Biotransformation of eicosapentaenoic acid into the dihydroxyeicosapentaenoic acids resolvin E4 and its enantiomer by 15S- and 15R-lipoxygenases expressed in Escherichia coli

Biotransformation of eicosapentaenoic acid into the dihydroxyeicosapentaenoic acids resolvin E4 and its enantiomer by 15S- and 15R-lipoxygenases expressed in Escherichia coli | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biotransformation of eicosapentaenoic acid into the dihydroxyeicosapentaenoic acids resolvin E4 and its enantiomer by 15 S - and 15 R -lipoxygenases expressed in Escherichia coli Jin Lee, Hyun-Ah Park, Kyung-Chul Shin, Deok-Kun Oh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4121438/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives To enhance the production of resolvin E4 (RvE4) or its enantiomer from eicosapentaenoic acid (EPA), Archangium violaceum 15 S -lipoxygenase (15 S -LOX) or Sorangium cellulosum 15 R -LOX was expressed in Escherichia coli with solvent, polymer, and adsorbent resin, respectively. Results The concentrations of cells and substrate and the types and concentrations of solvent, polymer, and resin were optimized for the biotransformation of EPA into RvE4 (5 S ,15 S -dihydroxyeicosapentaenoic acid) and its enantiomer (5 R ,15 R -dihydroxyeicosapentaenoic acid). Under optimized conditions, A. violaceum 15 S -LOX and S. cellulosum 15 R -LOX expressed in E. coli converted 6.0 mM (1.8 g L −1 ) EPA into 4.3 mM (1.4 g L −1 ) RvE4 and 5.8 mM (1.9 g L −1 ) RvE4 enantiomer in 60 min, with productivities of 4.3 and 5.8 mM h −1 and molar conversions of 72 and 97%, respectively. The concentrations of RvE4 and its enantiomer resulting from the conversion of EPA with solvent, polymer, and resin were 3.1- and 5.3-fold higher than those without additives, respectively. Conclusions The concentrations, productivities, and conversions of RvE4 and its enantiomer were increased by optimizing the concentrations of cells and substrate and the types and concentrations of solvent, polymer, and adsorbent resin. Biotransformation Eicosapentaenoic acid Lipoxygenase Optimization Resolvin E4 Resolvin E4 enantiomer Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Specialized pro-resolving mediators (SPMs) consist of trihydroxy fatty acids (TriHFAs) derived from arachidonic acid (ARA) and dihydroxy fatty acids (DiHFAs) and TriHFAs derived from eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA). However, TriHFA like trioxilins, which are produced from the epoxyhydroxy fatty acids hepoxilins by epoxide hydrolase, do not belong to SPMs. These mediators, present in trace amounts in humans, play a crucial role in resolving damage caused by infection and tissue injury (Serhan 2014 ). SPMs are categorized into lipoxins, maresins, protectins, and resolvins (Rvs) based on the type of starting fatty acid and carbon position and number of hydroxyl groups (An et al. 2021 ). Rvs are synthesized in vivo through a combination of 5-, 12-, and 15-lipoxygenases (LOXs); cyclooxygenase; and cytochrome P450. They are further subdivided into three subclasses: RvE-, RvD-, and RvT-series, which originate from EPA, DHA, and DPA, respectively (Serhan et al. 2022 ). Single-oxygenating LOXs catalyze regio- and stereoselective dioxygenation of C20- and C22-polyunsaturated fatty acids (PUFAs) containing two or three 1,4- Z , Z -pentadienes. This catalysis forms monohydroperoxy fatty acids with a Z , E -conjugated diene, which are subsequently converted into monohydroxy fatty acids by glutathione peroxidase in cells. They can also be readily reduced under physiological conditions or by reducing agents such as tris(2-carboxyethyl)phosphine and cysteine. On the other hand, double-oxygenating LOXs convert PUFAs into DiHFAs containing two Z , E -conjugated dienes through a two-step dioxygenation under reduction conditions (An et al. 2021 ). Resolvin E4 (RvE4), also known as 5 S ,15 S -dihydroxyeicosapentaenoic acid (5 S ,15 S -DiHEPA), has been shown to stimulate macrophage phagocytosis and efferocytosis, which ultimately contributes to the resolution of inflammation (Libreros et al. 2020 ). The RvE4 enantiomer (5 R ,15 R -DiHEPA) also exhibits anti-inflammatory activity (Serhan et al. 2022 ). RvE4 and its enantiomer are derived from EPA through distinct pathways: RvE4 is synthesized by Escherichia coli expressing double-oxygenating ARA 15 S -LOX from Archangium violaceum (Lee et al. 2020 ), while its enantiomer is generated by Escherichia coli expressing double-oxygenating ARA 15 R -LOX from Sorangium cellulosum (Lee et al. 2024 ). Nevertheless, the levels of RvE4 and its enantiomer produced by the recombinant cells were found to be low, indicating that the enhanced production via whole-cell biotransformation is needed to facilitate the industrial-scale synthesis of SPMs. Whole-cell biotransformation can be improved by increasing substrate solubility via the addition of solvents and polymers (Kim et al. 2023 ) and by decreasing the toxicity of the substrate and product at high concentrations via the addition of an adsorbent resin (Kim et al. 2020 ). To improve the biotransformation of EPA into RvE4 and its enantiomer, the addition of solvent, polymer, and resin of optimized concentrations and types is required. In this study, reaction conditions, including the concentrations of cells and substrate, and the concentrations and types of solvent, polymer, and resin, were optimized for the production of RvE4 and its enantiomer from EPA by A. violaceum 15 S -LOX and S . cellulosum 15 R -LOX expressed in E. coli , respectively. Under the optimized reaction conditions, biotransformation of EPA into RvE4 and its enantiomer was enhanced. Materials and Methods Materials EPA and 15 S -hydroxyeicosapentaenoic acid (HEPA) standards were purchased from Sigma-Aldrich and Cayman Chemicals, respectively. 15 R -HEPA, 5 S ,15 S -DiHEPA, 5 R ,15 R -DiHEPA, 14,15-hepoxilin B4 (14,15-HXB4, 13-hydroxy-14,15-epoxyeicosatetraenoic acid), and 13,14,15-trioxilin B4 (13,14,15-TrXB4, 13,14,15-trihydroxyeicosatetraenoic acid) were prepared as described previously (Lee et al. 2020 ). Polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP) were purchased from Sigma-Aldrich. Absorbent resin SP825 was purchased from Ion Technology (Sungnam, Republic of Korea). Bacterial strains, plasmids, gene cloning, and culture conditions The sources of 15 S - and 15 R -LOX genes, host cells, and expression vectors were A. violaceum DSM 52838 and S. cellulosum DSM 14627, E . coli C2566, and pET-28a plasmid, respectively. The genes were cloned for the production of 5 S ,15 S - and 5 R ,15 R -DiHEPAs, as described previously (Lee et al. 2020 ). Recombinant E. coli C2566 was incubated at 37°C in a 2-L baffled flask containing 400 mL of LB medium mixed with 0.1 mM kanamycin at 200 rpm on a shaker. The culture medium was supplemented with 0.1 mM IPTG to induce LOX expression due to the optical density of the culture suspension being 0.7 at 600 nm. Subsequently, additional incubation was conducted at 16°C for 18 h at 160 rpm. The recombinant cells were harvested from the culture broth via centrifugation at 3000 × g and 4°C for 20 min, and the resulting cells were utilized for biotransformation. Solvent and polymer optimization The effects of solvent and polymer types on the biotransformation of EPA as a substrate into RvE4 and its enantiomer by A. violaceum 15 S -LOX and S. cellulosum 15 R -LOX, respectively, expressed in E. coli were investigated using 5% (v/v) solvents, such as dimethyl sulfoxide (DMSO), ethyl acetate, ethanol (EtOH), isopropyl alcohol, and methanol, without polymer, and 5% (w/v) polymers, such as PEG (0.4, 6, 10, 20, and 35 kDa), PVA (89 kDa), and PVP (40 kDa) with 5% (v/v) DMSO or EtOH. The effects of DMSO and EtOH concentrations on RvE4 and its epimer production were investigated using 5% (w/v) PVA and PVP, respectively. Additionally, the effects of PVA and PVP concentrations were assessed using 2.5% (v/v) DMSO and 1% (v/v) EtOH, respectively. The solvent and polymer concentrations varied from 1–10%. The biotransformation of EPA into RvE4 and