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Biosynthesis of ethyl caffeate via caffeoyl-CoA acyltransferase expression in Escherichia coli

Biosynthesis of ethyl caffeate via caffeoyl-CoA acyltransferase expression in Escherichia coli Hydroxycinnamic acids (HCs) are natural compounds that form conjugates with diverse compounds in nature. Ethyl caffeate (EC) is a conjugate of caffeic acid (an HC) and ethanol. It has been found in several plants, including Prunus yedoensis, Polygonum amplexicaule, and Ligularia fischeri. Although it exhibits anticancer, anti‑inflammatory, and antifibrotic activities, its biosynthetic pathway in plants still remains unknown. This study aimed to design an EC syn‑ thesis pathway and clone genes relevant to the same. Genes involved in the caffeic acid synthesis pathway (tyrosine ammonia‑lyase ( TAL) and p‑ coumaric acid hydroxylase (HpaBC)) were introduced into Escherichia coli along with 4‑ coumaroyl CoA ligase (4CL) and acyltransferases (AtCAT ) cloned from Arabidopsis thaliana. In presence of ethanol, E. coli harboring the above genes successfully synthesized EC. Providing more tyrosine through the overexpression of shikimate‑pathway gene ‑module construct and using E. coli mutant enhanced EC yield; approximately 116.7 mg/L EC could be synthesized in the process. Synthesis of four more alkyl caffeates was confirmed in this study; these might potentially possess novel biological properties, which would require further investigation. Keywords: Acyltransferase, Alkyl caffeate, Hydroxycinnamic acid conjugate, Metabolic engineering Introduction yedoensis [6], Polygonum amplexicaule [7], and Ligularia Hydroxycinnamic acids (HCs) are abundant in nature. fischeri [8]. In particular, Ligularia fischeri is grown in They are synthesized via phenylpropanoid pathway in eastern Asia and is used in herbal medicine. The biologi - plants and serve as the building blocks for other phe- cal effects of L. fischeri are derived from EC. EC is known nolic compounds, such as flavonoids, coumarin, lignin, to exhibit anticancer [8], anti-inflammatory [9], and anti - proanthocyanidins, cutin, and suberin [1]. HCs form fibrotic activities [10]. In addition, it has the potential to conjugates with other molecules as well. The conjugate of regulate blood pressure by inhibiting aldosterone syn- caffeic acid (an HC) and quinic acid is called chlorogenic thase [11]. acid [2]. Avenanthramides are amides formed from HCs The synthetic pathway for EC in plants has not been and anthranilate derivatives [3]. Besides these, conju- fully elucidated yet. Caffeic acid is synthesized from gates of HCs with other compounds (spermine, puterine, tyrosine by deamination and hydroxylation, and needs tyramine, dopamine, tryptamine, and glycine) have also to be activated by the attachment of coenzyme A (CoA). been reported [4, 5]. Although high-molecular-weight alcohols, such as C18, Ethyl caffeate (EC) is a conjugate of caffeic acid and C20, and C22 alkan-1-ols, have been reported to be syn- ethanol, and is found in some plants, namely Prunus thesized from long-chain fatty acids in plants [12], how the ethyl group is provided during the synthesis of EC still remains a mystery. Ethanol is presumably synthesized *Correspondence: jhahn@konkuk.ac.kr in hypoxic condition [13], which is also suitable for the Department of Integrative Bioscience and Biotechnology, Bio/Molecular synthesis of EC; however, other ethyl group donors may Informatics Center, Konkuk University, Seoul 05029, Republic of Korea © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Lee et al. Appl Biol Chem (2021) 64:71 Page 2 of 6 also be present. The mechanism underlying the conjuga - an engineered E. coli strain, and providing ethanol, we tion of caffeoyl-CoA with ethyl group donor still remains could successfully synthesize EC. unknown. Previous study on the biosynthesis of suberin might provide a clue to the process of generating an EC Materials and methods conjugate. Suberin is an ester of long-chain primary alco- Constructs hols and HCs [14]. The conjugate reaction is mediated Caffeoyl-CoA transferases from A. thaliana (AtCAT1 by aliphatic suberin feruloyl transferase in Arabidopsis [AT5G63560.1] and AtCAT2 [At5g41040]) were cloned thaliana [15]. Therefore, an enzyme of this family might using reverse transcription-polymerase chain reaction catalyze the conjugation reaction to form EC, although (RT-PCR). The primers were as follows: aaacatATG GCC the origin of ethyl group in plants still remains unknown.GAC TCA TTC G and aaggtaccTCA TAT ATC CAT AAT Microbial systems (mainly Escherichia coli and Sac-CCC TTGGA for AtCAT1, and aaacatATG GTT GCT charomyces cerevisiae) have been used to synthesize phy-GAG AAC AATAA and aaggtaccTTA TAT CTG TAA AAA tochemicals [16]. Among the variety of