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

Applied Biological Chemistry volume 64, Article number: 71 (2021) Cite this article

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 synthesis 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.

Introduction

Hydroxycinnamic acids (HCs) are abundant in nature. They are synthesized via phenylpropanoid pathway in plants and serve as the building blocks for other phenolic compounds, such as flavonoids, coumarin, lignin, proanthocyanidins, cutin, and suberin [1]. HCs form conjugates with other molecules as well. The conjugate of caffeic acid (an HC) and quinic acid is called chlorogenic acid [2]. Avenanthramides are amides formed from HCs and anthranilate derivatives [3]. Besides these, conjugates of HCs with other compounds (spermine, puterine, tyramine, dopamine, tryptamine, and glycine) have also been reported [4, 5].

Ethyl caffeate (EC) is a conjugate of caffeic acid and ethanol, and is found in some plants, namely Prunus yedoensis [6], Polygonum amplexicaule [7], and Ligularia fischeri [8]. In particular, Ligularia fischeri is grown in eastern Asia and is used in herbal medicine. The biological effects of L. fischeri are derived from EC. EC is known to exhibit anticancer [8], anti-inflammatory [9], and antifibrotic activities [10]. In addition, it has the potential to regulate blood pressure by inhibiting aldosterone synthase [11].

The synthetic pathway for EC in plants has not been fully elucidated yet. Caffeic acid is synthesized from tyrosine by deamination and hydroxylation, and needs to be activated by the attachment of coenzyme A (CoA). Although high-molecular-weight alcohols, such as C18, C20, and C22 alkan-1-ols, have been reported to be synthesized 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 in hypoxic condition [13], which is also suitable for the synthesis of EC; however, other ethyl group donors may also be present. The mechanism underlying the conjugation of caffeoyl-CoA with ethyl group donor still remains unknown. Previous study on the biosynthesis of suberin might provide a clue to the process of generating an EC conjugate. Suberin is an ester of long-chain primary alcohols and HCs [14]. The conjugate reaction is mediated by aliphatic suberin feruloyl transferase in Arabidopsis thaliana [15]. Therefore, an enzyme of this family might catalyze the conjugation reaction to form EC, although the origin of ethyl group in plants still remains unknown.

Microbial systems (mainly Escherichia coli and Saccharomyces cerevisiae) have been used to synthesize phytochemicals [16]. Among the variety of phytochemicals, phenolic compounds are mostly synthesized in microbial systems, since the biological synthetic genes have been characterized in many plants and hosts are engineered to provide more substrates [17]. Introduction of caffeic acid synthesis pathway genes into E. coli and engineering of the latter to provide more tyrosine resulted in the successful synthesis of caffeic acid. Tyrosine-overexpressing E. coli strain, due to overexpression of tyrAfbr, ppsA, tktA, and aroGfbr and deletion of pheA, tyrA, and/or tyrR genes, was employed for the reaction, and TAL (Tyrosine ammonia-lyase) from either Rhodotorula glutinis or Saccharothrix espanaensis and p-coumaric acid hydroxylase genes (HpaBC from E. coli or Sam5 from S. espanaensis) were introduced into it. The final titers of caffeic acid, reported in various publications, are quite different; one report used tyrosine-overproducing E. coli by introducing tyrAfbr, ppsA, tktA, and aroGfbr while deleting pheLA and tyrA genes, and overexpressing TAL. Tyrosine from R. glutinis, together with HpaBC, enabled 766.7 mg/L titer of caffeic acid production [18]. Another report introduced TAL from S. espanaensis and Sam5 into tyrosine-overproducing E. coli strain, in which tyrR was disrupted and tyrAfbr and aroGfbr were overexpressed. The strain synthesized 150 mg/L caffeic acid [19]. In this article, we reported the successful synthesis of EC in E. coli. We selected a gene encoding a protein that would synthesize EC from caffeoyl-CoA and ethanol (Fig. 1). Subsequently, we manipulated the shikimate pathway in E. coli to increase the synthesis of caffeic acid. By introducing genes for the conjugation of caffeic acid and ethanol into an engineered E. coli strain, and providing ethanol, we could successfully synthesize EC.