its enantiomer was performed at 20 or 25°C in a 100-mL baffled flask containing 10 mL of reaction solution at 200 rpm on a shaker in 50 mM HEPPS buffer (pH 8.5 or 8.0) containing 1 g E. coli expressing 15 S -LOX and 15 R -LOX cells L − 1 , respectively, 3 mM EPA, solvent and/or polymer, and 200 mM cysteine as a reducing agent for 60 min. Optimization of cell, substrate, and resin concentrations The optimal ratio of cells to substrate for the biotransformation of EPA into RvE4 or its enantiomer from EPA as a substrate was investigated by varying the cell concentration from 0.5 to 5 g E. coli expressing 15 S -LOX or 15 R -LOX cells L − 1 with 3 mM EPA and the substrate concentration from 1 to 5 mM with 1 g E. coli expressing 15 S -LOX or 15 R -LOX cells L − 1 . The optimal ratio of ARA to SP825 adsorbent resin was determined to be 4:5 (mM per g L − 1 ). The optimal ratios of cells, substrate, and resin for the biotransformation of EPA into RvE4 or its enantiomer by A. violaceum 15 S -LOX or S. cellulosum 15 R -LOX expressed in E. coli were determined to be 1:4:5 (g L − 1 per mM per g L − 1 ) or 1:3:3.75 (g L − 1 per mM per g L − 1 ) by varying the concentrations from 0.75 or 1 g L − 1 , 3 mM, and 3.75 g L − 1 to 1.75 or 2.33 g L − 1 , 7 mM, and 8.75 g L − 1 , respectively. The biotransformation of EPA into RvE4 or its enantiomer was conducted at 20 or 25°C in 50 mM HEPPS buffer (pH 8.5 or 8.0) supplemented with 2.5% (v/v) DMSO or 1% (v/v) EtOH, 2.5% (w/v) PVA or 7.5% (w/v) PVP, and 200 mM cysteine for 60 min, respectively. Biotransformation of EPA with solvent, polymer, and resin into RvE4 and its enantiomer The biotransformation of EPA into RvE4 or its enantiomer was performed at 20 or 25°C in 50 mM HEPPS buffer (pH 8.5 or 8.0) containing 1.5 g E. coli expressing 15 S -LOX cells L − 1 or 2 g E. coli expressing 15 R -LOX cells L − 1 , respectively, 6 mM EPA, 7.5 g SP825 L − 1 , 2.5% (v/v) DMSO or 1% (v/v) EtOH, 2.5% (w/v) PVA or 7.5% (w/v) PVP, and 200 mM cysteine for 90 min. After biotransformation, the reaction solutions were extracted by adding an equal volume of ethyl acetate. The ethyl acetate layer was harvested and dried using a rotary evaporator. The dried residue was dissolved in methanol for HPLC analysis. HPLC analysis All reaction compounds, including EPA, 15 S - and 15 R -HEPAs, 5 S ,15 S - and 5 R ,15 R -DiHEPAs, 14,15-HXB4, and 13,14,15-TrXB4, were analyzed and quantified using HPLC (Agilent) with a Nucleosil C18 column at 202 nm by using a gradient of acetonitrile:water:acetic acid (v/v/v, 50:50:0.1) and acetonitrile:acetic acid (v/v, 100:0.1), as reported previously (An et al. 2018; Lee et al. 2020). RvE4 and its stereoselective enantiomer were separated and analyzed using HPLC with a Lux Amylose-1 column at 234 nm. The gradient used acetonitrile:methanol:water:glacial acetic acid (v/v/v/v) from 27:3:70:0.05 to 63:7:30:0.05, following a method previously reported (Blum et al. 2019). Results and Discussion Optimizing the types and concentrations of solvent and polymer to enhance the biotransformation of EPA into RvE4 and its enantiomer. The inhibitory effect of PUFA as a substrate at high concentrations on LOX activity was reduced by the addition of solvent and polymer, which increased the solubility of PUFA (Lee et al. 2023). The optimal solvent and polymer type for the biotransformation of EPA into RvE4 or its enantiomer by A. violaceum 15 S -LOX or S. cellulosum 15 R -LOX expressed in E. coli were selected using 5% (v/v) solvent without polymer and 5% (w/v) polymer with 5% DMSO or EtOH, respectively. The optimal solvent for biotransformation was DMSO or EtOH (Fig. 1A), whereas the optimal polymer was PVA or PVP, respectively (Fig. 1B). The optimal concentrations of the selected solvent and polymer were determined to maximize the production of RvE4 or its enantiomer. The optimal concentration of DMSO or EtOH was 2.5% or 1% (v/v), respectively (Fig. 2A), whereas that of PVA or PVP were 2.5% or 7.5% (w/v), respectively (Fig. 2B). The concentration of RvE4 (2.1 mM) using 2.5% (v/v) DMSO and 2.5% (w/v) PVA was 1.5-fold higher than that (1.4 mM) without DMSO and PVA. Similarly, the concentration of the RvE4 epimer (2.2 mM) using 1% (v/v) EtOH and 7.5% (w/v) PVP was 2-fold higher than that (1.1 mM) without EtOH and PVP. These results indicate that the solvent and polymer are effective additives for the production of RvE4 and its enantiomer. Optimization of cell, substrate, and resin concentrations to enhance the biotransformation of EPA into RvE4 and its enantiomer The optimization process for the biotransformation of EPA into RvE4 and its enantiomer involved adjusting the ratio of cells to substrate by varying the concentrations of cells and EPA (Supplementary Fig. 1). The cell concentrations were maximal at 1 g L −1 , whereas the production of RvE4 and its enantiomer increased with increasing concentration of EPA; however, the production plateaued above 4 and 3 mM, respectively. The findings suggested that the most effective ratios of cells to substrate for producing RvE4 and its enantiomer were 1:4 and 1:3 (g L −1 per mM), respectively. SP825 was chosen as the absorbent resin for converting EPA into RvE4 and its enantiomer due to its superior binding capacity for ARA compared to other tested resins, including HP20, SP2MG, SP207, SP825, and SP850 (Lee et al. 2024). The optimal ratio of ARA to SP825 was 4:5 (mM per g L −1 ). Therefore, the optimal ratios of cells, EPA, and SP825 for producing RvE4 and its enantiomer were determined 1:4:5 and 1:3:3.75 (g L −1 per mM per g L −1 ), respectively. At these ratios, the optimal concentrations of cells, substrate, and resin for the biotransformation of EPA into RvE4 or its enantiomer at each optimal ratio were varied, and their optimal concentrations for the maximal production of RvE4 or its enantiomer were 1.5 g E. coli expressing A. violaceum 15 S -LOX cells L −1 or 2 g E. coli expressing S. cellulosum 15 R -LOX cells L −1 , 6 mM EPA, and 7.5 g SP825 L −1 , respectively (Fig. 3). The optimal concentration of EPA as a substrate was increased to 6 mM by adding the resin, resulting in an increase in the production of RvE4 and its enantiomer. The resin reduced substrate inhibition above 4 mM EPA for RvE4 and 3 mM for the EPA enantiomer. Biotransformation of EPA with solvent, polymer, and resin into RvE4 and its enantiomer under optimized conditions The optimal reaction conditions for the biotransformation of EPA into RvE4 or its enantiomer included 1.5 g E. coli expressing 15 S -LOX cells L −1 or 2 g E. coli expressing 15 R -LOX cells L −1 , 6 mM EPA, 7.5 g SP825 L −1 , 2.5% (v/v) DMSO or 1% (v/v) EtOH, 2.5% PVA (w/v) or 7.5% PVP (w/v), and 200 mM cysteine, respectively. Under the optimized conditions, A. violaceum 15 S -LOX and S. cellulosum 15 R -LOX expressed in E. coli converted 6 mM (1.8 g L −1 ) EPA into 4.3 mM (1.4 g L −1 ) RvE4 and 5.8 mM (1.9 g L −1 ) RvE4 enantiomer in 60 min, respectively (Fig. 4). The specific and volumetric productivities and conversion for the biotransformation of EPA into RvE4 or its enantiomer were 2.9 mmol h −1 g −1 , 4.3 mM h −1 , and 72% or 2.9 mmol h −1 g −1 , 5.8 mM h −1 ,and 97%, respectively. The concentration, specific and volumetric productivities, and conversion rates for the biotransformation of EPA into RvE4 or its enantiomer in the presence of solvent, polymer, and resin were 3.1-, 1.3-, 2.3-, and 2.0-fold higher or 5.3-, 2.1-, 4.1-, and 2.6-fold higher, respectively, than those in the absence of solvent, polymer, and resin (Supplementary Fig. 2). These results indicate that the solvent, polymer, and resin were effective additives for the production of RvE4 and its enantiomer. Biotransformation of EPA into DiHEPAs by regio- and stereoselective double-oxygenating LOXsexpressed in E. coli The production of C20 and C22 DiHFAs from PUFAs by regio- and stereoselective double-oxygenating LOXs expressed in E. coli has been previously performed without optimization of the three additives solvent, polymer, and resin (Kim et al. 2021; Lee et al. 2020; Oh et al. 2022). In this study, these additives