Mutant of aromatic dioxygenase cpuado and application thereof

Abstract

The application discloses a mutant of aromatic dioxygenase CpuADO and application thereof, and the amino acid sequence of the mutant of the aromatic dioxygenase CpuADO is shown in SEQ ID NO:2 or SEQ ID NO:3. It is found by the application that the aromatic dioxygenase CpuADO has wide-spectrum substrate conversion capacity, can catalyze various lignin monomer derived aromatic olefin substrates to generate corresponding aromatic aldehyde or aromatic ketone products, and the catalytic efficiency of two mutants obtained after site-directed mutagenesis of the aromatic dioxygenase CpuADO is further improved. The problems of poor adaptability, narrow substrate spectrum and low catalytic efficiency of existing aromatic dioxygenases to lignin monomer derived aromatic olefin substrates are solved, the high-value utilization of various types of lignin monomer derived aromatic olefins is facilitated, and an efficient new enzyme tool is provided for green biological oxidation and conversion of lignin monomer derived aromatic compounds.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and biocatalysis technology, specifically relating to a mutant of the aromatic dioxygenase CpuADO with broad-spectrum substrate conversion ability and its application in catalyzing the oxidative cleavage of lignin monomer-derived aromatic olefins to prepare aromatic aldehydes / ketones. Background Technology

[0002] Aromatic aldehydes and ketones are important high-value-added fine chemicals, widely used in fragrances, pharmaceutical intermediates, and agrochemicals. Vanillin, eugenol, benzaldehyde, and their structural analogs, in particular, have excellent aroma properties and reactivity, resulting in significant market demand in the food, daily chemical, and pharmaceutical industries. Currently, the industrial preparation of aromatic aldehydes and ketones mainly relies on chemical oxidation routes, with typical methods including benzylic CH oxidation, alcohol oxidation, and ozone oxidation. These chemical synthesis methods often require strong oxidants, organic solvents, and high-temperature or high-pressure conditions, leading to poor reaction selectivity, numerous byproducts, high energy consumption, and severe pollution, which is inconsistent with the development direction of green chemistry.

[0003] With the advancement of bio-based resource development, lignin is considered an important source of renewable aromatic compounds. Lignin depolymerization yields a series of lignin monomer-derived aromatic olefins, such as sinigrin, coniferyl alcohol, p-coumaryl alcohol, 4-vinylguaiacol, and isoeugenol. These substrates provide a rich raw material base for the bio-based preparation of aromatic aldehydes and ketones. However, how to efficiently convert these lignin-based aromatic olefins into their corresponding aromatic aldehydes or ketones under mild conditions remains a key problem that urgently needs to be solved.

[0004] Aromatic dioxygenases (ADOs) are a class of heme iron-independent oxidases that catalyze the ozone-like cleavage of C=C double bonds in aromatic alkenes under mild conditions. They convert substrates into aromatic aldehydes or ketones without the need for exogenous cofactors, thus being considered a "bio-ozonolysis" tool enzyme. Since their first report in 2007, ADOs from various sources have been used in the biotransformation of lignin-derived p-hydroxystyrene compounds. Existing studies have shown that some ADOs exhibit activity towards substrates such as isoeugenol, but their substrate spectrum is narrow, and their catalytic efficiency for multi-substituted lignin monomer-derived aromatic alkenes (such as sinaponicol, pterostilbene, and 4,4'-dihydroxystilbene) is limited, restricting their application in the high-value utilization of lignin. Furthermore, the structural basis affecting the catalytic efficiency of ADOs remains unclear, hindering further optimization of the enzyme's catalytic performance and its engineering scale-up applications. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide an aromatic dioxygenase mutant with a broad substrate spectrum and high catalytic efficiency.

[0006] The technical solutions for achieving the above-mentioned objectives include the following.

[0007] In a first aspect, the present invention provides a mutant of the aromatic dioxygenase CpuADO, the amino acid sequence of which is shown in SEQ ID NO:2 or SEQ ID NO:3.