Fig. 1
figure 1

Scheme for the synthesis of ethyl caffeate from caffeic acid

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Materials and methods

Constructs

Caffeoyl-CoA transferases from A. thaliana (AtCAT1 [AT5G63560.1] and AtCAT2 [At5g41040]) were cloned using reverse transcription-polymerase chain reaction (RT-PCR). The primers were as follows: aaacatATGGCCGACTCATTCG and aaggtaccTCATATATCCATAATCCCTTGGA for AtCAT1, and aaacatATGGTTGCTGAGAACAATAA and aaggtaccTTATATCTGTAAAAACTGTTCTTGA for AtCAT2. The restriction enzyme recognition sites (NdeI and KpnI) are underlined in the primer sequences. The PCR product was sequenced to verify the nucleotide sequence. The previously cloned Os4CL [20] was subcloned into EcoRI and HindIII sites of pCDF-duet1 (Novagen) and the resulting construct was named pC-OS4CL. AtCAT1 and AtCAT2 were subcloned into NdeI/KpnI site of pC-OS4CL, respectively, and became pC-OS4CL-AtCAT1 and pC-OS4CL-AtCAT2, accordingly. The previously cloned SeTAL and HpaBC [21, 22] were subcloned into EcoRI and HindIII sites and NdeI and XhoI sites of pET-duet1 (Novagen), respectively, resulting in the construct named pE-SeTAL-HpaBC.

Synthesis of EC

Escherichia coli BL21 (DE3) transformant, containing pC-OS4CL-AtCAT1 (B-EC1 in Table 1), was grown in Luria–Bertani (LB) broth with 50 μg/mL spectinomycin overnight at 37 °C. The culture was inoculated into fresh LB medium and grown at 37 °C until the OD600 reached 1.0. Thereafter, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the medium to a final concentration of 1 mM and the cells were incubated at 18 °C for 16 h. The amount of cells, corresponding to OD600 of 3 in 1 mL, was harvested and resuspended in 1 mL of M9 medium containing 1% yeast extract, 2% glucose, 50 μg/mL spectinomycin, and 1 mM IPTG. Caffeic acid and ethanol were also added to the medium resulting in a final concentration of 100µM and 1%, respectively. The culture was incubated at 30 °C for 24 h. The reaction product was extracted by ethyl acetate and dried in speed vacuum. The sample was eventually dissolved in dimethyl sulfoxide (DMSO) and analyzed by high-performance liquid chromatography (HPLC).

Table 1 Plasmids and Escherichia coli strains used in this study
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Analysis of EC

The reaction product was purified using thin layer chromatography (TLC; TLC silica gel 60 F254; Millipore, Burlington, MA, USA). The mobile phase was a mixture of dichloromethane, ethyl acetate, and formic acid (9:1:0.25). The structure was determined using NMR [23]; 1H NMR of ethyl caffeate (400 MHz, Methanol-d4): δ 1.31 (3H, t, J = 7.1 Hz), 4.21 (2H, q, J = 7.1 Hz), 6.25 (1H, d, J = 15.9 Hz), 6.78 (1H, d, J = 8.3 Hz), 6.94 (1H, dd, J = 8.3, 1.9 Hz), 7.03 (1H, dd, J = 1.9 Hz), and 7.53 (1H, d, J = 15.9 Hz).

HPLC analysis of the product was performed as earlier [24].

Results and discussion

Screening of genes for EC synthesis

EC is an ester of caffeic acid and ethanol. Plants contain genes that encode an enzyme capable of forming alkyl hydroxycinnamate ester [25, 26]. These enzymes have been involved in suberin and cutin biosynthesis, and are known to use long-chain alcohol (i.e. dodecan-1-ol) as an alkyl group donor. We assumed these enzymes to possibly use low-molecular-weight alcohol. Two genes (AtCAT1 and AtCAT2), which encoded fatty alcohol caffeoyl-coenzyme A acyltransferase [15, 26], were cloned and tested for the production of EC. Either of AtCAT1 and AtCAT2 was subcloned, along with Os4CL encoding 4-coumarate CoA ligase, since caffeic acid needed to be activated. E. coli harboring either pC-OS4CL-AtCAT1 (B-EC1) or pC-OS4CL-AtCAT2 (B-EC2) were fed caffeic acid and ethanol. The formation of EC in the culture filtrate was examined using HPLC. Formation of a new product was observed in both culture filtrates. While the E. coli strain B-EC1 converted all the caffeic acid into the reaction product, the other strain (B-EC2) still had unreacted caffeic acid. This suggested that AtCAT1 had better catalytic efficiency than AtCAT2. The reaction product from the strain B-EC1 was purified, and was identified as EC by NMR. The results indicated that AtCAT1 could conjugate caffeoyl-CoA with ethanol to make EC (Fig. 2).