phytochemicals, CTG TTC TTG A for AtCAT2. The restriction enzyme phenolic compounds are mostly synthesized in microbial recognition sites  (NdeI and KpnI) are underlined in the systems, since the biological synthetic genes have been primer sequences. The PCR product was sequenced to characterized in many plants and hosts are engineered verify the nucleotide sequence. The previously cloned to provide more substrates [17]. Introduction of caffeic Os4CL [20] was subcloned into EcoRI and HindIII sites acid synthesis pathway genes into E. coli and engineer- of pCDF-duet1 (Novagen) and the resulting construct ing of the latter to provide more tyrosine resulted in the was named pC-OS4CL. AtCAT1 and AtCAT2 were successful synthesis of caffeic acid. Tyrosine-overexpress - subcloned into NdeI/KpnI site of pC-OS4CL, respec- fbr ing E. coli strain, due to overexpression of tyrA , ppsA, tively, and became pC-OS4CL-AtCAT1 and pC-OS4CL- fbr tktA, and aroG and deletion of pheA, tyrA, and/or tyrR AtCAT2, accordingly. The previously cloned SeTAL and genes, was employed for the reaction, and TAL (Tyrosine HpaBC [21, 22] were subcloned into EcoRI and Hin- ammonia-lyase) from either Rhodotorula glutinis or Sac- dIII sites and NdeI and XhoI sites of pET-duet1 (Nova- charothrix espanaensis and p-coumaric acid hydroxylase gen), respectively, resulting in the construct named genes (HpaBC from E. coli or Sam5 from S. espanaensis) pE-SeTAL-HpaBC. were introduced into it. The final titers of caffeic acid, reported in various publications, are quite different; one Synthesis of EC report used tyrosine-overproducing E. coli by introduc- Escherichia coli BL21 (DE3) transformant, containing fbr fbr ing tyrA , ppsA, tktA, and aroG while deleting pheLA pC-OS4CL-AtCAT1 (B-EC1 in Table  1), was grown in and tyrA genes, and overexpressing TAL. Tyrosine from Luria–Bertani (LB) broth with 50  μg/mL spectinomycin R. glutinis, together with HpaBC, enabled 766.7  mg/L overnight at 37 °C. The culture was inoculated into fresh titer of caffeic acid production [18]. Another report intro - LB medium and grown at 37 °C until the OD reached duced TAL from S. espanaensis and Sam5 into tyrosine- 1.0. Thereafter, isopropyl β-D-1-thiogalactopyranoside overproducing E. coli strain, in which tyrR was disrupted (IPTG) was added to the medium to a final concentra - fbr fbr and tyrA and aroG were overexpressed. The strain tion of 1  mM and the cells were incubated at 18  °C for synthesized 150  mg/L caffeic acid [19]. In this article, 16  h. The amount of cells, corresponding to OD of 3 we reported the successful synthesis of EC in E. coli. We in 1 mL, was harvested and resuspended in 1 mL of M9 selected a gene encoding a protein that would synthe- medium containing 1% yeast extract, 2% glucose, 50  μg/ size EC from caffeoyl-CoA and ethanol (Fig.  1). Subse- mL spectinomycin, and 1  mM IPTG. Caffeic acid and quently, we manipulated the shikimate pathway in E. coli ethanol were also added to the medium resulting in a to increase the synthesis of caffeic acid. By introducing final concentration of 100µM and 1%, respectively. The genes for the conjugation of caffeic acid and ethanol into culture was incubated at 30  °C for 24  h. The reaction O O O HO HO HO Os4CL AtCAT1 OH SCoA O HO HO HO Ethanol Caffeic acid Caffeoyl-CoA Ethyl caffeate Fig. 1 Scheme for the synthesis of ethyl caffeate from caffeic acid L ee et al. Appl Biol Chem (2021) 64:71 Page 3 of 6 Table 1 Plasmids and Escherichia coli strains used in this study Plasmids and E. coli strains Relevant properties or genetic markers Source Plasmids pCDFDuet CDF ori, Sm Novagen pETDuet F1 ori, Amp Novagen pACYCDuet P15A ori, Cm Novagen pC‑ OS4CL pCDFDuet carrying 4CL from Oryza sativa This study pC‑ OS4CL‑AtCAT1 pCDFDuet carrying 4CL from Oryza sativa and CAT from Arabidopsis thaliana This study pC‑ OS4CL‑AtCAT2 pCDFDuet carrying 4CL from Oryza sativa and CAT from Arabidopsis thaliana This study pE‑SeTAL ‑HpaBC pETDuet carrying TAL from Saccharothrix espanaensis and HpaBC from Escherichia coli This study pA‑aroG‑tyrA pACYCDuet carrying aroG and tyrA from E. coli [21] f f f f pA‑aroG ‑tyrA pACYCDuet carrying aroG and tyrA from E. coli [21] f f f f pA‑aroG ‑ppsA‑tktA‑tyrA pACYCDuet carrying aroG , ppsA, tktA, and tyrA from E. coli [21] f f f f pA‑aroL ‑aroG ‑ppsA‑tktA‑tyrA pACYCDuet carrying aroL, aroG , ppsA, tktA, and tyrA from E. coli [30] f f pA‑aroL ‑aroE‑aroD ‑aroB‑ pACYCDuet carrying aroL, aroE, aroD, aroB, aroG , ppsA, tktA, and tyrA from E. coli [28] f f aroG ‑ppsA‑tktA‑tyrA f f pA‑aroC‑aroA‑aroL ‑aroE‑aroD ‑ pACYCDuet carrying aroC, aroA, aroL, aroE, aroD, aroB, aroG , ppsA, tktA, and tyrA from E. coli [31] f f aroB‑aroG ‑ppsA‑tktA‑tyrA Strains BL21 (DE3) F‑ ompT hsdSB(rB- mB-) gal dcm lon (DE3) Novagen BT BL21(DE3) FRT-ΔtyrR::FRT-kan -FRT [21] BTP BL21(DE3) ΔtyrR::FRT- ΔPheA::FRT-kan [21] -FRT B‑EC1 BL21 harboring pC‑ OS4CL‑AtCAT1 This study B‑EC2 BL21 harboring pC‑ OS4CL‑AtCAT2 This study B‑EC3 BL21 harboring pC‑ OS4CL‑AtCAT1 and pE‑SeTAL ‑HpaBC This study B‑EC4 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑tyrA‑aroG This study f f B‑EC5 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑tyrA ‑aroG This study f f B‑EC6 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑aroG ‑ppsA‑tktA‑tyrA This study f f B‑EC7 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑aroL ‑aroG ‑ppsA‑tktA‑tyrA This study f f B‑EC8 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑aroL ‑aroE‑aroD ‑aroB‑aroG ‑ppsA‑tktA‑tyrA This study f f B‑EC9 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑tyrA ‑aroC‑aroA‑aroL ‑aroE‑aroD ‑aroB‑aroG ‑ This study ppsA‑tktA f f BT‑EC7 BT harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑aroL ‑aroG ‑ppsA‑tktA‑tyrA This study f f BTP‑EC7 BTP harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑aroL ‑aroG ‑ppsA‑tktA‑tyrA This study product was extracted by ethyl acetate and dried in speed J = 8.3, 1.9 Hz), 7.03 (1H, dd, J = 1.9 Hz), and 7.53 (1H, d, vacuum. The sample was eventually dissolved in dimethyl J = 15.9 Hz). sulfoxide (DMSO) and analyzed by high-performance HPLC analysis of the product was performed as earlier liquid chromatography (HPLC). [24]. Analysis of EC Results and discussion The reaction product was purified using thin layer chro - Screening of genes for EC synthesis matography (TLC; TLC silica gel 60 F254; Millipore, EC is an ester of caffeic acid and ethanol. Plants contain Burlington, MA, USA). The mobile phase was a mix - genes that encode an enzyme capable of forming alkyl ture of dichloromethane, ethyl acetate, and formic acid hydroxycinnamate ester [25, 26]. These enzymes have (9:1:0.25). The structure was determined using NMR been involved in suberin and cutin biosynthesis, and [23]; H NMR of ethyl caffeate (400 MHz, Methanol-d4): are known to use long-chain alcohol (i.e. dodecan-1-ol) δ 1.31 (3H, t, J = 7.1  Hz), 4.21 (2H, q, J = 7.1  Hz), 6.25 as an alkyl group donor. We assumed these enzymes to (1H, d, J = 15.9 Hz), 6.78 (1H, d, J = 8.3 Hz), 6.94 (1H, dd, possibly use low-molecular-weight alcohol. Two genes (AtCAT1 and AtCAT2), which encoded fatty alcohol Lee et al. Appl Biol Chem (2021) 64:71 Page 4 of 6 caffeoyl-coenzyme A acyltransferase [15, 26], were cloned with more than six carbon atoms, since the solubility of and tested for the production of EC. Either of AtCAT1 such alcohols would be significantly low (in case of hex - and AtCAT2 was subcloned, along with Os4CL encoding anol, the water solubility was 5.9 g/L). 4-coumarate CoA ligase, since caffeic acid needed to be We investigated the maximum amount of EC synthe- activated. E. coli harboring either pC-OS4CL-AtCAT1 sized when E. coli was fed 1% ethanol. E. coli harboring (B-EC1) or pC-OS4CL-AtCAT2 (B-EC2) were fed caf- pC-OS4CL-AtCAT1 (B-EC1) was fed different concen - feic acid and ethanol. The formation of EC in the culture trations of caffeic acid (0.5, 0.7, 1.0, 1.2, 1.5, and 1.8 mM) filtrate was examined using HPLC. Formation of a new and 1% ethanol. Caffeic acid was completely converted product was observed in both culture filtrates. While the into EC up to the concentration of 1.2 mM. However, at E. coli strain B-EC1 converted all the caffeic acid into the 1.5 mM caffeic acid, approximately 0.3 mM was not con - reaction product, the other strain (B-EC2) still had unre- verted into EC, which suggested that AtCAT1 could con- acted caffeic acid. This suggested that AtCAT1 had better vert more than 1.2 mM (216.2 mg/L) caffeic acid and 1% catalytic efficiency than AtCAT2. The reaction product ethanol (about 0.36 M) was not the limiting factor. from the strain B-EC1 was purified, and was identified as EC by NMR. The results indicated that AtCAT1 could Synthesis of EC without feeding caffeic acid conjugate caffeoyl-CoA with ethanol to make EC (Fig. 2). We attempted to synthesize EC without feeding caffeic We further tested other low-molecular-weight alcohols acid to E. coli. Caffeic acid was synthesized from tyros - as substrates. E. coli strain B-EC1 was fed caffeic acid and ine using SeTAL and HpaBC [27]. SeTAL converts tyros- various low-molecular-weight alcohols, including metha- ine into p-coumaric acid, which is converted to caffeic nol, ethanol, propanol, butanol, and pentanol. Analysis acid by HpaBC. The two genes were introduced into E. of the supernatant showed that all the alcohols tested coli harboring pC-OS4CL-AtCAT1. The resulting E. coli were substrates for the synthesis of corresponding alkyl strain B-EC3 was fed ethanol thereafter. The culture fil - caffeates (data not shown). Propanol turned out to be the trate from the strain