Semi‑rational design and modification of CciUPO for the efficient biosynthesis of 25-hydroxyvitamin D3

Semi‑rational design and modification of CciUPO for the efficient biosynthesis of 25-hydroxyvitamin D3 | 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 Semi‑rational design and modification of Cci UPO for the efficient biosynthesis of 25-hydroxyvitamin D 3 Jingyi Zhou, Yingjia Tong, Fan He, Kai Chu, Hang Zhong, Jingsong Shi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9459508/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 25-Hydroxyvitamin D 3 (25-OH-VD 3 ) is the main active form of Vitamin D 3 and has broad applications in clinical treatment and agriculture. Although current industrial production predominantly relies on chemical synthesis, the growing demand for green and efficient manufacturing has accelerated the development of microbial enzymatic conversion methods. Due to its high reaction specificity, the unspecific peroxygenase from Coprinopsis cinerea ( Cci UPO) represents a promising biocatalyst for the synthesis of 25-OH-VD 3 . However, its low catalytic efficiency limits further application in the biosynthesis of 25-OH-VD 3 . To address this, semi-rational design was employed to modify the substrate-binding pocket and non-conserved residues of Cci UPO. The resulting triple mutant I73M/P108K/G245A increased the 25-OH-VD 3 concentration to 87.83 mg/L, representing a 41.18% increase over the wild type (62.21 mg/L). The mechanism for enhanced catalytic efficiency was elucidated through analysis of the substrate-binding pocket, enzyme-substrate interactions, and molecular dynamics simulations. Subsequently, the fermentation conditions and multi-enzyme cascade reaction were optimized. Under optimized conditions with a substrate VD 3 concentration of 0.5 g/L, the 25-OH-VD 3 concentration further increased to 152.50 mg/L. The combination of semi-rational engineering and process optimization of Cci UPO offers a feasible, green and efficient strategy for the biosynthesis of 25-OH-VD 3 . 25-OH-VD3 CciUPO Biosynthesis Semi-rational design and modification multi-enzyme cascade reaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction As the bioactive form of Vitamin D 3 (VD 3 ), 25-hydroxyvitamin D 3 (25-OH-VD 3 ) exerts pharmacological effects on regulating calcium and phosphorus metabolism, alleviating osteoporosis and rickets, and is therefore widely used in clinical treatment [1–4] . In addition, 25-OH-VD 3 has high application value in agriculture [5–7] . Currently, the traditional chemical synthesis of 25-OH-VD 3 suffers from several limitations in industrial production, including complex reaction steps, environmental pollution, and the generation of by-products [8–9] . Whereas, enzymatic synthesis of 25-OH-VD 3 with mild reaction conditions, high specificity and environmentally friendliness has attracted increasing attention [10–12] . According to the literature, the enzymes catalyzing the conversion of VD 3 to 25-OH-VD 3 are primarily cytochromes P450 (CYPs) and unspecific peroxygenases (UPOs) [13–17] . CYP-mediated C25 hydroxylation of VD 3 suffers from NADPH dependency, complex electron transfer, and low catalytic efficiency, which limit its industrial application. UPOs resemble CYPs in structure, both containing a cysteine-coordinated heme unit [18–20] . Instead of relying on expensive NAD(P)H to supply electrons, UPOs use hydrogen peroxide (H 2 O 2 ) as the oxidant and oxygen donor, which is more economical [21–25] . Notably, unspecific peroxygenase from Coprinopsis cinerea ( Cci UPO) catalyzes the regioselective hydroxylation of VD 3 at the C25 position, without the formation of other by-product [26] . Semi-rational design and modification has proven highly effective for improving enzyme catalytic efficiency, primarily through engineering substrate-binding pockets and non-conserved residues. The substrate-binding pocket is a key region where the enzyme directly interacts with the substrate and participates in catalysis. Modification of this pocket has been shown to greatly increase catalytic efficiency, as exemplified by 3-ketoacyl-CoA thiolase and aldo-keto reductase. Liu et al. rationally engineered the substrate-binding pocket, increasing the catalytic efficiency of 3-ketoacyl-CoA thiolase for its substrate by 6.67-fold [27] . Niu et al. reshaped the substrate-binding pocket of aldo-keto reductase, yielding the mutant W21A/G53N with 330-fold higher catalytic activity [28] . In addition, non-conserved residues, predominantly located in surface loop regions and around the active center, offer untapped potential for improving catalytic efficiency. Mutations in these regions can synergize with mutations of key residues in the substrate-binding pocket to enhance catalytic efficiency. Zou et al. combined computational design with engineering of non-conserved residues to generate a focused mutation library, yielding the mutant A169P with a 52.04% increase in the catalytic efficiency of alpha-galactosidase [29] . Therefore, applying similar strategies to engineer the substrate-binding pocket and non-conserved residues of Cci UPO is expected to significantly enhance its catalytic efficiency for 25-OH-VD 3 production. In our previous research, Cci UPO was successfully secreted extracellularly in Pichia pastoris X33. However, the C25 hydroxylation of VD 3 catalyzed by Cci UPO yielded only 62.21 mg/L of 25-OH-VD 3 [30] . In this study, to enhance the efficiency of 25-OH-VD 3 biosynthesis, semi-rational design and modification were performed on the substrate-binding pocket and non-conserved residues of Cci UPO. The mechanism underlying the improved performance was then elucidated by analyzing the substrate-binding pocket, molecular interactions, and molecular dynamics (MD) simulations. Furthermore, optimization of fermentation conditions and the multi-enzyme cascade reaction system further increased the 25-OH-VD 3 concentration. Semi-rational engineering of Cci UPO combined with process optimization significantly enhanced 25-OH-VD 3 biosynthesis, providing a feasible strategy for green and efficient production. 2. Materials and methods 2.1 Reagents Restriction endonucleases Sac I and Dpn I, 4 × Loading Buffer were purchased from Takara (Dalian, China). Peptone, yeast extract, and glucose were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). VD 3 and biotin were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). VD 3 , 25-OH-VD 3 standard substances and glucose oxidase were purchased from Yuan Ye Biotechnology Co., Ltd. (Shanghai, China). Zeocin was purchased from Solarbio Technology Co., Ltd. (Beijing, China). Unless otherwise specified, all reagents used in this study were sourced from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Primer synthesis and DNA sequencing were performed by Talen-bio Biological Technology Co., Ltd. (Shanghai, China). 2.2 Strains and cultivation E. coli JM109 was used for cloning and plasmid propagation. Pichia pastoris X33 was used for the heterologous expression of variants. The recombinant plasmid pPICZA-P AOX1 -P DAS2 -SP α factor - Cci UPO was previously constructed in our laboratory. The recombinant Pichia pastoris strain was cultured at 30℃ for 16 h in 10 mL of YPD liquid medium. Then, the cells were transferred to 50 mL of BMGY liquid medium at an inoculation volume of 2% (v/v) and cultured at 30℃ for 24 h. The cells were collected by centrifugation at 5000 rpm for 5 min, and the supernatant was discarded. The cells were washed with sterile ddH 2 O to remove glycerol and resuspended in 50 mL of BMMY medium. Methanol (1.5%, v/v) and the heme precursor 5-aminolevulinic acid hydrochloride (5-ALA) at a final concentration of 45 µmol/L were added every 24 h, and continuous induction was carried out for 72 h at 25°C with shaking at 120 rpm. After induction, the fermentation supernatant containing Cci UPO was collected. 2.3 Construction of the Cci UPO mutants The plasmid pPICZA-P AOX1 -P DAS2 -SP α factor - Cci UPO carrying the gene Cci UPO (GenBank ID: XP_001831910.1) was used as the template, and reverse PCR primers were designed for amplification (Table S1 ). Subsequently, the reverse PCR product was digested with Dpn I at 37°C for 30 min to remove the template plasmid. After digestion, the reverse PCR products were purified using a gel extraction kit and transformed into E. coli JM109 by heat shock method, followed by overnight incubation at 37°C. Single colonies were selected for colony PCR and sequencing to confirm the desired mutations. After successful sequencing, the recombinant plasmids were isolated and linearized using the restriction enzyme Sac I, and then transformed into Pichia pastoris X33 cells via electroporation. 2.4 Multi-enzyme cascade reaction system A multi-enzyme cascade reaction system was constructed. In this system, glucose oxidase utilizes glucose and O 2 to generate H 2 O 2 in situ, which subsequently drives Cci UPO to catalyze the C25 hydroxylation of VD 3 . The reaction system consisted of 25 mmol/L glucose, 40 U/mL glucose oxidase, 8 mmol/L Na 2 HPO 4 buffer (pH 5.0), 22.5 mg/mL 2-hydroxypropyl-β-cyclodextrin, 0.5 g/L substrate VD 3 , with Cci UPO crude enzyme added to a final volume of 2 mL. The reaction was carried out at 30℃ and 220 rpm for 24 h. 2.5 Identification of catalytic products The reaction mixture was extracted three times with 70% (v/v) ethyl acetate, and the upper organic phase was collected. After evaporation of the combined organic phases, the residue was redissolved in 1 mL of methanol and filtered through a 0.22 µm organic membrane to obtain the sample for HPLC analysis. HPLC analysis was performed on a Sunfire™ C18 column at 40°C with UV detection at 265 nm. The mobile phase was acetonitrile/water solution and the injection volume was 20 µL. The flow rate was maintained at 1.0 mL/min. A linear gradient of 60–100% aqueous acetonitrile was applied over 15 min, followed by an isocratic hold at 100% acetonitrile for an additional 20 min. Under these conditions, the retention times of 25-OH-VD 3 and VD 3 were 15.44 min and 25.28 min, respectively. 