Fig. 2
figure 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

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We further tested other low-molecular-weight alcohols as substrates. E. coli strain B-EC1 was fed caffeic acid and various low-molecular-weight alcohols, including methanol, ethanol, propanol, butanol, and pentanol. Analysis of the supernatant showed that all the alcohols tested were substrates for the synthesis of corresponding alkyl caffeates (data not shown). Propanol turned out to be the best substrate among all alcohols tested, based on the unreacted caffeic acid remaining. The different permeability of each alcohol into the E. coli, and other factors, such as volatility, were found to have an influence on the titer of each alkyl caffeate. Together, the results indicated that different alkyl caffeates could be synthesized by providing different alcohols. We did not explore the alcohols with more than six carbon atoms, since the solubility of such alcohols would be significantly low (in case of hexanol, the water solubility was 5.9 g/L).

We investigated the maximum amount of EC synthesized when E. coli was fed 1% ethanol. E. coli harboring pC-OS4CL-AtCAT1 (B-EC1) was fed different concentrations of caffeic acid (0.5, 0.7, 1.0, 1.2, 1.5, and 1.8 mM) and 1% ethanol. Caffeic acid was completely converted into EC up to the concentration of 1.2 mM. However, at 1.5 mM caffeic acid, approximately 0.3 mM was not converted into EC, which suggested that AtCAT1 could convert more than 1.2 mM (216.2 mg/L) caffeic acid and 1% ethanol (about 0.36 M) was not the limiting factor.

Synthesis of EC without feeding caffeic acid

We attempted to synthesize EC without feeding caffeic acid to E. coli. Caffeic acid was synthesized from tyrosine using SeTAL and HpaBC [27]. SeTAL converts tyrosine into p-coumaric acid, which is converted to caffeic acid by HpaBC. The two genes were introduced into E. coli harboring pC-OS4CL-AtCAT1. The resulting E. coli strain B-EC3 was fed ethanol thereafter. The culture filtrate from the strain B-EC3 showed a peak that had the same retention time as EC. Furthermore, no detectable caffeic acid was observed, indicating that all the synthesized caffeic acid had been converted to EC, and synthesis of more caffeic acid could increase the final titer of EC in E. coli. Therefore, we engineered E. coli to synthesize more tyrosine, which is a precursor of caffeic acid, by introducing genes, which are known to be involved in the synthesis of tyrosine in E. coli. We tested six constructs (pA-aroG-tyrA, pA-aroGf-tyrAf, pA-aroGf-ppsA-tktA-tyrAf, pA-aroL-aroGf-ppsA-tktA-tyrAf, pA-aroL-aroE-aroD-aroB-aroGf-ppsA-tktA-tyrAf, and pA-aroC-aroA-aroL-aroE-aroD-aroB-aroGf-ppsA-tktA-tyrAf) with various combination of genes relevant to the shikimate pathway of E. coli, including tyrA, aroG, tyrAf, aroGf, aroC, aroA, aroL, aroE, aroD, aroB, ppsA, and tktA [24, 28]. Each construct was transformed into E. coli strain B-EC3 and the resulting strains (B-EC4 – B-EC9) were tested for the synthesis of EC. The E. coli strains overexpressing any of these constructs showed better titer than B-EC3, which did not overexpress the shikimate pathway gene-module construct. The E. coli strain B-EC7, containing five genes, namely aroL, aroGfbr, ppsA, tktA, and tyrAfbr, produced the highest titer of EC (78.8 mg/L), followed by B-EC8 (68.6 mg/L), B-EC6 (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 overexpression 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].

Fig. 3
figure 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. PT7, T7 promoter; RBS, ribosome-binding site

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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 EC, we used E. coli mutants with deletion of genes from the shikimate pathway, for further production of tyrosine. The BT strain, in which tyrR was deleted, and the BTP strain, in which tyrR and tyrA were deleted, were used to increase the production of tyrosine [21]. We introduced pC-OS4CL-AtCAT1 and pA-aroL-aroGfbr-ppsA-tktA-tyrAfbr into BT and BTP (BT-EC7, BTP-EC7) to examine the titer of EC. Both the mutant strains showed better titer than the wild type. The E. coli strain BT-EC7 synthesized approximately 116.7 mg/L EC, which was 1.48-fold higher than in the wild-type strain (Fig. 4).

Fig. 4
figure 4

Synthesis of ethyl caffeate in E. coli mutants expressing Os4CL, AtCAT1, SeTAL HpaBC, aroL, aroGfbr, ppsA, tktA, and tyrAfbr

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EC is a natural compound found in some plants, and its biosynthetic pathway is not yet known. Here, we designed the biosynthetic pathway, assembled genes for the synthesis of EC, and successfully synthesized the compound. Rational design of the biosynthetic pathway and selection of genes from diverse sources can make the synthesis of diverse compounds feasible. Furthermore, this could lead to the synthesis of unnatural compounds, derived from natural compounds. For example, we could synthesize at least four other alkyl caffeates, depending on the alcohol, which have not been found in nature yet, and the compounds could potentially have different biological activities.