B-EC3 showed a peak that had the best substrate among all alcohols tested, based on the same retention time as EC. Furthermore, no detect- unreacted caffeic acid remaining. The different perme - able caffeic acid was observed, indicating that all the ability of each alcohol into the E. coli, and other factors, synthesized caffeic acid had been converted to EC, and such as volatility, were found to have an influence on the synthesis of more caffeic acid could increase the final titer of each alkyl caffeate. Together, the results indicated titer of EC in E. coli. Therefore, we engineered E. coli to that different alkyl caffeates could be synthesized by pro - synthesize more tyrosine, which is a precursor of caf- viding different alcohols. We did not explore the alcohols feic acid, by introducing genes, which are known to be -200 -50 0.00 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 Time (min) Fig. 2 High performance liquid chromatography (HPLC) analysis. a HPLC profile of standard caffeic acid (S); b HPLC profile of reaction product (P) from B‑EC1 strain supplied with 100 μM caffeic acid. P was identified to be ethyl caffeate Absorbance (mAU) L ee et al. Appl Biol Chem (2021) 64:71 Page 5 of 6 involved in the synthesis of tyrosine in E. coli. We tested strain, in which tyrR and tyrA were deleted, were used to f f six constructs (pA-aroG-tyrA, pA-aroG -tyrA, pA- increase the production of tyrosine [21]. We introduced f f f f fbr aroG -ppsA-tktA-tyrA , pA-aroL-aroG -ppsA-tktA-tyrA , pC-OS4CL-AtCAT1 and pA-aroL-aroG -ppsA-tktA- f f fbr pA-aroL-aroE-aroD-aroB-aroG -ppsA-tktA-tyrA, and tyrA into BT and BTP (BT-EC7, BTP-EC7) to examine pA-aroC-aroA-aroL-aroE-aroD-aroB-aroG -ppsA-tktA- the titer of EC. Both the mutant strains showed better tyrA ) with various combination of genes relevant to the titer than the wild type. The E. coli strain BT-EC7 synthe - shikimate pathway of E. coli, including tyrA, aroG, tyrA , sized approximately 116.7 mg/L EC, which was 1.48-fold aroG , aroC, aroA, aroL, aroE, aroD, aroB, ppsA, and higher than in the wild-type strain (Fig. 4). tktA [24, 28]. Each construct was transformed into E. coli EC is a natural compound found in some plants, and strain B-EC3 and the resulting strains (B-EC4  –  B-EC9) its biosynthetic pathway is not yet known. Here, we were tested for the synthesis of EC. The E. coli strains designed the biosynthetic pathway, assembled genes for overexpressing any of these constructs showed better the synthesis of EC, and successfully synthesized the titer than B-EC3, which did not overexpress the shiki- compound. Rational design of the biosynthetic pathway mate pathway gene-module construct. The E. coli strain and selection of genes from diverse sources can make the fbr B-EC7, containing five genes, namely aroL, aroG , synthesis of diverse compounds feasible. Furthermore, fbr ppsA, tktA, and tyrA , produced the highest titer of this could lead to the synthesis of unnatural compounds, EC (78.8  mg/L), followed by B-EC8 (68.6  mg/L), B-EC6 derived from natural compounds. For example, we could (68.5  mg/L), B-EC5 (65.2  mg/L), B-EC9 (59.1  mg/L), B-EC4 (44.8 mg/L), and B-EC3 (38.6 mg/L) (Fig.  3). This result agreed with the previous studies in which the over- expression of shikimate gene module increased the final titer of the synthesized compound; however, the best gene-module construct was different depending on the compound synthesized [24, 28, 29]. Our feeding study had shown that approximately 216.2  mg/L (1.2  mL) caffeic acid was converted to EC. The current EC titer was approximately 78.8  mg/L (0.38  mM), which indicated that the final titer could be increased further. In order to increase the final titer of B-EC7BT-EC7BTP-EC7 EC, we used E. coli mutants with deletion of genes from Strains the shikimate pathway, for further production of tyrosine. Fig. 4 Synthesis of ethyl caffeate in E. coli mutants expressing Os4CL, fbr fbr The BT strain, in which tyrR was deleted, and the BTP AtCAT1, SeTAL HpaBC, aroL, aroG , ppsA, tktA, and tyrA T7 RBS B-EC3 P P T7 T7 aroG tyrA B-EC4 P P T7 T7 f f aroG tyrA B-EC5 P P T7 T7 f f aroG tyrA ppsA tktA B-EC6 P P T7 T7 f f aroG tyrA aroL ppsA tktA B-EC7 P T7 T7 f f aroG tyrA aroL aroE aroD aroB ppsA tktA B-EC8 P P T7 T7 f f aroG tyrA aroC aroA aroL aroE aroD aroB ppsA tktA B-EC9 020406080 100 Titer (mg/L) Fig. 3 Constructs containing shikimate pathway genes, used for the overproduction of tyrosine (left), and synthesis of ethyl caffeate in E. coli expressing corresponding constructs (right). All strains had genes Os4CL, AtCAT1, SeTAL, and HpaBC. P , T7 promoter; RBS, ribosome‑binding site T7 Titer (mg/L) Lee et al. Appl Biol Chem (2021) 64:71 Page 6 of 6 13. 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Biosynthesis of ethyl caffeate via caffeoyl-CoA acyltransferase expression in Escherichia coli

