The strain Monascus purpureus YJX-8 was isolated from the fermentation starter of Baijiu in our previous work (Xu et al., 2021). LIP05 was identified as the key enzyme of ester synthesis in aqueous phase and encoded by the gene numbered GME7409. Escherichia coli BL21 (DE3) were used as the host for heterologous expression of LIP05. Butyric acid, pentanoic acid, hexanoic acid, octanoic acid, decanoic acid, ethyl butyrate, ethyl pentanoate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate standards were purchased from Sigma Aldrich (St Louis, MO). Luria Bertani (LB) medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl) was used to cultivate the recombinant strains. Agarose (2%) was added to generate the solid LB medium. All other chemicals were analytical grade reagents and commercially available.

Template 4PSC from Trichoderma reesei (sequence identity of 46.40% and query coverage of 92.82%) was selected as the reference for homologous modeling (Roussel et al., 2014). Discovery Studio (V2019) was used to build the 3D protein model evaluated by Ramachandran plot. Molecular docking of LIP05 with butyric acid, pentanoic acid, hexanoic acid, octanoic acid, decanoic acid, ethyl butyrate, ethyl pentanoate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate was performed using AutoDock software (Goodsell et al., 1996). The structures of these small molecule compounds were downloaded from the ZINC database¹ and defined as the ligands. LIP05 was defined as the receptor. The input file of the receptor and ligands in PDB format were converted into the AutoDock-compatible PDBQT format using MGLTools. The active site of LIP05 was selected as the docking region. The grid center was set at the O^γ atom of the catalytic residue Ser150. The conformation with the lowest affinity energy was selected as the most probable binding conformation and visualized by PyMOL.

Molecular dynamics (MD) simulation of the LIP05 was performed using GROMACS version 2022² with the OPLS-AA force field (Kaminski et al., 2001). PDBFIXER³ was used to structure preparation of LIP05, added missing amino acids and side chain atoms and formed a protonated state of charged amino acids. Ions with opposite charges were added to neutralize the charge of the system. Additional Na⁺ and Cl⁺ were added to create a salt concentration of 150 mM to simulate the actual experiments. The steepest descent method was used to minimize the energy of the above constructed system to eliminate spatial conflicts. The system was equilibrated at 300 K for 100 ps in the constant temperature and volume. Afterwards, the system was equilibrated at 1.0 bar for 100 ps in the constant pressure and temperature (Páll et al., 2014; Abraham et al., 2015). MD simulation was running for 100 ns. The LINCS algorithm was used to impose rigid constraints on the hydrogen bonds (Hess, 2008a). Electrostatic interactions were treated using the particle-mesh Ewald method (Darden et al., 1993). The Berendsen thermostat (Berendsen et al., 1984) and Parrinello-Rahman barostat (Parrinello and Rahman, 1981) were used to maintain the temperature and pressure. The coordinates and energy were saved every 1.0 ps. The result was analyzed by GROMACS tools (Van Der Spoel et al., 2005; Hess et al., 2008b; Pronk et al., 2013).

The expression vector pCold-TF was amplified using the primer pair pCold-TF.f/ pCold-TF.r through polymerase chain reaction (PCR) to generate the linearized vector (Supplementary Table S1). The gene encoding LIP05 was added with the 18 or 20 bp homologous recombinant sequence region by PCR using the respective primer pairs to generate the DNA fragment (Supplementary Table S1). The fragment was integrated with linearized vector DNA to produce the expression vector according to the protocol of the Vazyme ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China). The constructed vectors were transferred into the host cell E. coli BL21 (DE3) to generate the respective transformants. Using the expression vector carried the gene encoding LIP05 as a template, mutants were constructed by whole plasmid PCR using the respective designed primer pairs as shown in Supplementary Table S1, and transferred into E. coli BL21 (DE3) to produce the respective transformants.

The recombinant E. coli strains were inoculated into LB medium containing 100 μg/ml Ampicillin sodium and kept at 37°C with stirring at 200 r/min for about 3 h until the OD₆₀₀ value reached 0.6–0.8. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and then recombinant E. coli strains were kept at 16°C and 200 r/min for 20 h. The cells were collected by centrifugation at 4°C and 8,000 × g for 5 min, and were washed twice with Tris–HCl buffer (50 mM, pH 7.5). The cells were disrupted using an ultrasonic cell disrupter and were centrifuged at 4°C and 9,000 × g for 20 min to obtain the crude enzyme solution, which was used for purification and esterification in aqueous phase.

The crude enzyme was purified on a Ni Sepharose HP column (1 ml, GE, Uppsala, Sweden) using ÄKTA Fast Protein Liquid Chromatography purification system (GE, Uppsala, Sweden) with phosphate buffer (50 mM, pH 7.4) containing 400 mM NaCl and different concentrations of imidazole. The purified enzyme band was verified by SDS-PAGE, the protein concentration was determined using a BCA protein assay kit (Thermo Fischer Scientific Inc., Rockford, United States).

The aqueous phase reaction system contained the enzyme solution (1 ml), the organic acids of butyric acid, pentanoic acid, hexanoic acid, octanoic acid, decanoic acid (a final concentration of 10 mM for each chemical), and ethanol (a final concentration of 1 M) in citric acid buffer (50 mM, pH 4.0) to a final volume of 10 ml. The reaction system was shaken in a water bath at 37°C with stirring at 150 r/min for about 24 h. Thereafter, 3 ml of n-hexane (with internal standard ethyl oleate added) was added into the reaction system, vortexed for 30 s, and centrifuged at 4°C and 12,000 × g for 5 min. The upper layer was filtered and used for qualitative detection of esters by gas chromatography (GC).