[0008] In a second aspect, the present invention provides a gene encoding a mutant of the aromatic dioxygenase CpuADO, the nucleotide sequence of which is shown in SEQ ID NO:5 or SEQ ID NO:6, or the amino acid sequence encoded by the nucleotide sequence of which is shown in SEQ ID NO:2 or SEQ ID NO:3.

[0009] A third aspect of the present invention provides a recombinant expression vector containing the coding gene of a mutant of the above-mentioned aromatic dioxygenase CpuADO.

[0010] In a fourth aspect, the present invention provides a recombinant engineered bacterium transformed into a recombinant expression vector containing the coding gene of the mutant of the aromatic dioxygenase CpuADO.

[0011] In a fifth aspect, the present invention provides the use of the above-mentioned mutant of the aromatic dioxygenase CpuADO, the coding gene of the above-mentioned mutant of the aromatic dioxygenase CpuADO, the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineered bacteria in catalyzing the derivatization of lignin monomers into aromatic olefins to produce aromatic aldehydes or aromatic ketones.

[0012] A sixth aspect of the present invention provides a method for preparing aromatic aldehydes or aromatic ketones, comprising the following steps: catalyzing a reaction of sinigrin with a mutant of the aromatic dioxygenase CpuADO, whose amino acid sequence is shown in SEQ ID NO:2, wherein the conditions for the catalytic reaction are 30°C to 45°C and pH 7.0 to 9.0; or comprising the following steps: catalyzing a reaction of 4-vinylphenol, 4-vinylguaiacol, isoeugenol, sinigrin, or coniferol with a mutant of the aromatic dioxygenase CpuADO, whose amino acid sequence is shown in SEQ ID NO:3, wherein the conditions for the catalytic reaction are 30°C to 45°C and pH 7.0 to 9.0.

[0013] The inventors of this invention discovered that the aromatic dioxygenase CpuADO, derived from the fungus *Coniochaeta pulveracea*, possesses a broad-spectrum substrate transformation capability, catalyzing the conversion of various lignin monomer-derived aromatic olefin substrates into corresponding aromatic aldehydes or ketones. Furthermore, the inventors found that replacing the phenylalanine (F) at position 349 of the aromatic dioxygenase CpuADO with tryptophan (W), resulting in the mutant CpuADO-F349W, significantly improves the catalytic efficiency for the substrate sinaponicum. Similarly, replacing the tryptophan (W) at position 338 of the aromatic dioxygenase CpuADO with aspartic acid (D), resulting in the mutant CpuADO-W338D, significantly improves the catalytic efficiency for substrates such as 4-vinylphenol, 4-vinylguaiacol, isoeugenol, coniferyl alcohol, and sinaponicum. This invention solves the problems of poor adaptability and low catalytic efficiency of existing aromatic dioxygenases for lignin monomer-derived aromatic olefin substrates, thus facilitating the high-value utilization of various types of lignin monomer-derived aromatic olefins.

[0014] The mutant of the aromatic dioxygenase CpuADO of the present invention can catalyze the derivatization of aromatic olefins from lignin monomers to produce aromatic aldehydes or ketones. The reaction system does not require exogenous cofactors and uses only oxygen as an oxidant. It can be carried out efficiently in aqueous phase or in systems containing a small amount of organic cosolvent, avoiding the problems of using large amounts of strong oxidants, heavy metal catalysts and organic solvents in traditional chemical oxidation. It has the characteristics of being green, mild and efficient, and meets the requirements of green chemistry and sustainable development.

[0015] This invention elucidates the molecular mechanism of the aromatic dioxygenase CpuADO through crystal structure analysis and molecular dynamics simulation, providing a structural basis and design basis for further targeted or combinatorial mutations, which is beneficial for further engineering modification and the construction of higher-performance ADO mutants. Attached Figure Description

[0016] Figure 1 The results are from CpuADO's SDS-PAGE. Lane M: Protein Marker; Lane 1: Protein sample after total bacterial cell disruption; Lane 2: Protein sample from the supernatant after centrifugation of total bacterial cell disruption; Lane 3: Protein sample from the precipitate after centrifugation of total bacterial cell disruption; Lane 4: Protein sample passing through the nickel column during purification; Lane 5: Protein sample after purification with 55 mM imidazole during nickel column purification; Lane 6: Protein sample eluted with 200 mM imidazole.