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Abstract

Hydroxycinnamic acids (HCs) are natural compounds that form conjugates with diverse compounds in nature. Ethyl caffeate (EC) is a conjugate of caffeic acid (an HC) and ethanol. It has been found in several plants, including Prunus yedoensis, Polygonum amplexicaule, and Ligularia fischeri. Although it exhibits anticancer, anti‑inflammatory, and antifibrotic activities, its biosynthetic pathway in plants still remains unknown. This study aimed to design an EC syn‑ thesis pathway and clone genes relevant to the same. Genes involved in the caffeic acid synthesis pathway (tyrosine ammonia‑lyase ( TAL) and p‑ coumaric acid hydroxylase (HpaBC)) were introduced into Escherichia coli along with 4‑ coumaroyl CoA ligase (4CL) and acyltransferases (AtCAT ) cloned from Arabidopsis thaliana. In presence of ethanol, E. coli harboring the above genes successfully synthesized EC. Providing more tyrosine through the overexpression of shikimate‑pathway gene ‑module construct and using E. coli mutant enhanced EC yield; approximately 116.7 mg/L EC could be synthesized in the process. Synthesis of four more alkyl caffeates was confirmed in this study; these might potentially possess novel biological properties, which would require further investigation. Keywords: Acyltransferase, Alkyl caffeate, Hydroxycinnamic acid conjugate, Metabolic engineering Introduction yedoensis [6], Polygonum amplexicaule [7], and Ligularia Hydroxycinnamic acids (HCs) are abundant in nature. fischeri [8]. In particular, Ligularia fischeri is grown in They are synthesized via phenylpropanoid pathway in eastern Asia and is used in herbal medicine. The biologi - plants and serve as the building blocks for other phe- cal effects of L. fischeri are derived from EC. EC is known nolic compounds, such as flavonoids, coumarin, lignin, to exhibit anticancer [8], anti-inflammatory [9], and anti - proanthocyanidins, cutin, and suberin [1]. HCs form fibrotic activities [10]. In addition, it has the potential to conjugates with other molecules as well. The conjugate of regulate blood pressure by inhibiting aldosterone syn- caffeic acid (an HC) and quinic acid is called chlorogenic thase [11]. acid [2]. Avenanthramides are amides formed from HCs The synthetic pathway for EC in plants has not been and anthranilate derivatives [3]. Besides these, conju- fully elucidated yet. Caffeic acid is synthesized from gates of HCs with other compounds (spermine, puterine, tyrosine by deamination and hydroxylation, and needs tyramine, dopamine, tryptamine, and glycine) have also to be activated by the attachment of coenzyme A (CoA). been reported [4, 5]. Although high-molecular-weight alcohols, such as C18, Ethyl caffeate (EC) is a conjugate of caffeic acid and C20, and C22 alkan-1-ols, have been reported to be syn- ethanol, and is found in some plants, namely Prunus thesized from long-chain fatty acids in plants [12], how the ethyl group is provided during the synthesis of EC still remains a mystery. Ethanol is presumably synthesized *Correspondence: jhahn@konkuk.ac.kr in hypoxic condition [13], which is also suitable for the Department of Integrative Bioscience and Biotechnology, Bio/Molecular synthesis of EC; however, other ethyl group donors may Informatics Center, Konkuk University, Seoul 05029, Republic of Korea © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Lee et al. Appl Biol Chem (2021) 64:71 Page 2 of 6 also be present. The mechanism underlying the conjuga - an engineered E. coli strain, and providing ethanol, we tion of caffeoyl-CoA with ethyl group donor still remains could successfully synthesize EC. unknown. Previous study on the biosynthesis of suberin might provide a clue to the process of generating an EC Materials and methods conjugate. Suberin is an ester of long-chain primary alco- Constructs hols and HCs [14]. The conjugate reaction is mediated Caffeoyl-CoA transferases from A. thaliana (AtCAT1 by aliphatic suberin feruloyl transferase in Arabidopsis [AT5G63560.1] and AtCAT2 [At5g41040]) were cloned thaliana [15]. Therefore, an enzyme of this family might using reverse transcription-polymerase chain reaction catalyze the conjugation reaction to form EC, although (RT-PCR). The primers were as follows: aaacatATG GCC the origin of ethyl group in plants still remains unknown.GAC TCA TTC G and aaggtaccTCA TAT ATC CAT AAT Microbial systems (mainly Escherichia coli and Sac-CCC TTGGA for AtCAT1, and aaacatATG GTT GCT charomyces cerevisiae) have been used to synthesize phy-GAG AAC AATAA and aaggtaccTTA TAT CTG TAA AAA tochemicals [16]. Among the variety of phytochemicals, CTG TTC TTG A for AtCAT2. The restriction enzyme phenolic compounds are mostly synthesized in microbial recognition sites  (NdeI and KpnI) are underlined in the systems, since the biological synthetic genes have been primer sequences. The PCR product was sequenced to characterized in many plants and hosts are engineered verify the nucleotide sequence. The previously cloned to provide more substrates [17]. Introduction of caffeic Os4CL [20] was subcloned into EcoRI and HindIII sites acid synthesis pathway genes into E. coli and engineer- of pCDF-duet1 (Novagen) and the resulting construct ing of the latter to provide more tyrosine resulted in the was named pC-OS4CL. AtCAT1 and AtCAT2 were successful synthesis of caffeic acid. Tyrosine-overexpress - subcloned into NdeI/KpnI site of pC-OS4CL, respec- fbr ing E. coli strain, due to overexpression of tyrA , ppsA, tively, and became pC-OS4CL-AtCAT1 and pC-OS4CL- fbr tktA, and aroG and deletion of pheA, tyrA, and/or tyrR AtCAT2, accordingly. The previously cloned SeTAL and genes, was employed for the reaction, and TAL (Tyrosine HpaBC [21, 22] were subcloned into EcoRI and Hin- ammonia-lyase) from either Rhodotorula glutinis or Sac- dIII sites and NdeI and XhoI sites of pET-duet1 (Nova- charothrix espanaensis and p-coumaric acid hydroxylase gen), respectively, resulting in the construct named genes (HpaBC from E. coli or Sam5 from S. espanaensis) pE-SeTAL-HpaBC. were introduced into it. The final titers of caffeic acid, reported in various publications, are quite different; one Synthesis of EC report used tyrosine-overproducing E. coli by introduc- Escherichia coli BL21 (DE3) transformant, containing fbr fbr ing tyrA , ppsA, tktA, and aroG while deleting pheLA pC-OS4CL-AtCAT1 (B-EC1 in Table  1), was grown in and tyrA genes, and overexpressing TAL. Tyrosine from Luria–Bertani (LB) broth with 50  μg/mL spectinomycin R. glutinis, together with HpaBC, enabled 766.7  mg/L overnight at 37 °C. The culture was inoculated into fresh titer of caffeic acid production [18]. Another report intro - LB medium and grown at 37 °C until the OD reached duced TAL from S. espanaensis and Sam5 into tyrosine- 1.0. Thereafter, isopropyl β-D-1-thiogalactopyranoside overproducing E. coli strain, in which tyrR was disrupted (IPTG) was added to the medium to a final concentra - fbr fbr and tyrA and aroG were overexpressed. The strain tion of 1  mM and the cells were incubated at 18  °C for synthesized 150  mg/L caffeic acid [19]. In this article, 16  h. The amount of cells, corresponding to OD of 3 we reported the successful synthesis of EC in E. coli. We in 1 mL, was harvested and resuspended in 1 mL of M9 selected a gene encoding a protein that would synthe- medium containing 1% yeast extract, 2% glucose, 50  μg/ size EC from caffeoyl-CoA and ethanol (Fig.  1). Subse- mL spectinomycin, and 1  mM IPTG. Caffeic acid and quently, we manipulated the shikimate pathway in E. coli ethanol were also added to the medium resulting in a to increase the synthesis of caffeic acid. By introducing final concentration of 100µM and 1%, respectively. The genes for the conjugation of caffeic acid and ethanol into culture was incubated at 30  °C for 24  h. The reaction O O O HO HO HO Os4CL AtCAT1 OH SCoA O HO HO HO Ethanol Caffeic acid Caffeoyl-CoA Ethyl caffeate Fig. 1 Scheme for the synthesis of ethyl caffeate from caffeic acid L ee et al. Appl Biol Chem (2021) 64:71 Page 3 of 6 Table 1 Plasmids and Escherichia coli strains used in this study Plasmids and E. coli strains Relevant properties or genetic markers Source Plasmids pCDFDuet CDF ori, Sm Novagen pETDuet F1 ori, Amp Novagen pACYCDuet P15A ori, Cm Novagen pC‑ OS4CL pCDFDuet carrying 4CL from Oryza sativa This study pC‑ OS4CL‑AtCAT1 pCDFDuet carrying 4CL from Oryza sativa and CAT from Arabidopsis thaliana This study pC‑ OS4CL‑AtCAT2 pCDFDuet carrying 4CL from Oryza sativa and CAT from Arabidopsis thaliana This study pE‑SeTAL ‑HpaBC pETDuet carrying TAL from Saccharothrix espanaensis and HpaBC from Escherichia coli This study pA‑aroG‑tyrA pACYCDuet carrying aroG and tyrA from E. coli [21] f f f f pA‑aroG ‑tyrA pACYCDuet carrying aroG and tyrA from E. coli [21] f f f f pA‑aroG ‑ppsA‑tktA‑tyrA pACYCDuet carrying aroG , ppsA, tktA, and tyrA from E. coli [21] f f f f pA‑aroL ‑aroG ‑ppsA‑tktA‑tyrA pACYCDuet carrying aroL, aroG , ppsA, tktA, and tyrA from E. coli [30] f f pA‑aroL ‑aroE‑aroD ‑aroB‑ pACYCDuet carrying aroL, aroE, aroD, aroB, aroG , ppsA, tktA, and tyrA from E. coli [28] f f aroG ‑ppsA‑tktA‑tyrA f f pA‑aroC‑aroA‑aroL ‑aroE‑aroD ‑ pACYCDuet carrying aroC, aroA, aroL, aroE, aroD, aroB, aroG , ppsA, tktA, and tyrA from E. coli [31] f f aroB‑aroG ‑ppsA‑tktA‑tyrA Strains BL21 (DE3) F‑ ompT hsdSB(rB- mB-) gal dcm lon (DE3) Novagen BT BL21(DE3) FRT-ΔtyrR::FRT-kan -FRT [21] BTP BL21(DE3) ΔtyrR::FRT- ΔPheA::FRT-kan [21] -FRT B‑EC1 BL21 harboring pC‑ OS4CL‑AtCAT1 This study B‑EC2 BL21 harboring pC‑ OS4CL‑AtCAT2 