2.6 Structure modeling and Molecular Dynamics Simulations Homology modeling of Cci UPO was performed using AlphaFold2, and the model was evaluated using the SAVES 6.0 website ( https://saves.mbi.ucla.edu/ ). The substrate VD 3 was docked into the active center of Cci UPO using AutoDock 4.0. PyMol software was used to visualize the protein structure. FireProt ( https://loschmidt.chemi.muni.cz/fireprot/ ) was used to design more mutants. The Consurf web server ( http://consurf.tau.ac.il ) was used to analyze the conservation of Cci UPO. The substrate-binding pocket volumes of wild-type and mutants were predicted with the DoGSiteScorer ( https://proteins.plus/ ). The PLIP website ( https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index ) was used to predict the interaction forces between the protein and the substrate VD 3 . Molecular dynamics (MD) simulations were carried out using GROMACS 2021 software. The protein was placed in a dodecahedral box under the CHARMM36 force field and TIP3P water model. The system was neutralized with 0.15 mol/L NaCl. Energy minimization was performed, followed by 100 ps of NVT and NPT equilibration at 300 K and 101.3 kPa, respectively. Finally, three independent 100 ns simulations were conducted. After simulation, the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) were calculated. 2.7 Fermentation optimization of the recombinant P. pastoris strains The optimal Pichia pastoris strain X33/pPICZA-P AOX1 -P DAS2 -SP α factor - Cci UPO I73M/P108K/G245A , designated as X-M1, was used for subsequent fermentation optimization. These parameters included induction temperature, methanol concentration, induction pH, 5-aminolevulinic acid (5-ALA) concentration, induction time, and the types and concentrations of carbon and nitrogen sources in the fermentation medium. Each condition was optimized sequentially based on the optimal condition obtained in the previous step. The initial methanol concentration, pH, 5-ALA concentration and induction time were set at 1.5%(v/v), 5.0, 45 µmol/L and 72 h, respectively. Meanwhile, 1%(v/v) glycerol was used as the carbon source of BMGY medium. Yeast extract and peptone (1:2, w/w) were used as the compound nitrogen source in both BMGY and BMMY, with a total concentration of 30 g/L. After induction, the multi-enzyme cascade reaction was performed, and the concentration of product 25-OH-VD 3 was analyzed by HPLC. 2.8 Optimization of the multi-enzyme cascade reaction The reaction system was scaled up to 50 mL, and the multi-enzyme cascade reaction was systematically optimized. Each condition was optimized sequentially based on the optimal condition obtained from the previous step. During optimization of glucose concentration, the concentrations of glucose oxidase and the co-solvent 2-hydroxypropyl-β-cyclodextrin were set at 40 U/mL and 22.5 mg/mL, respectively, while the reaction temperature and time were fixed at 30℃ and 24 h. 2.9 Statistical methodology analysis Experimental data were processed using SPSS statistical software and Microsoft Office Excel. Analysis of variance (ANOVA) was used to analyze the results, and the data are presented as mean ± standard deviation (mean ± SD). 3 Results and Discussion 3.1 Semi-rational design and modification of the substrate-binding pocket The homology model of Cci UPO was constructed using AlphaFold2 (Fig. 1 a), and the model quality was evaluated using the SAVES 6.0 server. Ramachandran plot analysis revealed that 88.1% of residues in the Cci UPO model occupied the most favored regions, 11.9% occupied allowed regions, and none were found in disallowed regions (Fig. 1 b). The model achieved an ERRAT score of 89.362 (Fig. 1 c), and 94.07% of residues exhibited a 3D-1D score ≥ 0.1 (Fig. 1 d), collectively indicating that the Cci UPO model was of high quality. AutoDock 4.0 software was used to dock the substrate VD 3 into the active center of the Cci UPO homology model. As shown in Fig. 2 a, the ligand VD 3 was located above the heme active center of Cci UPO, with a distance of 2.7 Å between the C25 atom of VD₃ and the heme, suggesting that Cci UPO is more likely to catalyze the C25 hydroxylation of VD 3 . The Cci UPO-VD₃ docking complex was visualized using PyMOL software. A total of twenty-two amino acid residues located within 6 Å of VD 3 were identified as potential substrate-binding sites, including M69, I73, F76, V77, F121, E122, P188, R189, F191, T192, Y194, E196, S197, F199, G245, L249, D250, V251, F280, F283, L286, and P322 (Fig. 2 b). Alanine scanning mutagenesis was conducted to evaluate the functional roles of the 22 amino acid residues identified as potential substrate-binding sites. As illustrated in Fig. 3 a, the mutant G245A exhibited superior catalytic performance, producing 71.99 mg/L of 25-OH-VD 3 , representing a 15.72% increase over the wild type (62.21 mg/L). Therefore, G245 was identified as a key amino acid residue affecting the catalytic efficiency of Cci UPO. Saturation mutagenesis was subsequently performed at residue G245. As illustrated in Fig. 3 b, the G245Y mutant increased the concentration of 25-OH-VD 3 to 65.63 mg/L, representing a 5.50% increase over the wild type. To obtain more mutants with improved catalytic efficiency, FireProt was used for designing mutants [31] , including A195C, A195P, I73M, V77M, V77L, and V77P. As shown in Fig. 3 c, the I73M mutant exhibited higher catalytic efficiency for the C25 hydroxylation of VD 3 , with the concentration of 25-OH-VD 3 reaching 78.68 mg/L, which represented a 26.47% increase over the wild type. 3.2 Semi-rational design and modification of non-conserved residues Non-conserved residues serve as potential sites for enhancing enzymatic catalytic efficiency. The conservation of Cc iUPO was assessed using the ConSurf server [32] . According to the conservation analysis results in Fig. 4 a, a conservation score of 6 was set as the threshold to differentiate conserved residues from non-conserved ones of Cci UPO [33] . Eleven non-conserved residues around the active center were selected for alanine scanning mutagenesis. As illustrated in Fig. 4 b, P108A and H133A mutants exhibited higher catalytic efficiency than the wild type, producing 25-OH-VD 3 at concentrations of 69.02 mg/L and 80.78 mg/L, respectively. P108 and H133 were selected for further saturation mutagenesis to screen for dominant mutants. As shown in Fig. 4 c, the P108K mutant exhibited significantly improved catalytic performance, with the concentration of 25-OH-VD 3 reaching 82.11 mg/L, which was 31.99% higher than that of the wild type. The remaining mutations had little positive effect on the catalytic efficiency. The H133G mutant also exhibited enhanced catalytic efficiency, yielding 25-OH-VD 3 at a concentration of 83.13 mg/L, representing a 33.63% increase over the wild type (Fig. 4 d). 3.3 Construction and screening of combined mutants To further enhance catalytic efficiency, combined mutants were generated by combining the dominant mutants G245A, I73M, P108K, H133A, and H133G. As illustrated in Fig. 5 , seven combination mutants exhibited superior catalytic performance than the wild type. Among all dominant mutants constructed in this work, the triple mutant I73M/P108K/G245A (M1), combining substrate-binding pocket modifications (I73M/G245A) with a non-conserved substitution (P108K), exhibited the highest catalytic efficiency for the biosynthesis of 25-OH-VD 3 , yielding a concentration of 87.83 mg/L. This represented a 41.18% increase compared with that of the wild type. The recombinant P. pastoris strain X33/pPICZA-P AOX1 -P DAS2 -SP α factor - Cci UPO I73M/P108K/G245A was designated as X-M1. 