[0017] Figure 2 This is the substrate spectrum for CpuADO.

[0018] Figure 3 Gas chromatogram results for the formation of the corresponding aromatic aldehydes from sinigrin catalyzed by CpuADO.

[0019] Figure 4 Gas chromatogram results for CpuADO-catalyzed isoeugenol to the corresponding aromatic ketones.

[0020] Figure 5 Gas chromatogram results for the CpuADO-catalyzed conversion of resveratrol to the corresponding aromatic aldehydes.

[0021] Figure 6 This is a schematic diagram of the crystal structure of CpuADO.

[0022] Figure 7 This is a three-dimensional structural diagram of the substrate channel, catalytic active site, and structure-directed mutation site of CpuADO. Figure (A) shows the internal cavity structure of CpuADO viewed from the direction of the active site; light purple represents the protein molecule surface, and orange spheres represent the active site Fe. 2+ The yellow bars represent histidines H195, H312, and H513, while the blue bars represent key residues W338, F349, and R352 located near the substrate channel. The red dashed lines and arrows indicate the substrate channel path and entry point from the protein surface to the active site. Figure (B) is a magnified view from the channel entry direction, showing the spatial configuration of residues F349, R352, N42, M247, and H249 near the entry point and the channel.

[0023] Figure 8 This is a structural comparison diagram of CpuADO and MapADO.

[0024] Figure 9 Fe in the last frame of a molecular dynamics simulation of the substrate sinapoxetine for mutants CpuADO-F349W(A) and CpuADO-W338D(B). 2+ -C=C distance.

[0025] Figure 10 The active sites and structural kinetics of CpuADO WT-sinopeptidyl complex, mutant CpuADO-F349W-sinopeptidyl complex, and mutant CpuADO-W338D-sinopeptidyl complex were analyzed in a 100 ns molecular dynamics simulation. Figure (A) shows the active sites and structural kinetics of Fe. 2+ -C=C Distance changes over time; Figure (B) shows the RMSD curve of the main chain.

[0026] Figure 11 Fe in the last frame of a molecular dynamics simulation of the mutant CpuADO-W338D against substrates 4-vinylguaiacol (A), isoeugenol (B), and coniferyl alcohol (C) 2+ -C=C distance.

[0027] Figure 12The active sites and structural dynamics of the complex of mutant CpuADO-W338D with 4-vinylguaiacol, isoeugenol, and coniferyl alcohol were analyzed in a 100 ns molecular dynamics simulation. Figures (A), (C), and (E) show the active sites and structural dynamics of Fe. 2+ -C=C Distance changes over time; Figures (B), (D), and (F) are the main chain RMSD curves. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0030] The inventors of this invention, during the crystal structure analysis of the aromatic dioxygenase CpuADO derived from the fungus *Coniochaeta pulveracea*, discovered that CpuADO possesses a typical heptal β-helical fold structure and contains Fe at its catalytic center. 2+ ions, the Fe 2+The CpuADO crystal structure, formed by the coordination of four histidine residues H195, H249, H312 and H513, has a crystal structure resolution of approximately 2.2 Å (PDB ID: 9LP3). Its structure contains substrate channels from the protein surface to the active site, which can accommodate various lignin monomer-derived aromatic olefin substrates such as sinigrin, coniferyl alcohol, isoeugenol, and 4-vinylguaiacol. Substrate screening results for CpuADO indicate that it can initiate C=C double bond oxidative cleavage reactions on various lignin monomer-derived aromatic olefin substrates, including monocyclic and dicyclic aromatic olefins (such as 4-vinylphenol, 4-isopropenylphenol, 4-vinylguaiacol, isoeugenol, sinaponicol, trans-4-hydroxystilbene, resveratrol, pterostilbene, and 4,4'-dihydroxystilbene), generating corresponding aromatic aldehydes or ketones. It exhibits high conversion rates of catalytic substrates containing both para-hydroxyl and methoxy substituents (such as 4-vinylguaiacol, isoeugenol, and isotannine), making it suitable for the high-value utilization of various types of lignin monomer-derived aromatic compounds. Based on CpuADO crystal structure and molecular dynamics simulation analysis, residues such as W338, F349, and R352 were identified as key amino acids located near the substrate channel entrance or substrate pocket, regulating substrate localization and electron distribution. Further site-directed mutagenesis of multiple substrate channel-related residues in CpuADO was conducted, resulting in two mutants, CpuADO-F349W and CpuADO-W338D, which significantly improved the catalytic performance for typical lignin monomer-derived aromatic olefin substrates. For the glucosinolate substrate, the wild-type CpuADO achieved a conversion rate of 6.21%, with syringaldehyde as the main product. The mutant CpuADO-F349W increased the conversion rate to 11.01%, a 77.3% improvement over the wild-type. The mutant CpuADO-W338D showed increased conversion rates of 4-vinylphenol, 4-vinylguaiacol, isoeugenol, coniferyl alcohol, and glucosinolate by 53.8%, 32.3%, 22.2%, 35.8%, and 36.9%, respectively.