This study B‑EC3 BL21 harboring pC‑ OS4CL‑AtCAT1 and pE‑SeTAL ‑HpaBC This study B‑EC4 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑tyrA‑aroG This study f f B‑EC5 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑tyrA ‑aroG This study f f B‑EC6 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑aroG ‑ppsA‑tktA‑tyrA This study f f B‑EC7 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑aroL ‑aroG ‑ppsA‑tktA‑tyrA This study f f B‑EC8 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑aroL ‑aroE‑aroD ‑aroB‑aroG ‑ppsA‑tktA‑tyrA This study f f B‑EC9 BL21 harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑tyrA ‑aroC‑aroA‑aroL ‑aroE‑aroD ‑aroB‑aroG ‑ This study ppsA‑tktA f f BT‑EC7 BT harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑aroL ‑aroG ‑ppsA‑tktA‑tyrA This study f f BTP‑EC7 BTP harboring pC‑ OS4CL‑AtCAT1, pE‑SeTAL ‑HpaBC, and pA‑aroL ‑aroG ‑ppsA‑tktA‑tyrA This study product was extracted by ethyl acetate and dried in speed J = 8.3, 1.9 Hz), 7.03 (1H, dd, J = 1.9 Hz), and 7.53 (1H, d, vacuum. The sample was eventually dissolved in dimethyl J = 15.9 Hz). sulfoxide (DMSO) and analyzed by high-performance HPLC analysis of the product was performed as earlier liquid chromatography (HPLC). [24]. Analysis of EC Results and discussion The reaction product was purified using thin layer chro - Screening of genes for EC synthesis matography (TLC; TLC silica gel 60 F254; Millipore, EC is an ester of caffeic acid and ethanol. Plants contain Burlington, MA, USA). The mobile phase was a mix - genes that encode an enzyme capable of forming alkyl ture of dichloromethane, ethyl acetate, and formic acid hydroxycinnamate ester [25, 26]. These enzymes have (9:1:0.25). The structure was determined using NMR been involved in suberin and cutin biosynthesis, and [23]; H NMR of ethyl caffeate (400 MHz, Methanol-d4): are known to use long-chain alcohol (i.e. dodecan-1-ol) δ 1.31 (3H, t, J = 7.1  Hz), 4.21 (2H, q, J = 7.1  Hz), 6.25 as an alkyl group donor. We assumed these enzymes to (1H, d, J = 15.9 Hz), 6.78 (1H, d, J = 8.3 Hz), 6.94 (1H, dd, possibly use low-molecular-weight alcohol. Two genes (AtCAT1 and AtCAT2), which encoded fatty alcohol Lee et al. Appl Biol Chem (2021) 64:71 Page 4 of 6 caffeoyl-coenzyme A acyltransferase [15, 26], were cloned with more than six carbon atoms, since the solubility of and tested for the production of EC. Either of AtCAT1 such alcohols would be significantly low (in case of hex - and AtCAT2 was subcloned, along with Os4CL encoding anol, the water solubility was 5.9 g/L). 4-coumarate CoA ligase, since caffeic acid needed to be We investigated the maximum amount of EC synthe- activated. E. coli harboring either pC-OS4CL-AtCAT1 sized when E. coli was fed 1% ethanol. E. coli harboring (B-EC1) or pC-OS4CL-AtCAT2 (B-EC2) were fed caf- pC-OS4CL-AtCAT1 (B-EC1) was fed different concen - feic acid and ethanol. The formation of EC in the culture trations of caffeic acid (0.5, 0.7, 1.0, 1.2, 1.5, and 1.8 mM) filtrate was examined using HPLC. Formation of a new and 1% ethanol. Caffeic acid was completely converted product was observed in both culture filtrates. While the into EC up to the concentration of 1.2 mM. However, at E. coli strain B-EC1 converted all the caffeic acid into the 1.5 mM caffeic acid, approximately 0.3 mM was not con - reaction product, the other strain (B-EC2) still had unre- verted into EC, which suggested that AtCAT1 could con- acted caffeic acid. This suggested that AtCAT1 had better vert more than 1.2 mM (216.2 mg/L) caffeic acid and 1% catalytic efficiency than AtCAT2. The reaction product ethanol (about 0.36 M) was not the limiting factor. from the strain B-EC1 was purified, and was identified as EC by NMR. The results indicated that AtCAT1 could Synthesis of EC without feeding caffeic acid conjugate caffeoyl-CoA with ethanol to make EC (Fig. 2). We attempted to synthesize EC without feeding caffeic We further tested other low-molecular-weight alcohols acid to E. coli. Caffeic acid was synthesized from tyros - as substrates. E. coli strain B-EC1 was fed caffeic acid and ine using SeTAL and HpaBC [27]. SeTAL converts tyros- various low-molecular-weight alcohols, including metha- ine into p-coumaric acid, which is converted to caffeic nol, ethanol, propanol, butanol, and pentanol. Analysis acid by HpaBC. The two genes were introduced into E. of the supernatant showed that all the alcohols tested coli harboring pC-OS4CL-AtCAT1. The resulting E. coli were substrates for the synthesis of corresponding alkyl strain B-EC3 was fed ethanol thereafter. The culture fil - caffeates (data not shown). Propanol turned out to be the trate from the strain B-EC3 showed a peak that had the best substrate among all alcohols tested, based on the same retention time as EC. Furthermore, no