3.4 Insights into the enhanced catalytic efficiency by structure analysis In order to explore the potential mechanism of Cci UPO and its mutants, the substrate-binding pocket and enzyme-substrate interactions were analyzed. As shown in Table 1 , the I73M mutation reduced the pocket volume from 548.42 Å 3 to 417.47 Å 3 , and the G245A, P108K, and H133G mutants exhibited slightly smaller pocket volumes. The triple mutant I73M/P108K/G245A showed a reduced volume of 425.73 Å 3 . The reduced substrate-binding pocket may enhance the binding affinity between the enzyme and the substrate, thereby contributing to the improved catalytic efficiency. Furthermore, enzyme-substrate interactions analysis revealed that the wild type formed hydrophobic interactions with VD 3 (Fig. 6 a), while the I73M mutant displayed similar interactions (Fig. 6 b). The G245A and P108K mutants exhibited increased hydrophobic contacts (Fig. 6 c and 6 d), and the H133G mutant formed additional hydrophobic interactions and a hydrogen bond (Fig. 6 e). Notably, the triple mutant I73M/P108K/G245A showed substantially enhanced hydrophobic interactions compared with the wild type and single mutants (Fig. 6 f). The progressive increase in hydrophobic interactions from the wild type to the triple mutant suggests a possible correlation with the improved catalytic efficiency. The synergistic pocket constriction by the three mutations is proposed to restrict the conformational freedom of VD 3 , more accurately aligning its C25-H bond with the heme iron-bound oxygen atom. This favorable binding posture may facilitate substrate-induced fit and stabilize the transition state, thereby contributing to the greatly enhanced catalytic efficiency of C25 hydroxylation. Table 1 Prediction of substrate-binding pocket volume Mutant Pocket volume (Å 3 ) WT 548.42 I73M 417.47 G245A 539.65 P108K 547.97 H133G 548.22 I73M/P108K/G245A 425.73 To further elucidate the catalytic mechanism, molecular dynamics (MD) simulations were performed on the dominant mutants. Compared with the wild type, the I73M, G245A, P108K, and H133G mutants exhibited lower RMSD fluctuations, indicating enhanced protein stability (Fig. 7 a, 7 c, 7 e, 7 g). At position I73, reduced RMSF was observed, and protein rigidity was enhanced (Fig. 7 b). As illustrated in Fig. 7 d, the RMSF at position G245 was increased, leading to enhanced protein flexibility, which may facilitate substrate entry, localization, and transition state stabilization. As shown in Fig. 7 f, the RMSF at position 108 was reduced, indicating enhanced enzyme rigidity, thereby facilitating transition state stabilization. The introduction of glycine at position 133 increased the RMSF (Fig. 7 h). This mutation enhanced protein flexibility, which may facilitate substrate-induced fit, allowing the catalytic groups to approach the reaction center with a more favorable spatial orientation, thereby promoting the formation and stability of the transition state and improving catalytic efficiency. Notably, the triple mutant I73M/P108K/G245A exhibited a lower RMSD value and reached the plateau phase earlier, indicating significantly enhanced overall structural stability (Fig. 7 i). Concurrently, the RMSF of the triple mutant decreased (Fig. 7 j), suggesting enhanced protein rigidity and better transition state stabilization, thereby contributing to the improved catalytic efficiency. 3.5 Optimization of fermentation conditions and multi-enzyme cascade reaction The recombinant P. pastoris strain X-M1, harboring methanol-inducible promoters for Cci UPO expression, served as the production host. Induction conditions of P. pastoris strain X-M1 such as temperature, methanol concentration, pH, 5-aminolevulinic acid (5-ALA) concentration and induction time directly influence both cell growth and heterologous protein expression [34–36] . Therefore, to maximize the production of 25-OH-VD 3 , the induction conditions were systematically optimized. As illustrated in Fig. 8 , the optimal induction conditions were determined as follows: temperature 25°C, methanol 1.5% (v/v), pH 5.0, 5-ALA 60 µmol/L, and induction time 72 h. Under these optimized conditions, with a substrate VD 3 concentration of 0.5 g/L, the concentration of 25-OH-VD 3 reached 88.66 mg/L. Subsequently, the fermentation medium composition of P. pastoris strain X-M1 was optimized. As shown in Fig. 9 a and Fig. 9 b, glucose was identified as the optimal carbon source, with an optimal concentration of 20 g/L. Regarding the nitrogen source, a mixture of yeast extract and peptone at a ratio of 1:2 (w/w) with a total concentration of 45 g/L exhibited the best performance (Fig. 9 c and Fig. 9 d). Following these optimizations, the concentration of 25-OH-VD 3 increased to 111.46 mg/L. UPOs are sensitive to the concentration of H 2 O 2 . Direct addition of H 2 O 2 may cause excessively high local concentrations, leading to irreversible damage to the heme center of UPO and resulting in enzyme inactivation [37,38] . Glucose oxidase utilizes glucose and oxygen as substrates to generate H 2 O 2 in situ. This reaction, together with Cci UPO, constitutes a multi-enzyme cascade reaction system. Based on the above optimizations of induction conditions and medium components, the reaction system was scaled up to 50 mL, and the multi-enzyme cascade reaction was systematically optimized. As illustrated in Fig. 10, the optimal multi-enzyme cascade reaction system consisted of 25 mmol/L glucose, 20 U/mL glucose oxidase, and 45 mg/mL 2-hydroxypropyl-β-cyclodextrin, with a reaction temperature of 30°C and a reaction time of 24 h. Under these optimized conditions, the concentration of 25-OH-VD₃ reached 152.50 mg/L, representing a 73.63% increase compared with the level prior to optimization (87.83 mg/L). Conclusion In summary, this study successfully enhanced the catalytic efficiency of Cci UPO for C25 hydroxylation of VD 3 through semi‑rational design and modification of its substrate‑binding pocket and non-conserved residues. Utilizing site-directed saturation mutagenesis coupled with product concentration-based screening, the resulting triple mutant I73M/P108K/G245A exhibited improved catalytic efficiency, with the 25-OH-VD 3 concentration reaching 87.83 mg/L. Mechanistic analysis demonstrated that the triple mutant exhibited a constricted substrate-binding pocket, strengthened hydrophobic interactions, and enhanced protein stability and rigidity, which synergistically contributed to the improvement in catalytic efficiency. Following systematic optimization of fermentation conditions and the multi-enzyme cascade reaction, the concentration of 25-OH-VD 3 reached 152.50 mg/L. This work provides a feasible design and modification strategy for Cci UPO, laying the foundation for the efficient and green synthesis of 25-OH-VD 3 . Declarations The authors declare that they have no conflict of interest. Conflict of interest Dr. Zhenghong Xu, a co-author of this study, is a member of the editorial board of Systems Microbiology and Biomanufacturing. However, this did not influence the peer review process or the decision-making for this manuscript. All authors declare no other conflicts of interest. Funding This work was supported by the National Key R & D Program of China (No. 2024YFA0917900) and the Fundamental Research Funds for the Central Universities (No. JUSRP202504023). Author Contribution Jingyi Zhou: Writing Original Draft, Investigation, Methodology, Data Curation, Visualization. Yingjia Tong: Investigation, Formal analysis. Fan He: Methodology, Formal analysis. Kai Chu: Investigation, Methodology. Hang Zhong: Methodology, Formal analysis. Jinsong Shi: Supervision, Validation, Formal analysis. Zhenghong Xu: Conceptualization, Supervision, Project administration. Hui Li: Conceptualization, Funding acquisition, Supervision, Methodology, Formal analysis, Writing - Review & Editing. Data Availability Data will be made available on request. References Wang Z Y, Zeng Y, Jia H M, et al. Bioconversion of Vitamin D 3 to bioactive Calcifediol and Calcitriol as high-value compounds. Biotechnology for Biofuels and Bioproducts, 2022, 15: 109-121. Park C Y, Shin S, Han S N. Multifaceted roles of Vitamin D for diabetes: from immunomodulatory functions to metabolic regulations. Nutrients, 2024, 16(18): 3185-3207. Koga G K C, Maeda S S, Lazaretti-Castro M，et al. Rapid and dose-dependent increase of 25(OH)D levels after Calcifediol supplementation in a woman with obesity, chronic liver disease, and osteoporosis. Archives of Endocrinology Metabolism, 2025, 69(6): 1-6, e240428. Gonnelli S，Pitinca M D T，Camarri S，et al. 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Niu J F, Zhu X Y, Chi H B, et al. Reshaping the binding pocket of Aldo-Keto Reductase for enhanced stereoselectivity and activity. Journal of Agricultural and Food Chemistry, 2026, 74: 9539−9551. Zou Y F, Zheng P, Chen P C, et al. Multidimensional computational strategies enhance the thermostability of alpha-galactosidase. International Journal of Biological Macromolecules, 2025, 314: 144316-144330. Zhou J Y, Tong Y J, Liang Y S, et al. Heterologous expression of peroxygenase from Coprinopsis cinerea and optimization of the reaction system for catalytic synthesis of 25-OH-VD 3 . China Biotechnolog; 2026, http://www/doi.org/10.13523/j.cb.202512008. Acessed 09 March 2026. Tian X Y, Long J Y, Chen B Q, et al. Synergistic enhancement of thermal stability and activity of Glycolaldehyde Synthase via computer-aided design and mechanistic analysis. Journal of Agricultural and Food Chemistry, 2026, 74(1): 1326–1338. Yariv B,Yariv E, Kessel A, et al. Using evolutionary data to make sense of macromolecules with a “face-lifted” ConSurf. Tools for Protein Science ,2023, 32 (3): No. e4582. Li Y, Li S F, Fu S Y，et al. Design of a Distal Site Saturation Test-Iterative Parallel Mutagenesis for Engineering Hydroxysteroid Dehydrogenase. Journal of Agricultural and Food Chemistry, 2025, 73(16): 9701–9713. Yu Y, Liu Z M, Chen M, et al. Enhancing the expression of recombinant κ-carrageenase in Pichia pastoris using dual promoters, co-expressing chaperones and transcription factors. Biocatalysis and Biotransformation, 2020, 38(2): 104-113. Zhang X Y, Yang Y X, Chen S T, et al. Engineering Pichia pastoris for high-level biosynthesis of squalene. Biochemical Engineering Journal, 2025, 217: 109677-109687. Parashar D, Satyanarayana T. Enhancing the production of recombinant acidic α-amylase and phytase in Pichia pastoris under dual promoters constitutive (GAP) and inducible (AOX) in mixed fed batch high cell density cultivation. Process Biochemistry, 2016, 51(10): 1315-1322. Alexander K, Katrin C, René U, et al. Exploring the catalase activity of unspecific peroxygenases and the mechanism of peroxide-dependent heme destruction. Journal of Molecular Catalysis B-Enzymatic, 2016, 134: 238-246. Valderrama B, Ayala M, Vazquez Duhalt R. Suicide inactivation of peroxidases and the challenge of engineering more robust enzymes. Chemistry & Biology, 2002, 9(5): 555-565. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9459508","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":632057773,"identity":"2e28d149-f9f3-4551-a642-4a23f8d3b0ef","order_by":0,"name":"Jingyi Zhou","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Jingyi","middleName":"","lastName":"Zhou","suffix":""},{"id":632057774,"identity":"c6f3f765-977a-4f19-b204-80461af1dfc7","order_by":1,"name":"Yingjia Tong","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Yingjia","middleName":"","lastName":"Tong","suffix":""},{"id":632057775,"identity":"289d9d01-bace-4c68-aeb2-c316fdf493c7","order_by":2,"name":"Fan He","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"He","suffix":""},{"id":632057776,"identity":"7eb285be-ff5c-4084-87bf-072e19cc4e1d","order_by":3,"name":"Kai Chu","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Chu","suffix":""},{"id":632057777,"identity":"c2b9808e-af5c-44b3-9aad-e926947d374c","order_by":4,"name":"Hang Zhong","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Zhong","suffix":""},{"id":632057778,"identity":"cc324ccc-11c7-4334-96ed-b7d6656007fb","order_by":5,"name":"Jingsong Shi","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Jingsong","middleName":"","lastName":"Shi","suffix":""},{"id":632057779,"identity":"c7aee07a-f35f-4b84-8862-52f7ca0ca586","order_by":6,"name":"Zhenghong Xu","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Zhenghong","middleName":"","lastName":"Xu","suffix":""},{"id":632057780,"identity":"5952eb3b-dd33-4b13-aeaf-1d60d1d0f2ca","order_by":7,"name":"Hui Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYBACPmYeBoaECjYehNABAlrYwFrOkKSFAaiYsQ1ZiKAWdt5jEg/n8cnwz26/9uDnDgY5vhsJjJ8L8DqML00icRsbj8SdM+WGvWcYjCVvJDBLz8DvFzOwFoYbOWkSvG0MiRtuJIA9SEDLHDYeeaAWyb9tDPVEamlg4zG4kX5MGmhLggERWowtEo6x8RjeyGGTlm2TMJx55mGzND4t/PxnDG/+qDlmL3cj/Znk2zYbeb7jyQc/49MCBceAmMcASEgAMWMDYQ0MDDVAzP6AGJWjYBSMglEwAgEAga1ATb/0tnUAAAAASUVORK5CYII=","orcid":"","institution":"Jiangnan University","correspondingAuthor":true,"prefix":"","firstName":"Hui","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-04-19 05:39:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9459508/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9459508/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108247477,"identity":"072d74f5-3424-4b8a-ab4f-bb44d6c0280f","added_by":"auto","created_at":"2026-05-01 00:52:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284578,"visible":true,"origin":"","legend":"\u003cp\u003eHomology modeling and quality assessment of \u003cem\u003eCci\u003c/em\u003eUPO. (a) The homology model of \u003cem\u003eCci\u003c/em\u003eUPO. (b) Ramachandran plot analysis. (c) ERRAT analysis. (d) Verify3D analysis.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/ad3d958e5d301ff53dccaf8a.png"},{"id":108491292,"identity":"578906ca-72e8-4156-a507-912f02f94559","added_by":"auto","created_at":"2026-05-05 09:53:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1251239,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking of \u003cem\u003eCci\u003c/em\u003eUPO. (a) Close-up view of the predicted binding pose of VD\u003csub\u003e3 \u003c/sub\u003e(yellow) within the heme(green). (b) Identification of potential substrate-binding residues.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/79989776ac8be7c6b6c887b7.png"},{"id":108247479,"identity":"4014e9f9-e944-4122-9345-7708cd72e325","added_by":"auto","created_at":"2026-05-01 00:52:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":994191,"visible":true,"origin":"","legend":"\u003cp\u003eThe 25-OH-VD\u003csub\u003e3 \u003c/sub\u003econcentrations of substrate-binding pocket mutants. (a) 25-OH-VD\u003csub\u003e3\u003c/sub\u003e concentrations of alanine scanning mutants. (b) 25-OH-VD\u003csub\u003e3\u003c/sub\u003e concentrations of saturation mutants at G245. (c) 25-OH-VD\u003csub\u003e3\u003c/sub\u003e concentrations of Fireprot designed mutants.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/6d02df1495dd34eef1db8669.png"},{"id":108492385,"identity":"196d6853-3e51-4476-beaa-423fd4746968","added_by":"auto","created_at":"2026-05-05 09:57:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1532115,"visible":true,"origin":"","legend":"\u003cp\u003eThe 25-OH-VD\u003csub\u003e3\u003c/sub\u003e concentrations of non-conserved residue mutants. (a) Conservation analysis of \u003cem\u003eCci\u003c/em\u003eUPO. (b) 25-OH-VD\u003csub\u003e3\u003c/sub\u003e concentrations of alanine scanning mutants. (c) 25-OH-VD\u003csub\u003e3 \u003c/sub\u003econcentrations of saturation mutants at P108. (d) 25-OH-VD\u003csub\u003e3\u003c/sub\u003e concentrations of saturation mutants at H133.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/45d58ebfb07bad505a567966.png"},{"id":108491401,"identity":"4af95c26-f211-4a1d-b698-d250568e0bea","added_by":"auto","created_at":"2026-05-05 09:53:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90022,"visible":true,"origin":"","legend":"\u003cp\u003eThe 25-OH-VD\u003csub\u003e3\u003c/sub\u003e concentrations of the combination mutants.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/12ccb0ac2d66cd97545b56d8.png"},{"id":108491572,"identity":"4bf77f43-8618-41ac-9f57-9c5b007b6dea","added_by":"auto","created_at":"2026-05-05 09:54:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":346405,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of intermolecular interaction forces. (a) The interaction force between \u003cem\u003eCci\u003c/em\u003eUPO and ligand VD\u003csub\u003e3\u003c/sub\u003e. (b) The interaction force between I73M mutant and ligand VD\u003csub\u003e3\u003c/sub\u003e. (c) The interaction force between G245A mutant and ligand VD\u003csub\u003e3\u003c/sub\u003e. (d) The interaction force between P108K mutant and ligand VD\u003csub\u003e3\u003c/sub\u003e. (e) The interaction force between H133G mutant and ligand VD\u003csub\u003e3\u003c/sub\u003e. (f) The interaction force between I73M/P108K/G245A mutant and ligand VD\u003csub\u003e3\u003c/sub\u003e. Note: Dashed lines represent hydrophobic interaction forces; Solid lines represent hydrogen bonds; orange-yellow molecular structure represents VD\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/972cad8cbb5d3d433a9e3ba1.png"},{"id":108491304,"identity":"abc079c0-ef6f-447f-b496-d3602b20fcd8","added_by":"auto","created_at":"2026-05-05 09:53:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3641762,"visible":true,"origin":"","legend":"\u003cp\u003eRMSD and RMSF analysis of dominant mutants. (a) RMSD of mutant I73M. (b) RMSF of mutant I73M. (c) RMSD of mutant G245A. (d) RMSF of mutant G245A. (e) RMSD of mutant P108K. (f) RMSF of mutant P108K. (g) RMSD of mutant H133G. (h) RMSF of mutant H133G. (i) RMSD of I73M/P108K/G245A mutant. (j) RMSF of I73M/P108K/G245A mutant.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/357418f1b0a772d0f5d24dcd.png"},{"id":108491465,"identity":"d81a10e0-2869-44b4-b6ae-c45eb82b1d95","added_by":"auto","created_at":"2026-05-05 09:54:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1405159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 7\u003c/strong\u003e The effects of different induction conditions on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. (a) The influence of different induction temperatures on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. (b) The influence of different induced methanol concentrations on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. (c) The influence of different induction pH on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. (d)The influence of different concentrations of 5-ALA on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. (e) The influence of different induction times on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/5b337e811720ef1f915fa9f8.png"},{"id":108247481,"identity":"7fb9e9d6-0dd4-4cec-b7cf-e24a8689f72c","added_by":"auto","created_at":"2026-05-01 00:52:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1859087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 8\u003c/strong\u003e The effects of different components of the culture medium on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. (a) The influence of different types of carbon sources on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. (b) The influence of different carbon source concentrations on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. (c) The influence of different types of nitrogen sources on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. (d) The influence of different nitrogen source concentrations on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/ad7ec3ae28864554cd671e8f.png"},{"id":108247482,"identity":"2689278c-3e1e-4274-b7c8-ed4ad89e7041","added_by":"auto","created_at":"2026-05-01 00:52:08","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":983281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 9\u003c/strong\u003e The effects of different multi-enzyme cascade catalytic reaction systems on the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. (a) The influence of the concentration of glucose on the multi-enzyme cascade reaction. (b) The influence of concentration of glucose oxidase on the multi-enzyme cascade reaction. (c) The influence of 2-hydroxypropyl-β-cyclodextrin concentration on the multi-enzyme cascade reaction. (d) The influence of reaction temperature on the multi-enzyme cascade reaction. (e) The influence of reaction time on the multi-enzyme cascade reaction.