were optimized for the biotransformation of EPA into DiHEPAs such as RvE4 and its enantiomer. The biotransformation of EPA into DiHEPAs by regioselective and stereoselective LOXsexpressed in E. coli is shown in Table 1. The concentration of DiHFAs produced from EPA by LOX expressed in E. coli followed the order: S. cellulosum 15 R -LOX with the three additives (5.8 mM) = S. macrogoltabida 9 S -LOX (5.8) > S. cellulosum 15 R -LOX with resin without solvent and polymer (5.1) > A. violaceum 15 S -LOX with the three additives (4.3) > E. numazuensis 12 S -LOX (1.5) > A. violaceum 15 S -LOX (1.4). The specific productivity of DiHEPA for EPA by recombinant cells followed the order: A. violaceum 15 S -LOX with the three additives (2.9 mmol h −1 g −1 ) = S. cellulosum 15 R -LOX with the three additives (2.9) > Sphingpyxis macrogoltabida 9 S -LOX (2.3) = S. cellulosum 15 R -LOX (2.3) > A. violaceum 15 S -LOX (1.9) > Endozoicomonas numazuensis 12 S -LOX (0.8), whereas the volumetric productivity followed the order: S. macrogoltabida 9 S -LOX (11.6 mM h −1 ) > S. cellulosum 15 R -LOX with the three additives (5.8) > A. violaceum 15 S -LOX with the three additives (4.3) > S. cellulosum 15 R -LOX (3.5) > E. numazuensis 12 S -LOX (1.6) > A. violaceum 15 S -LOX (0.9). The findings suggest that S. cellulosum 15 R -LOX expressed in E. coli with the three additives demonstrated the highest concentration of DiHEPA. Additionally, A. violaceum 15 S -LOX and S. cellulosum 15 R -LOX expressed in E. coli with the three additives exhibited the highest specific productivity of DiHFA from EPA among the reported LOXs expressed in E. coli . In summary, the concentrations of cells and substrate and types and concentrations of solvent, polymer, and resin were optimized for the biotransformation of EPA into RvE4 (5 S ,15 S -DiHEPA) and its enantiomer (5 R ,15 R -DiHEPA) by A. violaceum 15 S -LOX and S. cellulosum 15 R -LOX expressed in E. coli , respectively. The concentration and productivity for producing RvE4 or its enantiomer increased by 3.1- and 2.3-fold or 5.5- and 4.1-fold, respectively, by adding the optimal types and concentrations of solvent, polymer, and resin. Additionally, S. cellulosum 15 R -LOX expressed in E. coli with the three additives exhibited the highest specific productivity and concentration of DiHEPA from EPA among the reported DiHEPAs produced by regio- and stereoselective LOXs expressed in E. coli . These findings hold promise for the industrial production of SPMs. Declarations Funding This study was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry [RS-2024-00398879] of the Ministry of Agriculture, Food, and Rural Affairs, Republic of Korea. Conflict of interest The authors declare they have no conflict of interest. Ethical approval This article does not contain any studies involving human participants or animals. References An JU, Song YS, Kim KR, Ko YJ, Yoon DY, Oh DK (2018) Biotransformation of polyunsaturated fatty acids to bioactive hepoxilins and trioxilins by microbial enzymes. Nat Commun 9:128. https://doi.org/10.1038/s41467-017-02543-8 An JU, Kim SE, Oh DK (2021) Molecular insights into lipoxygenases for biocatalytic synthesis of diverse lipid mediators. Prog Lipid Res 83. https://doi.org/10.1016/j.plipres.2021.101110 Blum M, Dogan I, Karber M, Rothe M, Schunck WH (2019) Chiral lipidomics of monoepoxy and monohydroxy metabolites derived from long-chain polyunsaturated fatty acids. J Lipid Res 60:135-148. https://doi.org/10.1194/jlr.M089755 Kim MJ, Lee J, Kim SE, Shin KC, Oh DK (2023) Production of C20 9 S - and C22 11 S -hydroxy fatty acids by cells expressing Shewanella hanedai arachidonate 9 S -lipoxygenase. Appl Microbiol Biotechnol 107:247-260. https://doi.org/10.1007/s00253-022-12285-3 Kim TH, Kang SH, Han JE, Seo EJ, Jeon EY, Choi GE, Park JB, Oh DK (2020) Multilayer engineering of enzyme cascade catalysis for one-pot preparation of nylon monomers from renewable fatty acids. 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Bioresour Technol 388:129750. https://doi.org/10.1016/j.biortech.2023.129750 Lee J, Ko YJ, Park JB, Oh DK (2024) Biotransformation of C20- and C22-polyunsaturated fatty acids and fish oil hydrolyzates to R , R -dihydroxy fatty acids as lipid mediators by double-oxygenating 15 R -lipoxygenase. Green Chem. https://doi.org/10.1039/D4GC00308J Libreros S, Shay AE, Nshimiyimana R, Fichtner D, Martin MJ, Wourms N, Serhan CN (2020) A new E-series resolvin: RvE4 stereochemistry and function in efferocytosis of inflammation-resolution. Front Immunol 11:631319. https://doi.org/10.3389/fimmu.2020.631319 Oh CW, Kim SE, Lee J, Oh DK (2022) Bioconversion of C20- and C22-polyunsaturated fatty acids into 9 S ,15 S - and 11 S ,17 S -dihydroxy fatty acids by Escherichia coli expressing double-oxygenating 9 S -lipoxygenase from Sphingopyxis macrogoltabida . 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Biotransformation of EPA into DiHEPAs by regio- and stereoselective double-oxygenating LOXs expressed in E. coli LOX expressed in E. coli Substrate (mM) Product (mM) Specific productivity (mmol h −1 g −1 ) Volumetric productivity (mM h −1 ) Molar conversion (%) Reference A. violaceum 15 S -LOX EPA (3 mM) a RvE4 (1.4 mM) 1.4 1.4 47 This study (Lee et al. 2024) EPA (6 mM) b RvE4 (4.3 mM) 2.9 4.3 72 S. cellulosum 15 R -LOX EPA (3 mM) a RvE4 enantiomer (1.1 mM) 1.1 1.1 36 EPA (6 mM) b RvE4 enantiomer (5.8 mM) 2.9 5.8 97 S. macrogoltabida 9 S -LOX EPA (6 mM) c 9 S ,15 S -DiHEPA (5.8 mM) 2.3 11.6 97 (Oh et al. 2022) E. numazuensis 12 S -LOX EPA (3 mM) a 5 S ,12 S -DiHEPA (1.5 mM) 0.8 1.5 53 (Kim et al. 2021) A. violaceum 15 S -LOX EPA (1 mM) a RvE4 (0.4 mM) 1.9 0.9 48 (Lee et al. 2020) S. cellulosum 15 R -LOX EPA (6 mM) d RvE4 enantiomer (5.1 mM) 2.3 3.4 87 (Lee et al. 2024) RvE4. 5 S ,15 S -DiHEPA; Enantiomer of RvE4, 5 R ,15 R -DiHEPA. Specific productivity is defined as the initial production rate within a linear range. Volumetric productivity is defined as the maximum concentration per unit time. a Biotransformation without solvent, polymer, and resin. b Biotransformation with solvent, polymer, and resin. c Biotransformation with solvent and without polymer and resin. d Biotransformation without solvent and polymer and with resin. Supplementary Files RvE4SI.docx Supporting information Supplementary Fig. 1—Optimization of cell and substrate concentrations for the biotransformation of EPA into RvE4 and its enantiomer by A. violaceum 15 S -LOX and S. cellulosum 15 R -LOX expressed in E. coli , respectively. Supplementary Fig. 2—Whole-cell biotransformation of EPA into RvE4 and its enantiomer by A. violaceum 15 S -LOX and S. cellulosum 15 R -LOX, respectively, expressed in E. coli in the absence of solvent, polymer, and adsorbent resin. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4121438","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289385907,"identity":"15628d70-dcd3-40bd-b951-532a9f66ce67","order_by":0,"name":"Jin Lee","email":"","orcid":"","institution":"Konkuk University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Lee","suffix":""},{"id":289385908,"identity":"ccde8591-8e2e-4f82-a2d4-466b4199b5f5","order_by":1,"name":"Hyun-Ah Park","email":"","orcid":"","institution":"Konkuk University","correspondingAuthor":false,"prefix":"","firstName":"Hyun-Ah","middleName":"","lastName":"Park","suffix":""},{"id":289385909,"identity":"d3f95c9b-52dc-4832-bf74-5dab7fffd630","order_by":2,"name":"Kyung-Chul Shin","email":"","orcid":"","institution":"Hankuk University of Foreign Studies - Yongin Campus: Hankuk University of Foreign Studies - Global Campus","correspondingAuthor":false,"prefix":"","firstName":"Kyung-Chul","middleName":"","lastName":"Shin","suffix":""},{"id":289385910,"identity":"712ef558-c4e3-4057-a6bc-36ef7438ccd2","order_by":3,"name":"Deok-Kun Oh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDCCAwzMjA0MDHISDAzMIB4QsBGnxZh0LYkziNbCd7z5sOHMtjvpM9t7DxvznGGQ529gS/uAT4vkmWPJiRvbnuXO5jmXnMxzg8FwxgG2wzPwaTG4kWN88GHb4dx5EjnGh3k+MDBuYGBvxuswg/tvwFrS5aBa7AlrucFjDHTY4QRpoBaQwxI3MLAdxqtF8kxasuGMc8Ag6DljbDjnjETyjMNsyXi18B0/fFiyp+ywvMTxHmOJN8dsbPvb24zxakEHkBgdBaNgFIyCUUAhAAALrkv/JWLojgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6886-7589","institution":"Konkuk