detect- unreacted caffeic acid remaining. The different perme - able caffeic acid was observed, indicating that all the ability of each alcohol into the E. coli, and other factors, synthesized caffeic acid had been converted to EC, and such as volatility, were found to have an influence on the synthesis of more caffeic acid could increase the final titer of each alkyl caffeate. Together, the results indicated titer of EC in E. coli. Therefore, we engineered E. coli to that different alkyl caffeates could be synthesized by pro - synthesize more tyrosine, which is a precursor of caf- viding different alcohols. We did not explore the alcohols feic acid, by introducing genes, which are known to be -200 -50 0.00 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 Time (min) Fig. 2 High performance liquid chromatography (HPLC) analysis. a HPLC profile of standard caffeic acid (S); b HPLC profile of reaction product (P) from B‑EC1 strain supplied with 100 μM caffeic acid. P was identified to be ethyl caffeate Absorbance (mAU) L ee et al. Appl Biol Chem (2021) 64:71 Page 5 of 6 involved in the synthesis of tyrosine in E. coli. We tested strain, in which tyrR and tyrA were deleted, were used to f f six constructs (pA-aroG-tyrA, pA-aroG -tyrA, pA- increase the production of tyrosine [21]. We introduced f f f f fbr aroG -ppsA-tktA-tyrA , pA-aroL-aroG -ppsA-tktA-tyrA , pC-OS4CL-AtCAT1 and pA-aroL-aroG -ppsA-tktA- f f fbr pA-aroL-aroE-aroD-aroB-aroG -ppsA-tktA-tyrA, and tyrA into BT and BTP (BT-EC7, BTP-EC7) to examine pA-aroC-aroA-aroL-aroE-aroD-aroB-aroG -ppsA-tktA- the titer of EC. Both the mutant strains showed better tyrA ) with various combination of genes relevant to the titer than the wild type. The E. coli strain BT-EC7 synthe - shikimate pathway of E. coli, including tyrA, aroG, tyrA , sized approximately 116.7 mg/L EC, which was 1.48-fold aroG , aroC, aroA, aroL, aroE, aroD, aroB, ppsA, and higher than in the wild-type strain (Fig. 4). tktA [24, 28]. Each construct was transformed into E. coli EC is a natural compound found in some plants, and strain B-EC3 and the resulting strains (B-EC4  –  B-EC9) its biosynthetic pathway is not yet known. Here, we were tested for the synthesis of EC. The E. coli strains designed the biosynthetic pathway, assembled genes for overexpressing any of these constructs showed better the synthesis of EC, and successfully synthesized the titer than B-EC3, which did not overexpress the shiki- compound. Rational design of the biosynthetic pathway mate pathway gene-module construct. The E. coli strain and selection of genes from diverse sources can make the fbr B-EC7, containing five genes, namely aroL, aroG , synthesis of diverse compounds feasible. Furthermore, fbr ppsA, tktA, and tyrA , produced the highest titer of this could lead to the synthesis of unnatural compounds, EC (78.8  mg/L), followed by B-EC8 (68.6  mg/L), B-EC6 derived from natural compounds. For example, we could (68.5  mg/L), B-EC5 (65.2  mg/L), B-EC9 (59.1  mg/L), B-EC4 (44.8 mg/L), and B-EC3 (38.6 mg/L) (Fig.  3). This result agreed with the previous studies in which the over- expression of shikimate gene module increased the final titer of the synthesized compound; however, the best gene-module construct was different depending on the compound synthesized [24, 28, 29]. Our feeding study had shown that approximately 216.2  mg/L (1.2  mL) caffeic acid was converted to EC. The current EC titer was approximately 78.8  mg/L (0.38  mM), which indicated that the final titer could be increased further. In order to increase the final titer of B-EC7BT-EC7BTP-EC7 EC, we used E. coli mutants with deletion of genes from Strains the shikimate pathway, for further production of tyrosine. Fig. 4 Synthesis of ethyl caffeate in E. coli mutants expressing Os4CL, fbr fbr The BT strain, in which tyrR was deleted, and the BTP AtCAT1, SeTAL HpaBC, aroL, aroG , ppsA, tktA, and tyrA T7 RBS B-EC3 P P T7 T7 aroG tyrA B-EC4 P P T7 T7 f f aroG tyrA B-EC5 P P T7 T7 f f aroG tyrA ppsA tktA B-EC6 P P T7 T7 f f aroG tyrA aroL ppsA tktA B-EC7 P T7 T7 f f aroG tyrA aroL aroE aroD aroB ppsA tktA B-EC8 P P T7 T7 f f aroG tyrA aroC aroA aroL aroE aroD aroB ppsA tktA B-EC9 020406080 100 Titer (mg/L) Fig. 3 Constructs containing shikimate pathway genes, used for the overproduction of tyrosine (left), and synthesis of ethyl caffeate in E. coli expressing corresponding constructs (right). All strains had genes Os4CL, AtCAT1, SeTAL, and HpaBC. P , T7 promoter; RBS, ribosome‑binding site T7 Titer (mg/L) Lee et al. Appl Biol Chem (2021) 64:71 Page 6 of 6 13. 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Journal

Applied Biological ChemistrySpringer Journals

Published: Dec 1, 2021

Keywords: Acyltransferase; Alkyl caffeate; Hydroxycinnamic acid conjugate; Metabolic engineering

References