The ester concentration was determined by GC (Agilent 7890B, Santa Clara, CA) using a 19,091 N-213I column (30 m × 0.32 mm × 0.50 μm, Agilent, Santa Clara, CA). The column temperature was kept at 40°C for 5 min, increased to 170°C at 8°C/min, maintained for 10 min, and increased to 240°C at 8°C/min, maintained for 5 min. The carrier gas was nitrogen with a flow rate of 1.0 ml/min. The instrument was equipped with an autosampler and a flame ionization detector. The injection volume was 1.0 μl. Origin 8.0 software was used for statistical analysis. The sequences were aligned using the Clustal algorithm⁴, and the results were visualized using ENDscript/ESPript.⁵

Ester synthesis reactions catalyzed by α/β fold hydrolase are mostly occurred in organic phase, and few reports focus on enzymatic ester synthesis in aqueous phase (Xu et al., 2021). LIP05 could catalyze the synthesis of fatty acid ethyl esters in aqueous phase, but the catalytic mechanism was still unclear. LIP05 had two domains and belonged to α/β fold hydrolase, the core catalytic domain consisted of 5 β-sheets [β1 (Tyr66-Ala71), β2 (Leu101-Gly105), β3 (His143-Tyr149), β4 (Ser173-Phe177), and β5 (Val194-Ala197)] surrounded by 7 α-helices [α2 (Gly85-Val96), α3 (Ile113-Ala118), α4 (Asp121-Thr138), α5 (Gln151-Lys162), α6 (Ala165-Trp170), α7 (Asn203-Gln206), and α8 (Pro213-Ala232)], the lid domain consisted of 1 α-helix [α1 (Ile30-Thr48); Figures 1A,B]. The catalytic triad was Ser150-His215-Asp202 (Supplementary Figure S1), and belonged to the classical catalytic triad of α/β fold hydrolase (Rauwerdink and Kazlauskas, 2015; Zong et al., 2022). Ser150 was located on the “nucleophile elbow” connecting β3 and α5, His215 was located on the α8, and Asp202 was located on the loop between β5 and α7 (Figures 1A,B). The catalytic active center was covered by the lid domain (Figures 1C,D). The enzyme activity of the LIP05-50 (lid-removed mutant) was increased by 52.50% ± 18.90%, but the stability was decreased, indicating that the lid domain played an important role in maintaining the stability of the enzyme, and the removing of the lid domain was conducive to the improvement of enzyme activity (Xu et al., 2021). The enzymes 4PSC and 2CUT had similar structure with that of LIP05 (Figure 1E). 4PSC had 5 β-sheets surrounded by 8 α-helices, and the lid domain consisted of 2 α-helices (α1 and α2). Similarly, both LIP05 and 4PSC showed high enzyme activity in an acidic environment, which might be due to the strong interaction of the oil–water interface in a low pH environment and beneficial to the opening of the lid domain (Roussel et al., 2014). On the contrary, 2CUT had no lid domain, and the active center was exposed to the environment, and therefore, there was no interface activation phenomenon (Martinez et al., 1994).

The molecular dynamics simulation of LIP05 in aqueous phase was analyzed. The RMSD of the protein increased from 0.2 nm to 0.35 nm and finally stabilized at 0.3 nm (Supplementary Figure S2). The radius of gyration was increased significantly from 10 ns to 20 ns (Supplementary Figure S3), indicating that the tightness of protein folding was decreased, and the looseness of protein was increased. Furthermore, the solvent accessible surface area (SASA) also suggested that the hydrophobic area inside the protein was exposed by the opening of the lid domain from 10 ns to 30 ns (Supplementary Figure S4). At this time, the active center was exposed because of the opening of the lid domain, and it was covered again after 40 ns (Figure 2A). It was discovered that the distance between the Tyr116 and Ile204 played a critical role in the exposure of the active center, which determined the substrate spectrum by composing the substrate-entrance channel. The distance of carboxy oxygen atoms between Tyr116 and Ile204 from 10 ns to 30 ns was fluctuated between 6.90 Å – 10.60 Å (Figure 2B), which could accommodate the substrate entrance into the catalytic active pocket, and explained why LIP05 could catalyze the synthesis of fatty acid ethyl esters in aqueous phase. However, the substrate interacted with enzyme after entered the catalytic active pocket was still unclear. Molecular docking was used to predict the interactions between enzyme and substrates (butyric acid, pentanoic acid, hexanoic acid, octanoic acid, and decanoic acid) and products (ethyl butyrate, ethyl pentanoate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate; Supplementary Figure S5). All the docking results were summarized in Supplementary Table S2. Docking with hexanoic acid indicated that LIP05 could interact with the ligand by hydrogen bond through the Thr74 (2.60 Å; Supplementary Figure S5), and LIP05 could interact with the ligand ethyl hexanoate by hydrogen bonds through the Ser150 (2.60 Å) and His215 (2.20 Å; Supplementary Figure S5). In addition, the hydrophobic interaction was also a key force between the enzyme and the substrates. LIP05 could interact with substrates by hydrophobic interaction through the residues Ile30, Leu37, Gly73, Leu83, Tyr116, Tyr149, Ile204, Ile211 and Leu216 (Supplementary Figure S6).

Molecular docking indicated LIP05 could form hydrogen bonds and hydrophobic interactions with substrates under 10 residues (Ile30, Leu37, Gly73, Thr74, Leu83, Tyr116, Tyr149, Ile204, Ile211, and Leu216). Hydrogen bonds and hydrophobic interactions could determine protein folding and played an important role in maintaining protein structure (Brandl et al., 2001). In order to reveal how these residues affected the ester catalysis of LIP05, all these residues were mutated by different types of amino acids according to steric hindrance, hydrophilicity and hydrophobicity of the amino acids to explore the catalytic mechanism of LIP05 (Supplementary Figures S7, S8).