University","correspondingAuthor":true,"prefix":"","firstName":"Deok-Kun","middleName":"","lastName":"Oh","suffix":""}],"badges":[],"createdAt":"2024-03-18 08:38:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4121438/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4121438/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54583111,"identity":"8262e164-3ee9-4b41-8d24-02ace5ba9571","added_by":"auto","created_at":"2024-04-12 15:05:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64541,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of solvents and polymers on the biotransformation of EPA into RvE4 and its enantiomer by \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX\u003cem\u003e \u003c/em\u003eexpressed in \u003cem\u003eE. coli\u003c/em\u003e, respectively. (A) Effect of solvent type on the production of RvE4 and its enantiomer. (B) Effect of polymer type on the production of RvE4 and its enantiomer. The dark and gray bars indicate RvE4 and its enantiomer, respectively. Error bars indicate standard deviations for the average of three separate experimental data.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4121438/v1/d8ba6c46e0053150df7ba8ee.jpg"},{"id":54583104,"identity":"82b783a6-85cc-42d1-aaff-9fbd6cb6cc2a","added_by":"auto","created_at":"2024-04-12 15:05:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59900,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of concentrations of the solvents DMSO and EtOH and polymers PVA and PVP for the maximal biotransformation of EPA into RvE4 and its enantiomer by \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOXexpressed in \u003cem\u003eE. coli\u003c/em\u003e, respectively. (A) Optimization of DMSO and EtOH concentrations on the maximal production of RvE4 and its enantiomer, respectively. (B) Optimization of PVA and PVP concentrations on the maximal production of RvE4 enantiomer and its enantiomer, respectively. Error bars indicate standard deviations for the average of three separate experimental data.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4121438/v1/4641cb50757e3fa3bca2aa6f.jpg"},{"id":54583101,"identity":"a438671b-91bd-43e5-a44a-2615f369d39d","added_by":"auto","created_at":"2024-04-12 15:05:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":70315,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of cell, substrate, and resin concentrations at optimal ratios for the maximal biotransformation of EPA into RvE4 and its enantiomer by \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e, respectively. (A) Optimization of cell, substrate, and resin concentrations at optimal ratios for the maximal biotransformation of EPA into RvE4 by\u003cem\u003e A. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e. The optimal ratio of cells, substrate, and resin was 1:4:5 (g L\u003csup\u003e−1\u003c/sup\u003e per mM per g L\u003csup\u003e−1\u003c/sup\u003e). (B) Optimization of cell, substrate, and resin concentrations at optimal ratios for the maximal biotransformation of EPA into RvE4 enantiomer at optimal ratios by using\u003cem\u003e S. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e. The optimal ratio of cells, substrate, and resin was 1:3:3.75 (g L\u003csup\u003e−1\u003c/sup\u003e per mM per g L\u003csup\u003e−1\u003c/sup\u003e). Error bars indicate standard deviations for the average of three separate experimental data.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4121438/v1/2f39b0a233aaf604b28778cf.jpg"},{"id":54583110,"identity":"144cefc3-6954-4d3a-94e4-153e6f73c8cb","added_by":"auto","created_at":"2024-04-12 15:05:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":80778,"visible":true,"origin":"","legend":"\u003cp\u003eTime-course reactions for\u003cstrong\u003e \u003c/strong\u003ethe biotransformation of eicosapentaenoic acid (EPA) into resolvin E4 (RvE4) and its enantiomer in the presence of optimized solvent, polymer, and resin by \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOXexpressed in \u003cem\u003eE. coli\u003c/em\u003e, respectively. (A) Time-course reactions for the biotransformation of EPA into RvE4 in the presence of optimized solvent, polymer, and resin. (B) Biotransformation of EPA into RvE4 enantiomer in the presence of optimized solvent, polymer, and resin. Error bars indicate standard deviations for the average of three separate experimental data.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4121438/v1/6d6cb5bcf6abb6a4437e48ee.jpg"},{"id":57993255,"identity":"6d22bd46-7555-49ed-afb9-a486d05fc0ac","added_by":"auto","created_at":"2024-06-09 07:00:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":755050,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4121438/v1/1ea1d948-2abc-4c91-afd7-5b046ef79fcc.pdf"},{"id":54583107,"identity":"51eb1fbd-e07d-4cad-a9da-f3f35943cb08","added_by":"auto","created_at":"2024-04-12 15:05:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":103435,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting information\u003c/p\u003e\n\u003cp\u003eSupplementary Fig. 1—Optimization of cell and substrate concentrations for the biotransformation of EPA into RvE4 and its enantiomer by \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e, respectively.\u003c/p\u003e\n\u003cp\u003eSupplementary Fig. 2—Whole-cell biotransformation of EPA into RvE4 and its enantiomer by \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS. cellulosum\u003c/em\u003e15\u003cem\u003eR\u003c/em\u003e-LOX, respectively, expressed in \u003cem\u003eE. coli\u003c/em\u003e in the absence of solvent, polymer, and adsorbent resin.\u003c/p\u003e","description":"","filename":"RvE4SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-4121438/v1/6bdc8422a46efb8f67a13bc3.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cstrong\u003eBiotransformation of eicosapentaenoic acid into the dihydroxyeicosapentaenoic acids resolvin E4 and its enantiomer by 15\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e- and 15\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-lipoxygenases\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpressed in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia coli\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpecialized pro-resolving mediators (SPMs) consist of trihydroxy fatty acids (TriHFAs) derived from arachidonic acid (ARA) and dihydroxy fatty acids (DiHFAs) and TriHFAs derived from eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA). However, TriHFA like trioxilins, which are produced from the epoxyhydroxy fatty acids hepoxilins by epoxide hydrolase, do not belong to SPMs. These mediators, present in trace amounts in humans, play a crucial role in resolving damage caused by infection and tissue injury (Serhan \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). SPMs are categorized into lipoxins, maresins, protectins, and resolvins (Rvs) based on the type of starting fatty acid and carbon position and number of hydroxyl groups (An et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Rvs are synthesized \u003cem\u003ein vivo\u003c/em\u003e through a combination of 5-, 12-, and 15-lipoxygenases (LOXs); cyclooxygenase; and cytochrome P450. They are further subdivided into three subclasses: RvE-, RvD-, and RvT-series, which originate from EPA, DHA, and DPA, respectively (Serhan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSingle-oxygenating LOXs catalyze regio- and stereoselective dioxygenation of C20- and C22-polyunsaturated fatty acids (PUFAs) containing two or three 1,4-\u003cem\u003eZ\u003c/em\u003e,\u003cem\u003eZ\u003c/em\u003e-pentadienes. This catalysis forms monohydroperoxy fatty acids with a \u003cem\u003eZ\u003c/em\u003e,\u003cem\u003eE\u003c/em\u003e-conjugated diene, which are subsequently converted into monohydroxy fatty acids by glutathione peroxidase in cells. They can also be readily reduced under physiological conditions or by reducing agents such as tris(2-carboxyethyl)phosphine and cysteine. On the other hand, double-oxygenating