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/86adbae620519d2f55d6d16a.png"},{"id":108247476,"identity":"041cb71c-036a-4e6d-8046-2cbc97d5cb10","added_by":"auto","created_at":"2026-05-01 00:51:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":306170,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/ab70cfae-d749-4460-8605-df739e6b5f99.pdf"},{"id":108491635,"identity":"2575b056-bb1d-4b2e-8cbe-c121800c2a2c","added_by":"auto","created_at":"2026-05-05 09:54:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":29301,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9459508/v1/52964c473fa5977ce5efce96.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eSemi‑rational design and modification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCci\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eUPO for the efficient biosynthesis of 25-hydroxyvitamin D\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs the bioactive form of Vitamin D\u003csub\u003e3\u003c/sub\u003e (VD\u003csub\u003e3\u003c/sub\u003e), 25-hydroxyvitamin D\u003csub\u003e3\u003c/sub\u003e (25-OH-VD\u003csub\u003e3\u003c/sub\u003e) exerts pharmacological effects on regulating calcium and phosphorus metabolism, alleviating osteoporosis and rickets, and is therefore widely used in clinical treatment\u003csup\u003e[1\u0026ndash;4]\u003c/sup\u003e. In addition, 25-OH-VD\u003csub\u003e3\u003c/sub\u003e has high application value in agriculture\u003csup\u003e[5\u0026ndash;7]\u003c/sup\u003e. Currently, the traditional chemical synthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e suffers from several limitations in industrial production, including complex reaction steps, environmental pollution, and the generation of by-products\u003csup\u003e[8\u0026ndash;9]\u003c/sup\u003e. Whereas, enzymatic synthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e with mild reaction conditions, high specificity and environmentally friendliness has attracted increasing attention\u003csup\u003e[10\u0026ndash;12]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAccording to the literature, the enzymes catalyzing the conversion of VD\u003csub\u003e3\u003c/sub\u003e to 25-OH-VD\u003csub\u003e3\u003c/sub\u003e are primarily cytochromes P450 (CYPs) and unspecific peroxygenases (UPOs) \u003csup\u003e[13\u0026ndash;17]\u003c/sup\u003e. CYP-mediated C25 hydroxylation of VD\u003csub\u003e3\u003c/sub\u003e suffers from NADPH dependency, complex electron transfer, and low catalytic efficiency, which limit its industrial application. UPOs resemble CYPs in structure, both containing a cysteine-coordinated heme unit\u003csup\u003e[18\u0026ndash;20]\u003c/sup\u003e. Instead of relying on expensive NAD(P)H to supply electrons, UPOs use hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) as the oxidant and oxygen donor, which is more economical\u003csup\u003e[21\u0026ndash;25]\u003c/sup\u003e. Notably, unspecific peroxygenase from \u003cem\u003eCoprinopsis cinerea\u003c/em\u003e (\u003cem\u003eCci\u003c/em\u003eUPO) catalyzes the regioselective hydroxylation of VD\u003csub\u003e3\u003c/sub\u003e at the C25 position, without the formation of other by-product\u003csup\u003e[26]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSemi-rational design and modification has proven highly effective for improving enzyme catalytic efficiency, primarily through engineering substrate-binding pockets and non-conserved residues. The substrate-binding pocket is a key region where the enzyme directly interacts with the substrate and participates in catalysis. Modification of this pocket has been shown to greatly increase catalytic efficiency, as exemplified by 3-ketoacyl-CoA thiolase and aldo-keto reductase. Liu et al. rationally engineered the substrate-binding pocket, increasing the catalytic efficiency of 3-ketoacyl-CoA thiolase for its substrate by 6.67-fold\u003csup\u003e[27]\u003c/sup\u003e. Niu et al. reshaped the substrate-binding pocket of aldo-keto reductase, yielding the mutant W21A/G53N with 330-fold higher catalytic activity\u003csup\u003e[28]\u003c/sup\u003e. In addition, non-conserved residues, predominantly located in surface loop regions and around the active center, offer untapped potential for improving catalytic efficiency. Mutations in these regions can synergize with mutations of key residues in the substrate-binding pocket to enhance catalytic efficiency. Zou et al. combined computational design with engineering of non-conserved residues to generate a focused mutation library, yielding the mutant A169P with a 52.04% increase in the catalytic efficiency of alpha-galactosidase\u003csup\u003e[29]\u003c/sup\u003e. Therefore, applying similar strategies to engineer the substrate-binding pocket and non-conserved residues of \u003cem\u003eCci\u003c/em\u003eUPO is expected to significantly enhance its catalytic efficiency for 25-OH-VD\u003csub\u003e3\u003c/sub\u003e production.\u003c/p\u003e \u003cp\u003eIn our previous research, \u003cem\u003eCci\u003c/em\u003eUPO was successfully secreted extracellularly in \u003cem\u003ePichia pastoris\u003c/em\u003e X33. However, the C25 hydroxylation of VD\u003csub\u003e3\u003c/sub\u003e catalyzed by \u003cem\u003eCci\u003c/em\u003eUPO yielded only 62.21 mg/L of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e[30]\u003c/sup\u003e. In this study, to enhance the efficiency of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e biosynthesis, semi-rational design and modification were performed on the substrate-binding pocket and non-conserved residues of \u003cem\u003eCci\u003c/em\u003eUPO. The mechanism underlying the improved performance was then elucidated by analyzing the substrate-binding pocket, molecular interactions, and molecular dynamics (MD) simulations. Furthermore, optimization of fermentation conditions and the multi-enzyme cascade reaction system further increased the 25-OH-VD\u003csub\u003e3\u003c/sub\u003e concentration. Semi-rational engineering of \u003cem\u003eCci\u003c/em\u003eUPO combined with process optimization significantly enhanced 25-OH-VD\u003csub\u003e3\u003c/sub\u003e biosynthesis, providing a feasible strategy for green and efficient production.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents\u003c/h2\u003e \u003cp\u003eRestriction endonucleases \u003cem\u003eSac\u003c/em\u003eI and \u003cem\u003eDpn\u003c/em\u003eI, 4 \u0026times; Loading Buffer were purchased from Takara (Dalian, China). Peptone, yeast extract, and glucose were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). VD\u003csub\u003e3\u003c/sub\u003e and biotin were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). VD\u003csub\u003e3\u003c/sub\u003e, 25-OH-VD\u003csub\u003e3\u003c/sub\u003e standard substances and glucose oxidase were purchased from Yuan Ye Biotechnology Co., Ltd. (Shanghai, China). Zeocin was purchased from Solarbio Technology Co., Ltd. (Beijing, China). Unless otherwise specified, all reagents used in this study were sourced from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Primer synthesis and DNA sequencing were performed by Talen-bio Biological Technology Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Strains and cultivation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e JM109 was used for cloning and plasmid propagation. \u003cem\u003ePichia pastoris\u003c/em\u003e X33 was used for the heterologous expression of variants. The recombinant plasmid pPICZA-P\u003csub\u003eAOX1\u003c/sub\u003e-P\u003csub\u003eDAS2\u003c/sub\u003e-SP\u003csub\u003eα factor\u003c/sub\u003e-\u003cem\u003eCci\u003c/em\u003eUPO was previously constructed in our laboratory.