Availability of data and materials

All data generated or analyzed during the present study are included in this published article.

References

  1. Vogt T (2010) Phenylpropanoid biosynthesis. Mol Plant 3:2–20

    Article  CAS  Google Scholar 

  2. Clifford MN (1999) Chlorogenic acids and other cinnamates-nature, occurrence and dietary burden. J Sci Food Agric 79:362–372

    Article  CAS  Google Scholar 

  3. Lee SJ, Sim GY, Kang H, Yeo WS, Kim B-G, Ahn J-H (2018) Synthesis of avenanthramides using engineered Escherichia coli. Microb Cell Fact 17:46

    Article  Google Scholar 

  4. Martin-Tanguy J (1985) The occurrence and possible function of hydroxycinnamic acid amides in plants. Plant Growth Regul 3:381–399

    Article  CAS  Google Scholar 

  5. Facchini PJ, Hagel J, Zulak KG (2002) Hydroxycinnamic acid amide metabolism: physiology and biochemistry. Can J Bot 80:577–589

    Article  CAS  Google Scholar 

  6. Noshita T, Ai Sakaguchi A, Funayama S (2006) Isolation of ethyl caffeate from the petals of Prunus yedoensis. J Nat Med 60:266–267

    Article  CAS  Google Scholar 

  7. Xiang M, Su H, Hu J, Yan Y (2011) Isolation, identification and determination of methyl caffeate, ethyl caffeate and other phenolic compounds from Polygonum amplexicaule var. sinense. J Med Plant Res 5:1685–1691

    CAS  Google Scholar 

  8. Lee HN, Kim J-K, Kim JH, Lee S-J, Ahn E-K, Oh JS, Seo D-W (2014) A mechanistic study on the anti-cancer activity of ethyl caffeate in human ovarian cancer SKOV-3 cells. Chem Biol Interact 219:151–158

    Article  CAS  Google Scholar 

  9. Chiang Y, Lo C-P, Chen Y-P, Wang S-Y, Yang N-S, Kuo Y-H, Shyur L-F (2005) Ethyl caffeate suppresses NF-κB activation and its downstream inflammatory mediators, iNOS, COX-2, and PGE2 in vitro or in mouse skin. Br J Pharmacol 146:352–363

    Article  CAS  Google Scholar 

  10. Boselli E, Bendia E, Di Lecce G, Benedetti A, Frega NG (2009) Ethyl caffeate from Verdicchio wine: chromatographic purification and in vivo evaluation of its antifibrotic activity. J Sep Sci 32:3585–3590

    Article  CAS  Google Scholar 

  11. Luo Gg LuF, Qiao L, Chen X, Li G, Zhang Y (2016) Discovery of potential inhibitors of aldosterone synthase from Chinese herbs using pharmacophore modeling, molecular docking, and molecular dynamics simulation studies. BioMed Res Int 2016:1–8

    Google Scholar 

  12. Domergue F, Vishwanath SJ, Joubès J, Ono J, Lee JA, Bourdon M, Alhattab R, Lowe C, Pascal S, Lessire R, Rowland O (2010) Three Arabidopsis fatty acyl-coenzyme A reductases, FAR1, FAR4, and FAR5, generate primary fatty alcohols associated with suberin deposition. Plant Physiol 153:1539–1554

    Article  CAS  Google Scholar 

  13. Bui LT, Novi G, Lombardi L, Iannuzzi C, Rossi J, Santaniello A, Mensuali A, Corbineau F, Giuntoli B, Perata P, Zaffagnini M, Licausi F (2019) Conservation of ethanol fermentation and its regulation in land plants. J Exp Bot 70:1815–1827

    Article  CAS  Google Scholar 

  14. Kolattukudy PE (2001) Polyesters in higher plants. Adv Biochem Eng Biotechnol 71:1–49

    CAS  PubMed  Google Scholar 

  15. Kosma DK, Molina I, Ohlrogge JB, Pollard M (2012) Identification of an Arabidopsis fatty alcohol: caffeoyl-coenzyme A acyltransferase required for the synthesis of alkyl hydroxycinnamates in root waxes. Plant Physiol 160:237–248

    Article  CAS  Google Scholar 

  16. Carvens A, Payne J, Smolke CD (2019) Synthetic biology strategies for microbial biosynthesis of plant natural products. Nature Com 10:2142