LOXs convert PUFAs into DiHFAs containing two \u003cem\u003eZ\u003c/em\u003e,\u003cem\u003eE\u003c/em\u003e-conjugated dienes through a two-step dioxygenation under reduction conditions (An et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResolvin E4 (RvE4), also known as 5\u003cem\u003eS\u003c/em\u003e,15\u003cem\u003eS\u003c/em\u003e-dihydroxyeicosapentaenoic acid (5\u003cem\u003eS\u003c/em\u003e,15\u003cem\u003eS\u003c/em\u003e-DiHEPA), has been shown to stimulate macrophage phagocytosis and efferocytosis, which ultimately contributes to the resolution of inflammation (Libreros et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The RvE4 enantiomer (5\u003cem\u003eR\u003c/em\u003e,15\u003cem\u003eR\u003c/em\u003e-DiHEPA) also exhibits anti-inflammatory activity (Serhan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). RvE4 and its enantiomer are derived from EPA through distinct pathways: RvE4 is synthesized by \u003cem\u003eEscherichia coli\u003c/em\u003e expressing double-oxygenating ARA 15\u003cem\u003eS\u003c/em\u003e-LOX from \u003cem\u003eArchangium violaceum\u003c/em\u003e (Lee et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), while its enantiomer is generated by \u003cem\u003eEscherichia coli\u003c/em\u003e expressing double-oxygenating ARA 15\u003cem\u003eR\u003c/em\u003e-LOX from \u003cem\u003eSorangium cellulosum\u003c/em\u003e (Lee et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nevertheless, the levels of RvE4 and its enantiomer produced by the recombinant cells were found to be low, indicating that the enhanced production via whole-cell biotransformation is needed to facilitate the industrial-scale synthesis of SPMs. Whole-cell biotransformation can be improved by increasing substrate solubility via the addition of solvents and polymers (Kim et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and by decreasing the toxicity of the substrate and product at high concentrations via the addition of an adsorbent resin (Kim et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To improve the biotransformation of EPA into RvE4 and its enantiomer, the addition of solvent, polymer, and resin of optimized concentrations and types is required.\u003c/p\u003e \u003cp\u003eIn this study, reaction conditions, including the concentrations of cells and substrate, and the concentrations and types of solvent, polymer, and resin, were optimized for the production of RvE4 and its enantiomer from EPA by \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e, respectively. Under the optimized reaction conditions, biotransformation of EPA into RvE4 and its enantiomer was enhanced.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eMaterials\u003c/p\u003e \u003cp\u003eEPA and 15\u003cem\u003eS\u003c/em\u003e-hydroxyeicosapentaenoic acid (HEPA) standards were purchased from Sigma-Aldrich and Cayman Chemicals, respectively. 15\u003cem\u003eR\u003c/em\u003e-HEPA, 5\u003cem\u003eS\u003c/em\u003e,15\u003cem\u003eS\u003c/em\u003e-DiHEPA, 5\u003cem\u003eR\u003c/em\u003e,15\u003cem\u003eR\u003c/em\u003e-DiHEPA, 14,15-hepoxilin B4 (14,15-HXB4, 13-hydroxy-14,15-epoxyeicosatetraenoic acid), and 13,14,15-trioxilin B4 (13,14,15-TrXB4, 13,14,15-trihydroxyeicosatetraenoic acid) were prepared as described previously (Lee et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP) were purchased from Sigma-Aldrich. Absorbent resin SP825 was purchased from Ion Technology (Sungnam, Republic of Korea).\u003c/p\u003e \u003cp\u003eBacterial strains, plasmids, gene cloning, and culture conditions\u003c/p\u003e \u003cp\u003eThe sources of 15\u003cem\u003eS\u003c/em\u003e- and 15\u003cem\u003eR\u003c/em\u003e-LOX genes, host cells, and expression vectors were \u003cem\u003eA. violaceum\u003c/em\u003e DSM 52838 and \u003cem\u003eS. cellulosum\u003c/em\u003e DSM 14627, \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e C2566, and pET-28a plasmid, respectively. The genes were cloned for the production of 5\u003cem\u003eS\u003c/em\u003e,15\u003cem\u003eS\u003c/em\u003e- and 5\u003cem\u003eR\u003c/em\u003e,15\u003cem\u003eR\u003c/em\u003e-DiHEPAs, as described previously (Lee et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Recombinant \u003cem\u003eE. coli\u003c/em\u003e C2566 was incubated at 37\u0026deg;C in a 2-L baffled flask containing 400 mL of LB medium mixed with 0.1 mM kanamycin at 200 rpm on a shaker. The culture medium was supplemented with 0.1 mM IPTG to induce LOX expression due to the optical density of the culture suspension being 0.7 at 600 nm. Subsequently, additional incubation was conducted at 16\u0026deg;C for 18 h at 160 rpm. The recombinant cells were harvested from the culture broth via centrifugation at 3000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 20 min, and the resulting cells were utilized for biotransformation.\u003c/p\u003e \u003cp\u003eSolvent and polymer optimization\u003c/p\u003e \u003cp\u003eThe effects of solvent and polymer types on the biotransformation of EPA as a substrate into RvE4 and its enantiomer by \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX, respectively, expressed in \u003cem\u003eE. coli\u003c/em\u003e were investigated using 5% (v/v) solvents, such as dimethyl sulfoxide (DMSO), ethyl acetate, ethanol (EtOH), isopropyl alcohol, and methanol, without polymer, and 5% (w/v) polymers, such as PEG (0.4, 6, 10, 20, and 35 kDa), PVA (89 kDa), and PVP (40 kDa) with 5% (v/v) DMSO or EtOH. The effects of DMSO and EtOH concentrations on RvE4 and its epimer production were investigated using 5% (w/v) PVA and PVP, respectively. Additionally, the effects of PVA and PVP concentrations were assessed using 2.5% (v/v) DMSO and 1% (v/v) EtOH, respectively. The solvent and polymer concentrations varied from 1\u0026ndash;10%. The biotransformation of EPA into RvE4 and its enantiomer was performed at 20 or 25\u0026deg;C in a 100-mL baffled flask containing 10 mL of reaction solution at 200 rpm on a shaker in 50 mM HEPPS buffer (pH 8.5 or 8.0) containing 1 g \u003cem\u003eE. coli\u003c/em\u003e expressing 15\u003cem\u003eS\u003c/em\u003e-LOX and 15\u003cem\u003eR\u003c/em\u003e-LOX cells L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, 3 mM EPA, solvent and/or polymer, and 200 mM cysteine as a reducing agent for 60 min.\u003c/p\u003e \u003cp\u003eOptimization of cell, substrate, and resin concentrations\u003c/p\u003e \u003cp\u003eThe optimal ratio of cells to substrate for the biotransformation of EPA into RvE4 or its enantiomer from EPA as a substrate was investigated by varying the cell concentration from 0.5 to 5 g \u003cem\u003eE. coli\u003c/em\u003e expressing 15\u003cem\u003eS\u003c/em\u003e-LOX or 15\u003cem\u003eR\u003c/em\u003e-LOX cells L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with 3 mM EPA and the substrate concentration from 1 to 5 mM with 1 g \u003cem\u003eE. coli\u003c/em\u003e expressing 15\u003cem\u003eS\u003c/em\u003e-LOX or 15\u003cem\u003eR\u003c/em\u003e-LOX cells L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The optimal ratio of ARA to SP825 adsorbent resin was determined to be 4:5 (mM per g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The optimal ratios of cells, substrate, and resin for the biotransformation of EPA into RvE4 or its enantiomer by \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX or \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e were determined to be 1:4:5 (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e per mM per g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) or 1:3:3.75 (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e per mM per g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) by varying the concentrations from 0.75 or 1 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 3 mM, and 3.75 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1.75 or 2.33 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 7 mM, and 8.75 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The biotransformation of EPA into RvE4 or its enantiomer was conducted at 20 or 25\u0026deg;C in 50 mM HEPPS buffer (pH 8.5 or 8.0) supplemented with 2.5% (v/v) DMSO or 1% (v/v) EtOH, 2.5% (w/v) PVA or 7.5% (w/v) PVP, and 200 mM cysteine for 60 min, respectively.