\u003c/p\u003e \u003cp\u003eThe recombinant \u003cem\u003ePichia pastoris\u003c/em\u003e strain was cultured at 30℃ for 16 h in 10 mL of YPD liquid medium. Then, the cells were transferred to 50 mL of BMGY liquid medium at an inoculation volume of 2% (v/v) and cultured at 30℃ for 24 h. The cells were collected by centrifugation at 5000 rpm for 5 min, and the supernatant was discarded. The cells were washed with sterile ddH\u003csub\u003e2\u003c/sub\u003eO to remove glycerol and resuspended in 50 mL of BMMY medium. Methanol (1.5%, v/v) and the heme precursor 5-aminolevulinic acid hydrochloride (5-ALA) at a final concentration of 45 \u0026micro;mol/L were added every 24 h, and continuous induction was carried out for 72 h at 25\u0026deg;C with shaking at 120 rpm. After induction, the fermentation supernatant containing \u003cem\u003eCci\u003c/em\u003eUPO was collected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Construction of the \u003cem\u003eCci\u003c/em\u003eUPO mutants\u003c/h2\u003e \u003cp\u003eThe plasmid pPICZA-P\u003csub\u003eAOX1\u003c/sub\u003e-P\u003csub\u003eDAS2\u003c/sub\u003e-SP\u003csub\u003eα factor\u003c/sub\u003e-\u003cem\u003eCci\u003c/em\u003eUPO carrying the gene \u003cem\u003eCci\u003c/em\u003eUPO (GenBank ID: XP_001831910.1) was used as the template, and reverse PCR primers were designed for amplification (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Subsequently, the reverse PCR product was digested with \u003cem\u003eDpn\u003c/em\u003eI at 37\u0026deg;C for 30 min to remove the template plasmid. After digestion, the reverse PCR products were purified using a gel extraction kit and transformed into \u003cem\u003eE. coli\u003c/em\u003e JM109 by heat shock method, followed by overnight incubation at 37\u0026deg;C. Single colonies were selected for colony PCR and sequencing to confirm the desired mutations. After successful sequencing, the recombinant plasmids were isolated and linearized using the restriction enzyme \u003cem\u003eSac\u003c/em\u003eI, and then transformed into \u003cem\u003ePichia pastoris\u003c/em\u003e X33 cells via electroporation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Multi-enzyme cascade reaction system\u003c/h2\u003e \u003cp\u003eA multi-enzyme cascade reaction system was constructed. In this system, glucose oxidase utilizes glucose and O\u003csub\u003e2\u003c/sub\u003e to generate H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in situ, which subsequently drives \u003cem\u003eCci\u003c/em\u003eUPO to catalyze the C25 hydroxylation of VD\u003csub\u003e3\u003c/sub\u003e. The reaction system consisted of 25 mmol/L glucose, 40 U/mL glucose oxidase, 8 mmol/L Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e buffer (pH 5.0), 22.5 mg/mL 2-hydroxypropyl-β-cyclodextrin, 0.5 g/L substrate VD\u003csub\u003e3\u003c/sub\u003e, with \u003cem\u003eCci\u003c/em\u003eUPO crude enzyme added to a final volume of 2 mL. The reaction was carried out at 30℃ and 220 rpm for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Identification of catalytic products\u003c/h2\u003e \u003cp\u003eThe reaction mixture was extracted three times with 70% (v/v) ethyl acetate, and the upper organic phase was collected. After evaporation of the combined organic phases, the residue was redissolved in 1 mL of methanol and filtered through a 0.22 \u0026micro;m organic membrane to obtain the sample for HPLC analysis.\u003c/p\u003e \u003cp\u003eHPLC analysis was performed on a Sunfire\u0026trade; C18 column at 40\u0026deg;C with UV detection at 265 nm. The mobile phase was acetonitrile/water solution and the injection volume was 20 \u0026micro;L. The flow rate was maintained at 1.0 mL/min. A linear gradient of 60\u0026ndash;100% aqueous acetonitrile was applied over 15 min, followed by an isocratic hold at 100% acetonitrile for an additional 20 min. Under these conditions, the retention times of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e and VD\u003csub\u003e3\u003c/sub\u003e were 15.44 min and 25.28 min, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Structure modeling and Molecular Dynamics Simulations\u003c/h2\u003e \u003cp\u003eHomology modeling of \u003cem\u003eCci\u003c/em\u003eUPO was performed using AlphaFold2, and the model was evaluated using the SAVES 6.0 website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://saves.mbi.ucla.edu/\u003c/span\u003e\u003cspan address=\"https://saves.mbi.ucla.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The substrate VD\u003csub\u003e3\u003c/sub\u003e was docked into the active center of \u003cem\u003eCci\u003c/em\u003eUPO using AutoDock 4.0. PyMol software was used to visualize the protein structure. FireProt (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://loschmidt.chemi.muni.cz/fireprot/\u003c/span\u003e\u003cspan address=\"https://loschmidt.chemi.muni.cz/fireprot/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to design more mutants. The Consurf web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://consurf.tau.ac.il\u003c/span\u003e\u003cspan address=\"http://consurf.tau.ac.il\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze the conservation of \u003cem\u003eCci\u003c/em\u003eUPO. The substrate-binding pocket volumes of wild-type and mutants were predicted with the DoGSiteScorer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://proteins.plus/\u003c/span\u003e\u003cspan address=\"https://proteins.plus/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The PLIP website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plip-tool.biotec.tu-dresden.de/plip-web/plip/index\u003c/span\u003e\u003cspan address=\"https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict the interaction forces between the protein and the substrate VD\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eMolecular dynamics (MD) simulations were carried out using GROMACS 2021 software. The protein was placed in a dodecahedral box under the CHARMM36 force field and TIP3P water model. The system was neutralized with 0.15 mol/L NaCl. Energy minimization was performed, followed by 100 ps of NVT and NPT equilibration at 300 K and 101.3 kPa, respectively. Finally, three independent 100 ns simulations were conducted. After simulation, the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Fermentation optimization of the recombinant \u003cem\u003eP. pastoris\u003c/em\u003e strains\u003c/h2\u003e \u003cp\u003eThe optimal \u003cem\u003ePichia pastoris\u003c/em\u003e strain X33/pPICZA-P\u003csub\u003eAOX1\u003c/sub\u003e-P\u003csub\u003eDAS2\u003c/sub\u003e-SP\u003csub\u003eα factor\u003c/sub\u003e-\u003cem\u003eCci\u003c/em\u003eUPO\u003csup\u003eI73M/P108K/G245A\u003c/sup\u003e, designated as X-M1, was used for subsequent fermentation optimization. These parameters included induction temperature, methanol concentration, induction pH, 5-aminolevulinic acid (5-ALA) concentration, induction time, and the types and concentrations of carbon and nitrogen sources in the fermentation medium.\u003c/p\u003e \u003cp\u003eEach condition was optimized sequentially based on the optimal condition obtained in the previous step. The initial methanol concentration, pH, 5-ALA concentration and induction time were set at 1.5%(v/v), 5.0, 45 \u0026micro;mol/L and 72 h, respectively. Meanwhile, 1%(v/v) glycerol was used as the carbon source of BMGY medium. Yeast extract and peptone (1:2, w/w) were used as the compound nitrogen source in both BMGY and BMMY, with a total concentration of 30 g/L. After induction, the multi-enzyme cascade reaction was performed, and the concentration of product 25-OH-VD\u003csub\u003e3\u003c/sub\u003e was analyzed by HPLC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Optimization of the multi-enzyme cascade reaction\u003c/h2\u003e \u003cp\u003eThe reaction system was scaled up to 50 mL, and the multi-enzyme cascade reaction was systematically optimized. Each condition was optimized sequentially based on the optimal condition obtained from the previous step. During optimization of glucose concentration, the concentrations of glucose oxidase and the co-solvent 2-hydroxypropyl-β-cyclodextrin were set at 40 U/mL and 22.5 mg/mL, respectively, while the reaction temperature and time were fixed at 30℃ and 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical methodology analysis\u003c/h2\u003e \u003cp\u003eExperimental data were processed using SPSS statistical software and Microsoft Office Excel. Analysis of variance (ANOVA) was used to analyze the results, and the data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.1 Semi-rational design and modification of the substrate-binding pocket\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe homology model of \u003cem\u003eCci\u003c/em\u003eUPO was constructed using AlphaFold2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), and the model quality was evaluated using the SAVES 6.0 server. Ramachandran plot analysis revealed that 88.1% of residues in the \u003cem\u003eCci\u003c/em\u003eUPO model occupied the most favored regions, 11.9% occupied allowed regions, and none were found in disallowed regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The model achieved an ERRAT score of 89.362 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), and 94.07% of residues exhibited a 3D-1D score\u0026thinsp;\u0026ge;\u0026thinsp;0.1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), collectively indicating that the \u003cem\u003eCci\u003c/em\u003eUPO model was of high quality.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAutoDock 4.0 software was used to dock the substrate VD\u003csub\u003e3\u003c/sub\u003e into the active center of the \u003cem\u003eCci\u003c/em\u003eUPO homology model. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, the ligand VD\u003csub\u003e3\u003c/sub\u003e was located above the heme active center of \u003cem\u003eCci\u003c/em\u003eUPO, with a distance of 2.7 \u0026Aring; between the C25 atom of VD₃ and the heme, suggesting that \u003cem\u003eCci\u003c/em\u003eUPO is more likely to catalyze the C25 hydroxylation of VD\u003csub\u003e3\u003c/sub\u003e. The \u003cem\u003eCci\u003c/em\u003eUPO-VD₃ docking complex was visualized using PyMOL software. A total of twenty-two amino acid residues located within 6 \u0026Aring; of VD\u003csub\u003e3\u003c/sub\u003e were identified as potential substrate-binding sites, including M69, I73, F76, V77, F121, E122, P188, R189, F191, T192, Y194, E196, S197, F199, G245, L249, D250, V251, F280, F283, L286, and P322 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlanine scanning mutagenesis was conducted to evaluate the functional roles of the 22 amino acid residues identified as potential substrate-binding sites. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, the mutant G245A exhibited superior catalytic performance, producing 71.99 mg/L of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e, representing a 15.72% increase over the wild type (62.21 mg/L). Therefore, G245 was identified as a key amino acid residue affecting the catalytic efficiency of \u003cem\u003eCci\u003c/em\u003eUPO. Saturation mutagenesis was subsequently performed at residue G245. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, the G245Y mutant increased the concentration of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e to 65.63 mg/L, representing a 5.50% increase over the wild type. To obtain more mutants with improved catalytic efficiency, FireProt was used for designing mutants\u003csup\u003e[31]\u003c/sup\u003e, including A195C, A195P, I73M, V77M, V77L, and V77P. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, the I73M mutant exhibited higher catalytic efficiency for the C25 hydroxylation of VD\u003csub\u003e3\u003c/sub\u003e, with the concentration of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e reaching 78.68 mg/L, which represented a 26.47% increase over the wild type.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Semi-rational design and modification of non-conserved residues\u003c/h2\u003e \u003cp\u003eNon-conserved residues serve as potential sites for enhancing enzymatic catalytic efficiency. The conservation of \u003cem\u003eCc\u003c/em\u003eiUPO was assessed using the ConSurf server\u003csup\u003e[32]\u003c/sup\u003e. According to the conservation analysis results in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, a conservation score of 6 was set as the threshold to differentiate conserved residues from non-conserved ones of \u003cem\u003eCci\u003c/em\u003eUPO\u003csup\u003e[33]\u003c/sup\u003e. Eleven non-conserved residues around the active center were selected for alanine scanning mutagenesis. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, P108A and H133A mutants exhibited higher catalytic efficiency than the wild type, producing 25-OH-VD\u003csub\u003e3\u003c/sub\u003e at concentrations of 69.02 mg/L and 80.78 mg/L, respectively. P108 and H133 were selected for further saturation mutagenesis to screen for dominant mutants. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, the P108K mutant exhibited significantly improved catalytic performance, with the concentration of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e reaching 82.11 mg/L, which was 31.99% higher than that of the wild type. The remaining mutations had little positive effect on the catalytic efficiency. The H133G mutant also exhibited enhanced catalytic efficiency, yielding 25-OH-VD\u003csub\u003e3\u003c/sub\u003e at a concentration of 83.13 mg/L, representing a 33.63% increase over the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Construction and screening of combined mutants\u003c/h2\u003e \u003cp\u003eTo further enhance catalytic efficiency, combined mutants were generated by combining the dominant mutants G245A, I73M, P108K, H133A, and H133G. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, seven combination mutants exhibited superior catalytic performance than the wild type. Among all dominant mutants constructed in this work, the triple mutant I73M/P108K/G245A (M1), combining substrate-binding pocket modifications (I73M/G245A) with a non-conserved substitution (P108K), exhibited the highest catalytic efficiency for the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e, yielding a concentration of 87.83 mg/L. This represented a 41.18% increase compared with that of the wild type. The recombinant \u003cem\u003eP. pastoris\u003c/em\u003e strain X33/pPICZA-P\u003csub\u003eAOX1\u003c/sub\u003e-P\u003csub\u003eDAS2\u003c/sub\u003e-SP\u003csub\u003eα factor\u003c/sub\u003e-\u003cem\u003eCci\u003c/em\u003eUPO\u003csup\u003eI73M/P108K/G245A\u003c/sup\u003e was designated as X-M1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Insights into the enhanced catalytic efficiency by structure analysis\u003c/h2\u003e \u003cp\u003eIn order to explore the potential mechanism of \u003cem\u003eCci\u003c/em\u003eUPO and its mutants, the substrate-binding pocket and enzyme-substrate interactions were analyzed. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the I73M mutation reduced the pocket volume from 548.42 \u0026Aring;\u003csup\u003e3\u003c/sup\u003e to 417.47 \u0026Aring;\u003csup\u003e3\u003c/sup\u003e, and the G245A, P108K, and H133G mutants exhibited slightly smaller pocket volumes. The triple mutant I73M/P108K/G245A showed a reduced volume of 425.73 \u0026Aring;\u003csup\u003e3\u003c/sup\u003e. The reduced substrate-binding pocket may enhance the binding affinity between the enzyme and the substrate, thereby contributing to the improved catalytic efficiency. Furthermore, enzyme-substrate interactions analysis revealed that the wild type formed hydrophobic interactions with VD\u003csub\u003e3\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), while the I73M mutant displayed similar interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The G245A and P108K mutants exhibited increased hydrophobic contacts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), and the H133G mutant formed additional hydrophobic interactions and a hydrogen bond (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Notably, the triple mutant I73M/P108K/G245A showed substantially enhanced hydrophobic interactions compared with the wild type and single mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). The progressive increase in hydrophobic interactions from the wild type to the triple mutant suggests a possible correlation with the improved catalytic efficiency. The synergistic pocket constriction by the three mutations is proposed to restrict the conformational freedom of VD\u003csub\u003e3\u003c/sub\u003e, more accurately aligning its C25-H bond with the heme iron-bound oxygen atom. This favorable binding posture may facilitate substrate-induced fit and stabilize the transition state, thereby contributing to the greatly enhanced catalytic efficiency of C25 hydroxylation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrediction of substrate-binding pocket volume\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMutant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePocket volume (\u0026Aring;\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e548.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI73M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e417.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG245A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e539.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP108K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e547.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH133G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e548.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI73M/P108K/G245A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e425.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the catalytic mechanism, molecular dynamics (MD) simulations were performed on the dominant mutants. Compared with the wild type, the I73M, G245A, P108K, and H133G mutants exhibited lower RMSD fluctuations, indicating enhanced protein stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). At position I73, reduced RMSF was observed, and protein rigidity was enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ed, the RMSF at position G245 was increased, leading to enhanced protein flexibility, which may facilitate substrate entry, localization, and transition state stabilization. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ef, the RMSF at position 108 was reduced, indicating enhanced enzyme rigidity, thereby facilitating transition state stabilization. The introduction of glycine at position 133 increased the RMSF (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eh). This mutation enhanced protein flexibility, which may facilitate substrate-induced fit, allowing the catalytic groups to approach the reaction center with a more favorable spatial orientation, thereby promoting the formation and stability of the transition state and improving catalytic efficiency. Notably, the triple mutant I73M/P108K/G245A exhibited a lower RMSD value and reached the plateau phase earlier, indicating significantly enhanced overall structural stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ei). Concurrently, the RMSF of the triple mutant decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ej), suggesting enhanced protein rigidity and better transition state stabilization, thereby contributing to the improved catalytic efficiency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Optimization of fermentation conditions and multi-enzyme cascade reaction\u003c/h2\u003e \u003cp\u003eThe recombinant \u003cem\u003eP. pastoris\u003c/em\u003e strain X-M1, harboring methanol-inducible promoters for \u003cem\u003eCci\u003c/em\u003eUPO expression, served as the production host. Induction conditions of \u003cem\u003eP. pastoris\u003c/em\u003e strain X-M1 such as temperature, methanol concentration, pH, 5-aminolevulinic acid (5-ALA) concentration and induction time directly influence both cell growth and heterologous protein expression\u003csup\u003e[34\u0026ndash;36]\u003c/sup\u003e. Therefore, to maximize the production of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e, the induction conditions were systematically optimized. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the optimal induction conditions were determined as follows: temperature 25\u0026deg;C, methanol 1.5% (v/v), pH 5.0, 5-ALA 60 \u0026micro;mol/L, and induction time 72 h. Under these optimized conditions, with a substrate VD\u003csub\u003e3\u003c/sub\u003e concentration of 0.5 g/L, the concentration of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e reached 88.66 mg/L.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, the fermentation medium composition of \u003cem\u003eP. pastoris\u003c/em\u003e strain X-M1 was optimized. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003eb, glucose was identified as the optimal carbon source, with an optimal concentration of 20 g/L. Regarding the nitrogen source, a mixture of yeast extract and peptone at a ratio of 1:2 (w/w) with a total concentration of 45 g/L exhibited the best performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003ec and Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003ed). Following these optimizations, the concentration of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e increased to 111.46 mg/L.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUPOs are sensitive to the concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Direct addition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e may cause excessively high local concentrations, leading to irreversible damage to the heme center of UPO and resulting in enzyme inactivation\u003csup\u003e[37,38]\u003c/sup\u003e. Glucose oxidase utilizes glucose and oxygen as substrates to generate H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in situ. This reaction, together with \u003cem\u003eCci\u003c/em\u003eUPO, constitutes a multi-enzyme cascade reaction system. Based on the above optimizations of induction conditions and medium components, the reaction system was scaled up to 50 mL, and the multi-enzyme cascade reaction was systematically optimized. As illustrated in Fig.\u0026nbsp;10, the optimal multi-enzyme cascade reaction system consisted of 25 mmol/L glucose, 20 U/mL glucose oxidase, and 45 mg/mL 2-hydroxypropyl-β-cyclodextrin, with a reaction temperature of 30\u0026deg;C and a reaction time of 24 h. Under these optimized conditions, the concentration of 25-OH-VD₃ reached 152.50 mg/L, representing a 73.63% increase compared with the level prior to optimization (87.83 mg/L).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study successfully enhanced the catalytic efficiency of \u003cem\u003eCci\u003c/em\u003eUPO for C25 hydroxylation of VD\u003csub\u003e3\u003c/sub\u003e through semi‑rational design and modification of its substrate‑binding pocket and non-conserved residues. Utilizing site-directed saturation mutagenesis coupled with product concentration-based screening, the resulting triple mutant I73M/P108K/G245A exhibited improved catalytic efficiency, with the 25-OH-VD\u003csub\u003e3\u003c/sub\u003e concentration reaching 87.83 mg/L. Mechanistic analysis demonstrated that the triple mutant exhibited a constricted substrate-binding pocket, strengthened hydrophobic interactions, and enhanced protein stability and rigidity, which synergistically contributed to the improvement in catalytic efficiency. Following systematic optimization of fermentation conditions and the multi-enzyme cascade reaction, the concentration of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e reached 152.50 mg/L. This work provides a feasible design and modification strategy for \u003cem\u003eCci\u003c/em\u003eUPO, laying the foundation for the efficient and green synthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eDr. Zhenghong Xu, a co-author of this study, is a member of the editorial board of Systems Microbiology and Biomanufacturing. However, this did not influence the peer review process or the decision-making for this manuscript. All authors declare no other conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Key R \u0026amp; D Program of China (No. 2024YFA0917900) and the Fundamental Research Funds for the Central Universities (No. JUSRP202504023).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJingyi Zhou: Writing Original Draft, Investigation, Methodology, Data Curation, Visualization. Yingjia Tong: Investigation, Formal analysis. Fan He: Methodology, Formal analysis. Kai Chu: Investigation, Methodology. Hang Zhong: Methodology, Formal analysis. Jinsong Shi: Supervision, Validation, Formal analysis. Zhenghong Xu: Conceptualization, Supervision, Project administration. Hui Li: Conceptualization, Funding acquisition, Supervision, Methodology, Formal analysis, Writing - Review \u0026amp; Editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang Z Y, Zeng Y, Jia H M, et al. Bioconversion of Vitamin D\u003csub\u003e3\u003c/sub\u003e to bioactive Calcifediol and Calcitriol as high-value compounds. Biotechnology for Biofuels and Bioproducts, 2022, 15: 109-121.\u003c/li\u003e\n\u003cli\u003ePark C Y, Shin S, Han S N. Multifaceted roles of Vitamin D for diabetes: from immunomodulatory functions to metabolic regulations. Nutrients, 2024, 16(18): 3185-3207.\u003c/li\u003e\n\u003cli\u003eKoga G K C, Maeda S S, Lazaretti-Castro M，et al. Rapid and dose-dependent increase of 25(OH)D levels after Calcifediol supplementation in a woman with obesity, chronic liver disease, and osteoporosis. 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Engineering \u003cem\u003ePichia pastoris\u003c/em\u003e for high-level biosynthesis of squalene. Biochemical Engineering Journal, 2025, 217: 109677-109687.\u003c/li\u003e\n\u003cli\u003eParashar D, Satyanarayana T. Enhancing the production of recombinant acidic \u0026alpha;-amylase and phytase in \u003cem\u003ePichia pastoris\u003c/em\u003e under dual promoters constitutive (GAP) and inducible (AOX) in mixed fed batch high cell density cultivation. Process Biochemistry, 2016, 51(10): 1315-1322. \u003c/li\u003e\n\u003cli\u003eAlexander K, Katrin C, Ren\u0026eacute; U, et al. Exploring the catalase activity of unspecific peroxygenases and the mechanism of peroxide-dependent heme destruction. Journal of Molecular Catalysis B-Enzymatic, 2016, 134: 238-246.\u003c/li\u003e\n\u003cli\u003eValderrama B, Ayala M, Vazquez Duhalt R. Suicide inactivation of peroxidases and the challenge of engineering more robust enzymes. Chemistry \u0026amp; Biology, 2002, 9(5): 555-565.\u003c/li\u003e\n\u003c/ol\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":"25-OH-VD3, CciUPO, Biosynthesis, Semi-rational design and modification, multi-enzyme cascade reaction","lastPublishedDoi":"10.21203/rs.3.rs-9459508/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9459508/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e25-Hydroxyvitamin D\u003csub\u003e3\u003c/sub\u003e (25-OH-VD\u003csub\u003e3\u003c/sub\u003e) is the main active form of Vitamin D\u003csub\u003e3\u003c/sub\u003e and has broad applications in clinical treatment and agriculture. Although current industrial production predominantly relies on chemical synthesis, the growing demand for green and efficient manufacturing has accelerated the development of microbial enzymatic conversion methods. Due to its high reaction specificity, the unspecific peroxygenase from \u003cem\u003eCoprinopsis cinerea\u003c/em\u003e (\u003cem\u003eCci\u003c/em\u003eUPO) represents a promising biocatalyst for the synthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. However, its low catalytic efficiency limits further application in the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e. To address this, semi-rational design was employed to modify the substrate-binding pocket and non-conserved residues of \u003cem\u003eCci\u003c/em\u003eUPO. The resulting triple mutant I73M/P108K/G245A increased the 25-OH-VD\u003csub\u003e3\u003c/sub\u003e concentration to 87.83 mg/L, representing a 41.18% increase over the wild type (62.21 mg/L). The mechanism for enhanced catalytic efficiency was elucidated through analysis of the substrate-binding pocket, enzyme-substrate interactions, and molecular dynamics simulations. Subsequently, the fermentation conditions and multi-enzyme cascade reaction were optimized. Under optimized conditions with a substrate VD\u003csub\u003e3\u003c/sub\u003e concentration of 0.5 g/L, the 25-OH-VD\u003csub\u003e3\u003c/sub\u003e concentration further increased to 152.50 mg/L. The combination of semi-rational engineering and process optimization of \u003cem\u003eCci\u003c/em\u003eUPO offers a feasible, green and efficient strategy for the biosynthesis of 25-OH-VD\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e","manuscriptTitle":"Semi‑rational design and modification of CciUPO for the efficient biosynthesis of 25-hydroxyvitamin D3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-01 00:51:53","doi":"10.21203/rs.3.rs-9459508/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":"bcb23f32-211d-4478-b6bd-3b6ca1648573","owner":[],"postedDate":"May 1st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-04-30T00:15:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T21:08:29+00:00","index":33,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T01:54:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-01 00:51:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9459508","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9459508","identity":"rs-9459508","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}