    Article  Google Scholar 

  17. Chouhan S, Sharma K, Zha J, Guleria S, Koffas MAG (2017) Recent advances in the recombinant biosynthesis of polyphenols. Front Microbiol 8:2259

    Article  Google Scholar 

  18. Huang Q, Lin Y, Yan Y (2013) Caffeic acid production enhancement by engineering a phenylalanine over-producing Escherichia coli strain. Biotechnol Bioeng 110:3188–3196

    Article  CAS  Google Scholar 

  19. Kang SY, Choi O, Lee JK, Hwang BY, Uhm TB (2012) Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain. Microb Cell Fact. 11:153

    Article  CAS  Google Scholar 

  20. Lee YJ, Jeon Y, Lee JS, Kim BG, Lee CH, Ahn J-H (2007) Enzymatic synthesis of phenolic CoAs using 4-coumarate:coenzyme A ligase (4CL) from rice. Bull Kor Chem Soc 28:365–366

    Article  CAS  Google Scholar 

  21. Kim MJ, Kim B-G, Ahn J-H (2013) Biosynthesis of bioactive O-methylated flavonoids in Escherichia coli. Appl Microbiol Biotechnol 97:7195–7204

    Article  CAS  Google Scholar 

  22. Chung D, Kim SY, Ahn J-H (2017) Production of three phenylethanoids, tyrosol, hydroxytyrosol, and salidroside, using plant genes expressing in Escherichia coli. Sci Rep 7:2578

    Article  Google Scholar 

  23. Yoon J-A, Kim B-G, Lee WJ, Lim Y, Chong Y, Ahn J-H (2012) Production of a novel quercetin glycoside through metabolic engineering of Escherichia coli. Appl Env Microbiol 78:4256–4262

    Article  CAS  Google Scholar 

  24. Kim H, Kim SY, Sim GY, Ahn J-H (2020) Synthesis of 4-hydroxybenzoic acid derivatives in Escherichia coli. J Agric Food Chem 68:9743–9749

    Article  CAS  Google Scholar 

  25. Gou JY, Yu XH, Liu CJ (2009) A hydroxycinnamoyltransferase responsible for synthesizing suberin aromatics in Arabidopsis. Proc Natl Acad Sci USA 106:18855–18860

    Article  CAS  Google Scholar 

  26. Rautengarten C, Ebert B, Ouellet M, Nafisi M, Baidoo EE, Benke P, Stranne M, Mukhopadhyay A, Keasling JD, Sakuragi Y, Scheller HV (2012) Arabidopsis deficient in cutin ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester. Plant Physiol 158:654–666

    Article  CAS  Google Scholar 

  27. An DG, Cha MN, Nadarajan SP, Kim BG, Ahn J-H (2016) Bacterial synthesis of four hydroxycinnamic acids. Appl Biol Chem 59:173–179

    Article  CAS  Google Scholar 

  28. Choo HJ, Ahn J-H (2019) Synthesis of three bioactive aromatic compounds by introducing polyketide synthase genes into engineered Escherichia coli. J Agric Food Chem 67:8581–8589

    Article  CAS  Google Scholar 

  29. Choi G-S, Choo HJ, Kim B-G, Ahn J-H (2020) Synthesis of acridone derivatives via heterologous expression of a plant type III polyketide synthase in Escherichia coli. Microb Cell Fact 19:73

    Article  CAS  Google Scholar 

  30. Kim S-Y, Kim H, Kim BG, Ahn J-H (2019) Biosynthesis of two hydroxybenzoic acid-amine conjugates in engineered Escherichia coli. J Microbiol Biotechnol 29:839–844

    Article  Google Scholar 

  31. Chong YJ, Lee HL, Song JH, Lee Y, Kim B-G, Mok H, Ahn J-H (2021) Biosynthesis of resveratrol derivatives and evaluation of their anti-inflammatory activity. Appl Biol Chem 64:33

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Integrative Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul, 05029, Republic of Korea

    Shin-Won Lee, Han Kim & Joong-Hoon Ahn

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  1. Shin-Won Lee
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Contributions

SWL and JHA designed the experiments. SWL and HK performed the experiments. SWL, HK, and JHA analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Joong-Hoon Ahn.

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Lee, SW., Kim, H. & Ahn, JH. Biosynthesis of ethyl caffeate via caffeoyl-CoA acyltransferase expression in Escherichia coli. Appl Biol Chem 64, 71 (2021). https://doi.org/10.1186/s13765-021-00643-0

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