\u003c/p\u003e \u003cp\u003eBiotransformation of EPA with solvent, polymer, and resin into RvE4 and its enantiomer\u003c/p\u003e \u003cp\u003eThe biotransformation of EPA into RvE4 or its enantiomer was performed at 20 or 25\u0026deg;C in 50 mM HEPPS buffer (pH 8.5 or 8.0) containing 1.5 g \u003cem\u003eE. coli\u003c/em\u003e expressing 15\u003cem\u003eS\u003c/em\u003e-LOX cells L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e or 2 g \u003cem\u003eE. coli\u003c/em\u003e expressing 15\u003cem\u003eR\u003c/em\u003e-LOX cells L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, 6 mM EPA, 7.5 g SP825 L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2.5% (v/v) DMSO or 1% (v/v) EtOH, 2.5% (w/v) PVA or 7.5% (w/v) PVP, and 200 mM cysteine for 90 min. After biotransformation, the reaction solutions were extracted by adding an equal volume of ethyl acetate. The ethyl acetate layer was harvested and dried using a rotary evaporator. The dried residue was dissolved in methanol for HPLC analysis.\u003c/p\u003e \u003cp\u003eHPLC analysis\u003c/p\u003e \u003cp\u003eAll reaction compounds, including EPA,\u0026nbsp;15\u003cem\u003eS\u003c/em\u003e- and 15\u003cem\u003eR\u003c/em\u003e-HEPAs,\u0026nbsp;5\u003cem\u003eS\u003c/em\u003e,15\u003cem\u003eS\u003c/em\u003e- and 5\u003cem\u003eR\u003c/em\u003e,15\u003cem\u003eR\u003c/em\u003e-DiHEPAs, 14,15-HXB4, and 13,14,15-TrXB4, were analyzed and quantified using HPLC (Agilent) with a Nucleosil C18 column at 202 nm by using a gradient of acetonitrile:water:acetic acid (v/v/v, 50:50:0.1) and acetonitrile:acetic acid (v/v, 100:0.1), as reported previously (An et al. 2018; Lee et al. 2020). RvE4 and its stereoselective enantiomer were separated and analyzed using HPLC with a Lux Amylose-1 column at 234 nm. The gradient used acetonitrile:methanol:water:glacial acetic acid (v/v/v/v) from 27:3:70:0.05 to 63:7:30:0.05, following a method previously reported (Blum et al. 2019).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eOptimizing the types and concentrations of solvent and polymer to enhance the biotransformation of EPA into RvE4 and its enantiomer.\u003c/p\u003e\n\u003cp\u003eThe inhibitory effect of PUFA as a substrate at high concentrations on LOX activity was reduced by the addition of solvent and polymer, which increased the solubility of PUFA (Lee et al. 2023). The optimal solvent and polymer type for the biotransformation of EPA into RvE4 or its enantiomer by \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX or \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e were selected using 5% (v/v) solvent without polymer and 5% (w/v) polymer with 5% DMSO or EtOH, respectively. The optimal solvent for biotransformation was DMSO or EtOH (Fig. 1A), whereas the optimal polymer was PVA or PVP, respectively (Fig. 1B). The optimal concentrations of the selected solvent and polymer were determined to maximize the production of RvE4 or its enantiomer. The optimal concentration of DMSO or EtOH was 2.5% or 1% (v/v), respectively (Fig. 2A), whereas that of PVA or PVP were 2.5% or 7.5% (w/v), respectively (Fig. 2B). The concentration of RvE4 (2.1 mM) using 2.5% (v/v) DMSO and 2.5% (w/v) PVA was 1.5-fold higher than that (1.4 mM) without DMSO and PVA. Similarly, the concentration of the RvE4 epimer (2.2 mM) using 1% (v/v) EtOH and 7.5% (w/v) PVP was 2-fold higher than that (1.1 mM) without EtOH and PVP. These results indicate that the solvent and polymer are effective additives for the production of RvE4 and its enantiomer.\u003c/p\u003e\n\u003cp\u003eOptimization of cell, substrate, and resin concentrations to enhance the biotransformation of EPA into RvE4 and its enantiomer\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe optimization process for the\u0026nbsp;biotransformation of EPA into RvE4 and its enantiomer involved adjusting the\u0026nbsp;ratio of cells to substrate by varying the concentrations of cells and EPA\u0026nbsp;(Supplementary Fig. 1). The cell concentrations were maximal at\u0026nbsp;1 g L\u003csup\u003e−1\u003c/sup\u003e, whereas the production of\u0026nbsp;RvE4 and its enantiomer increased with increasing concentration of EPA; however, the production plateaued above 4 and 3 mM, respectively. The findings suggested that\u0026nbsp;the most effective ratios of\u0026nbsp;cells\u0026nbsp;to substrate for\u0026nbsp;producing\u0026nbsp;RvE4 and its enantiomer were 1:4 and\u0026nbsp;1:3 (g L\u003csup\u003e−1\u003c/sup\u003e per mM), respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSP825 was chosen as the absorbent resin for converting EPA into RvE4 and its enantiomer due to its superior binding capacity for ARA compared to other tested resins, including HP20, SP2MG, SP207, SP825, and SP850 (Lee et al. 2024). The optimal ratio of ARA to SP825 was 4:5 (mM per g L\u003csup\u003e−1\u003c/sup\u003e). Therefore, the optimal\u0026nbsp;ratios of\u0026nbsp;cells, EPA, and\u0026nbsp;SP825\u0026nbsp;for\u0026nbsp;producing\u0026nbsp;RvE4 and its enantiomer were determined 1:4:5 and\u0026nbsp;1:3:3.75 (g L\u003csup\u003e−1\u003c/sup\u003e per mM per g L\u003csup\u003e−1\u003c/sup\u003e), respectively. At these ratios, the optimal concentrations of cells, substrate, and resin for the biotransformation of EPA into RvE4 or its enantiomer at each optimal ratio were varied, and their optimal concentrations for the maximal production of RvE4 or its enantiomer were 1.5 g \u003cem\u003eE. coli\u003c/em\u003e expressing \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX cells L\u003csup\u003e−1\u003c/sup\u003e or 2 g\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e expressing \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX cells\u0026nbsp;L\u003csup\u003e−1\u003c/sup\u003e, 6 mM\u0026nbsp;EPA,\u0026nbsp;and 7.5 g\u0026nbsp;SP825\u0026nbsp;L\u003csup\u003e−1\u003c/sup\u003e, respectively (Fig. 3). The optimal concentration of EPA as a substrate was increased to 6 mM by adding the resin, resulting in an increase in the production of RvE4 and its enantiomer. The resin reduced substrate inhibition above 4 mM EPA for RvE4 and 3 mM for the EPA enantiomer.\u003c/p\u003e\n\u003cp\u003eBiotransformation of EPA with solvent, polymer, and resin into RvE4 and its enantiomer under optimized conditions\u003c/p\u003e\n\u003cp\u003eThe optimal reaction conditions for the biotransformation of EPA into RvE4 or its enantiomer included 1.5 g \u003cem\u003eE. coli\u003c/em\u003e expressing 15\u003cem\u003eS\u003c/em\u003e-LOX cells\u0026nbsp;L\u003csup\u003e−1\u003c/sup\u003e or 2 g \u003cem\u003eE. coli\u003c/em\u003e expressing 15\u003cem\u003eR\u003c/em\u003e-LOX cells\u0026nbsp;L\u003csup\u003e−1\u003c/sup\u003e,\u0026nbsp;6 mM EPA, 7.5 g SP825\u0026nbsp;L\u003csup\u003e−1\u003c/sup\u003e, 2.5% (v/v) DMSO or 1% (v/v) EtOH, 2.5% PVA (w/v) or 7.5% PVP (w/v), and 200 mM cysteine, respectively. Under the optimized conditions, \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e converted 6 mM (1.8 g L\u003csup\u003e−1\u003c/sup\u003e)\u0026nbsp;EPA into 4.3 mM (1.4 g L\u003csup\u003e−1\u003c/sup\u003e)\u0026nbsp;RvE4 and 5.8 mM (1.9 g L\u003csup\u003e−1\u003c/sup\u003e) RvE4 enantiomer in 60 min, respectively (Fig. 4). The specific and volumetric productivities and conversion for the biotransformation of EPA into\u0026nbsp;RvE4 or its enantiomer\u0026nbsp;were 2.9 mmol h\u003csup\u003e−1\u003c/sup\u003e g\u003csup\u003e−1\u003c/sup\u003e, 4.3 mM h\u003csup\u003e−1\u003c/sup\u003e, and 72% or 2.9\u0026nbsp;mmol h\u003csup\u003e−1\u003c/sup\u003e g\u003csup\u003e−1\u003c/sup\u003e,\u0026nbsp;5.8 mM h\u003csup\u003e−1\u003c/sup\u003e,and 97%, respectively. The concentration, specific and volumetric productivities, and conversion rates for the biotransformation of EPA into RvE4 or its enantiomer in the presence of solvent, polymer, and resin were 3.1-, 1.3-, 2.3-, and 2.0-fold higher or 5.3-, 2.1-, 4.1-, and 2.6-fold higher, respectively, than those in the absence of solvent, polymer, and resin (Supplementary Fig. 2). These results indicate that the solvent, polymer, and \u0026nbsp;resin were effective additives for the production of RvE4 and its enantiomer.