4HBT-2BL21 (DE3) harboring pC-hbad-CaSHT and pE-PsTyDC-ubiCThis study
B-4HBT-3BL21 (DE3) harboring pACYCDuet-1, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-4HBT-4BL21 (DE3) harboring pA- aroGf-tyrAf, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-4HBT-5BL21 (DE3) harboring pA- aroGf-ppsA-tktA-tyrAf, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-4HBT-6BL21 (DE3) harboring pA-aroL-aroGf-ppsA-tktA-tyrAf, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-4HBT-7B-tyrR/pheA harboring pA-aroL-aroGf-ppsA-tktA-tyrAf, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-4HBT-8B-trpEG/pheA harboring pA-aroL-aroGf-ppsA-tktA-tyrAf, pC-hbad-CaSHT, and pE-PsTyDC-ubiCThis study
B-2-HBT-1BL21 (DE3) harboring pC-hbad and pE-OsHCTThis study
B-2-HBT-2BL21 (DE3) harboring pC-hbad and pE-OsHCT-BaTDCThis study
B-2-HBT-3BL21 (DE3) harboring pACYCDuet-1, pC-hbad-entC-pchB, and pE-OsHCT-BaTDCThis study
B-2-HBT-4B-tryR harboring pACYCDuet-1, pC-hbad-entC-pchB, and pE-OsHCT-BaTDCThis study
B-2-HBT-5BT7P harboring pACYCDuet-1, pC-hbad-entC-pchB, and pE-OsHCT-BaTDCThis study
B-2-HBT-6BT7P harboring pA-aroL-aroGf-ppsA-tktA, pC-hbad-entC-pchB, and pE-OsHCT-BaTDCThis study

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