\u003c/p\u003e\n\u003cp\u003eBiotransformation of EPA into DiHEPAs by\u0026nbsp;regio- and stereoselective\u0026nbsp;double-oxygenating\u0026nbsp;LOXsexpressed in \u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe production of C20 and C22 DiHFAs from PUFAs by regio- and stereoselective double-oxygenating LOXs expressed in \u003cem\u003eE. coli\u003c/em\u003e has been previously performed without optimization of the three additives solvent, polymer, and resin (Kim et al. 2021; Lee et al. 2020; Oh et al. 2022). In this study, these additives were optimized for the biotransformation of EPA into DiHEPAs such as RvE4 and its enantiomer. The biotransformation of EPA into DiHEPAs by regioselective and stereoselective LOXsexpressed in\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e is shown in Table 1. The concentration of DiHFAs produced from EPA by LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e followed the order: \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX with the three additives (5.8 mM) = \u003cem\u003eS. macrogoltabida\u003c/em\u003e 9\u003cem\u003eS\u003c/em\u003e-LOX (5.8) \u0026gt; \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX with resin without solvent and polymer (5.1) \u0026gt; \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX with the three additives (4.3) \u0026gt; \u003cem\u003eE. numazuensis\u003c/em\u003e 12\u003cem\u003eS\u003c/em\u003e-LOX (1.5) \u0026gt; \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX (1.4). The specific productivity of DiHEPA for EPA by recombinant cells followed the order:\u003cem\u003e\u0026nbsp;A. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX with the three additives (2.9 mmol h\u003csup\u003e−1\u003c/sup\u003e g\u003csup\u003e−1\u003c/sup\u003e) = \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX with the three additives (2.9) \u0026gt; \u003cem\u003eSphingpyxis macrogoltabida\u003c/em\u003e 9\u003cem\u003eS\u003c/em\u003e-LOX (2.3) =\u003cem\u003e\u0026nbsp;S. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX (2.3) \u0026gt; \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX (1.9) \u0026gt; \u003cem\u003eEndozoicomonas numazuensis\u003c/em\u003e 12\u003cem\u003eS\u003c/em\u003e-LOX (0.8), whereas the volumetric productivity followed the order: \u003cem\u003eS. macrogoltabida\u003c/em\u003e 9\u003cem\u003eS\u003c/em\u003e-LOX (11.6 mM h\u003csup\u003e−1\u003c/sup\u003e) \u0026gt; \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX with the three additives (5.8) \u0026gt; \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX with the three additives (4.3) \u0026gt; \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX (3.5) \u0026gt; \u003cem\u003eE. numazuensis\u003c/em\u003e 12\u003cem\u003eS\u003c/em\u003e-LOX (1.6) \u0026gt; \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX (0.9). The findings suggest that \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e with the three additives demonstrated the highest concentration of DiHEPA. Additionally, \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e with the three additives exhibited the highest specific productivity of DiHFA from EPA among the reported LOXs expressed in \u003cem\u003eE. coli\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In summary, the concentrations of cells and substrate and types and concentrations of solvent, polymer, and resin were optimized for the biotransformation of EPA into RvE4 (5\u003cem\u003eS\u003c/em\u003e,15\u003cem\u003eS\u003c/em\u003e-DiHEPA) and its enantiomer (5\u003cem\u003eR\u003c/em\u003e,15\u003cem\u003eR\u003c/em\u003e-DiHEPA) by \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e, respectively. The concentration and productivity for producing RvE4 or its enantiomer increased by 3.1- and 2.3-fold or 5.5- and 4.1-fold, respectively, by adding the optimal types and concentrations of solvent, polymer, and resin. Additionally, \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e with the three additives exhibited the highest specific productivity and concentration of DiHEPA from EPA among the reported DiHEPAs produced by regio- and stereoselective LOXs expressed in \u003cem\u003eE. coli\u003c/em\u003e. These findings hold promise for the industrial production of SPMs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Korea Institute of Planning and Evaluation for Technology\u0026nbsp;in Food, Agriculture, and Forestry [RS-2024-00398879] of the Ministry of Agriculture, Food, and Rural Affairs, Republic of Korea.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies involving human participants or animals.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAn JU, Song YS, Kim KR, Ko YJ, Yoon DY, Oh DK (2018) Biotransformation of polyunsaturated fatty acids to bioactive hepoxilins and trioxilins by microbial enzymes. 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Appl Microbiol Biotechnol 107:247-260. https://doi.org/10.1007/s00253-022-12285-3\u003c/li\u003e\n\u003cli\u003eKim TH, Kang SH, Han JE, Seo EJ, Jeon EY, Choi GE, Park JB, Oh DK (2020) Multilayer engineering of enzyme cascade catalysis for one-pot preparation of nylon monomers from renewable fatty acids. ACS Cat 10:4871-4878. https://doi.org/10.1021/acscatal.9b05426\u003c/li\u003e\n\u003cli\u003eKim TH, Lee J, Kim SE, Oh DK (2021) Biocatalytic synthesis of dihydroxy fatty acids as lipid mediators from polyunsaturated fatty acids by double dioxygenation of the microbial 12\u003cem\u003eS\u003c/em\u003e-lipoxygenase. Biotechnol Bioeng 118:3094-3104. https://doi.org/10.1002/bit.27820\u003c/li\u003e\n\u003cli\u003eLee J, An JU, Kim TH, Ko YJ, Park JB, Oh DK (2020) Discovery and engineering of a microbial double-oxygenating lipoxygenase for synthesis of dihydroxy fatty acids as specialized proresolving mediators. ACS Sustain Chem Eng 8:16172-16183. https://doi.org/10.1021/acssuschemeng.0c04793\u003c/li\u003e\n\u003cli\u003eLee J, Park HA, Shin KC, Park JB, Oh DK (2023) Efficient biotransformation of docosahexaenoic acid-rich oils into the lipid mediator resolvin D5 by cells expressing 15\u003cem\u003eS\u003c/em\u003e-lipoxygenase using a bioreactor. Bioresour Technol 388:129750. https://doi.org/10.1016/j.biortech.2023.129750\u003c/li\u003e\n\u003cli\u003eLee J, Ko YJ, Park JB, Oh DK (2024) Biotransformation of C20- and C22-polyunsaturated fatty acids and fish oil hydrolyzates to \u003cem\u003eR\u003c/em\u003e,\u003cem\u003eR\u003c/em\u003e-dihydroxy fatty acids as lipid mediators by double-oxygenating 15\u003cem\u003eR\u003c/em\u003e-lipoxygenase. Green Chem. https://doi.org/10.1039/D4GC00308J\u003c/li\u003e\n\u003cli\u003eLibreros S, Shay AE, Nshimiyimana R, Fichtner D, Martin MJ, Wourms N, Serhan CN (2020) A new E-series resolvin: RvE4 stereochemistry and function in efferocytosis of inflammation-resolution. Front Immunol 11:631319. https://doi.org/10.3389/fimmu.2020.631319\u003c/li\u003e\n\u003cli\u003eOh CW, Kim SE, Lee J, Oh DK (2022) Bioconversion of C20- and C22-polyunsaturated fatty acids into 9\u003cem\u003eS\u003c/em\u003e,15\u003cem\u003eS\u003c/em\u003e- and 11\u003cem\u003eS\u003c/em\u003e,17\u003cem\u003eS\u003c/em\u003e-dihydroxy fatty acids by \u003cem\u003eEscherichia coli \u003c/em\u003eexpressing double-oxygenating 9\u003cem\u003eS\u003c/em\u003e-lipoxygenase from \u003cem\u003eSphingopyxis macrogoltabida\u003c/em\u003e. J Biosci Bioeng 134:14-20. https://doi.org/10.1016/j.jbiosc.2022.04.001\u003c/li\u003e\n\u003cli\u003eSerhan CN (2014) Pro-resolving lipid mediators are leads for resolution physiology. Nature 510:92-101. https://doi.org/10.1038/nature13479\u003c/li\u003e\n\u003cli\u003eSerhan CN, Libreros S, Nshimiyimana R (2022) E-series resolvin metabolome, biosynthesis and critical role of stereochemistry of specialized pro-resolving mediators (SPMs) in inflammation-resolution: Preparing SPMs for long COVID-19, human clinical trials, and targeted precision nutrition. Semin Immunol 59:101597. https://doi.org/10.1016/j.smim.2022.101597\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Biotransformation of EPA into DiHEPAs by regio-\u0026nbsp;and stereoselective\u0026nbsp;double-oxygenating LOXs\u0026nbsp;expressed in \u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"98%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLOX expressed in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubstrate (mM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\"\u003e\n \u003cp\u003e\u003cstrong\u003eProduct (mM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecific productivity\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mmol h\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e\u0026minus;1\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eg\u003csup\u003e\u0026minus;1\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003e\u003cstrong\u003eVolumetric\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;productivity\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mM h\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMolar conversion (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eA. violaceum\u0026nbsp;\u003c/em\u003e15\u003cem\u003eS\u003c/em\u003e-LOX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003eEPA (3 mM)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003eRvE4 (1.4 mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\" rowspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(Lee et al. 2024)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.176470588235293%\" valign=\"top\"\u003e\n \u003cp\u003eEPA (6 mM)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.941176470588236%\" valign=\"top\"\u003e\n \u003cp\u003eRvE4 (4.3 mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.11764705882353%\" valign=\"top\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003e4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.930232558139537%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.790697674418604%\" valign=\"top\"\u003e\n \u003cp\u003eEPA (3 mM)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.093023255813954%\" valign=\"top\"\u003e\n \u003cp\u003eRvE4 enantiomer (1.1 mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.116279069767442%\" valign=\"top\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.441860465116278%\" valign=\"top\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.627906976744185%\" valign=\"top\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.176470588235293%\" valign=\"top\"\u003e\n \u003cp\u003eEPA (6 mM)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.941176470588236%\" valign=\"top\"\u003e\n \u003cp\u003eRvE4 enantiomer (5.8 mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.11764705882353%\" valign=\"top\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003e5.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS. macrogoltabida\u0026nbsp;\u003c/em\u003e9\u003cem\u003eS\u003c/em\u003e-LOX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003eEPA (6 mM)\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003e9\u003cem\u003eS\u003c/em\u003e,15\u003cem\u003eS\u003c/em\u003e-DiHEPA (5.8 mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e11.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\" valign=\"top\"\u003e\n \u003cp\u003e(Oh et al. 2022)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eE. numazuensis\u003c/em\u003e 12\u003cem\u003eS\u003c/em\u003e-LOX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003eEPA (3 mM)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003cem\u003eS\u003c/em\u003e,12\u003cem\u003eS\u003c/em\u003e-DiHEPA (1.5 mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\" valign=\"top\"\u003e\n \u003cp\u003e(Kim et al. 2021)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eA. violaceum\u0026nbsp;\u003c/em\u003e15\u003cem\u003eS\u003c/em\u003e-LOX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003eEPA (1 mM)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003eRvE4 (0.4 mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\" valign=\"top\"\u003e\n \u003cp\u003e(Lee et al. 2020)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003eEPA (6 mM)\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.387755102040817%\" valign=\"top\"\u003e\n \u003cp\u003eRvE4 enantiomer (5.1 mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\" valign=\"top\"\u003e\n \u003cp\u003e(Lee et al. 2024)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eRvE4. 5\u003cem\u003eS\u003c/em\u003e,15\u003cem\u003eS\u003c/em\u003e-DiHEPA; Enantiomer of RvE4, 5\u003cem\u003eR\u003c/em\u003e,15\u003cem\u003eR\u003c/em\u003e-DiHEPA. Specific productivity is defined as the initial production rate within a linear range. Volumetric productivity is defined as the maximum concentration per unit time.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Biotransformation without solvent, polymer, and resin. \u003csup\u003eb\u003c/sup\u003e Biotransformation with solvent, polymer, and resin.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003e Biotransformation with solvent and without polymer and resin. \u003csup\u003ed\u003c/sup\u003e Biotransformation without solvent and polymer and with resin.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biotransformation, Eicosapentaenoic acid, Lipoxygenase, Optimization, Resolvin E4, Resolvin E4 enantiomer","lastPublishedDoi":"10.21203/rs.3.rs-4121438/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4121438/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo enhance the production of resolvin E4 (RvE4) or its enantiomer from eicosapentaenoic acid (EPA), \u003cem\u003eArchangium violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-lipoxygenase (15\u003cem\u003eS\u003c/em\u003e-LOX) or \u003cem\u003eSorangium cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX was expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e with solvent, polymer, and adsorbent resin, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentrations of cells and substrate and the types and concentrations of solvent, polymer, and resin were optimized for the biotransformation of EPA into RvE4 (5\u003cem\u003eS\u003c/em\u003e,15\u003cem\u003eS\u003c/em\u003e-dihydroxyeicosapentaenoic acid) and its enantiomer (5\u003cem\u003eR\u003c/em\u003e,15\u003cem\u003eR\u003c/em\u003e-dihydroxyeicosapentaenoic acid). Under optimized conditions, \u003cem\u003eA. violaceum\u003c/em\u003e 15\u003cem\u003eS\u003c/em\u003e-LOX and \u003cem\u003eS. cellulosum\u003c/em\u003e 15\u003cem\u003eR\u003c/em\u003e-LOX expressed in \u003cem\u003eE. coli\u003c/em\u003e converted 6.0 mM (1.8 g L\u003csup\u003e−1\u003c/sup\u003e) EPA into 4.3 mM (1.4 g L\u003csup\u003e−1\u003c/sup\u003e) RvE4 and 5.8 mM (1.9 g L\u003csup\u003e−1\u003c/sup\u003e) RvE4 enantiomer in 60 min, with productivities of 4.3 and 5.8 mM h\u003csup\u003e−1\u003c/sup\u003e and molar conversions of 72 and 97%, respectively. The concentrations of RvE4 and its enantiomer resulting from the conversion of EPA with solvent, polymer, and resin were 3.1- and 5.3-fold higher than those without additives, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentrations, productivities, and conversions of RvE4 and its enantiomer were increased by optimizing the concentrations of cells and substrate and the types and concentrations of solvent, polymer, and adsorbent resin.\u003c/p\u003e","manuscriptTitle":"Biotransformation of eicosapentaenoic acid into the dihydroxyeicosapentaenoic acids resolvin E4 and its enantiomer by 15S- and 15R-lipoxygenases expressed in Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-12 15:05:25","doi":"10.21203/rs.3.rs-4121438/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2c62e26e-b172-4c74-8f9c-029bfbd65fd5","owner":[],"postedDate":"April 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-09T06:52:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-12 15:05:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4121438","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4121438","identity":"rs-4121438","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}