[0031] In some embodiments of the present invention, a mutant of the aromatic dioxygenase CpuADO is disclosed, the amino acid sequence of which is shown in SEQ ID NO:2 or SEQ ID NO:3.

[0032] In other embodiments of the present invention, the coding gene of the above-mentioned mutant aromatic dioxygenase CpuADO is disclosed, the nucleotide sequence of which is shown in SEQ ID NO:5 or SEQ ID NO:6, or the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO:2 or SEQ ID NO:3. The nucleotide sequence of the coding gene of the mutant aromatic dioxygenase CpuADO can be deduced by those skilled in the art from the amino acid sequence based on codon degeneracy.

[0033] In other embodiments of the present invention, a recombinant expression vector containing the coding gene of the aforementioned aromatic dioxygenase CpuADO mutant is disclosed. This recombinant expression vector includes a promoter, a coding gene, and a terminator.

[0034] In one embodiment, the expression vector is a pET vector. In this invention, the expression vector may be a pET vector, such as pET-28a(+), pET-15b, pET-21a(+), pET-22b(+), pET-32a(+), pET-Duet series, etc.

[0035] In one embodiment, the expression vector is the pET Duet-1 vector.

[0036] In other embodiments of the present invention, recombinant engineered bacteria transformed with an expression vector containing the coding gene of the above-mentioned aromatic dioxygenase CpuADO mutant are disclosed. After induction of expression by the recombinant engineered bacteria, the aromatic dioxygenase CpuADO mutant can be obtained by Ni-NTA affinity chromatography purification.

[0037] In one embodiment, the engineered bacterium is E. coli.

[0038] In one embodiment, the engineered bacterium is E. coli BL21(DE3).

[0039] In other embodiments of the present invention, the use of the above-mentioned aromatic dioxygenase CpuADO mutant, the coding gene of the above-mentioned aromatic dioxygenase CpuADO mutant, the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineered bacteria in catalyzing the derivatization of lignin monomers into aromatic olefins to produce aromatic aldehydes or aromatic ketones is disclosed.

[0040] In one embodiment, the lignin monomer-derived aromatic olefin is 4-vinylphenol, 4-isopropenylphenol, 4-vinylguaiacol, isoeugenol, sinigrin, coniferyl alcohol, 4,4'-dihydroxystilbene, trans-4-hydroxystilbene, resveratrol, isopanthene, oxidized resveratrol, and / or pterostilbene.

[0041] In some other embodiments of the present invention, a method for preparing aromatic aldehydes is disclosed, comprising the following steps: using a mutant of aromatic dioxygenase CpuADO with an amino acid sequence as shown in SEQ ID NO:2 to catalyze a reaction of sinapoxetine, wherein the conditions of the catalytic reaction are 30°C to 45°C and pH 7.0 to 9.0.

[0042] In some other embodiments of the present invention, a method for preparing aromatic aldehydes is disclosed, comprising the following steps: using a mutant of the aromatic dioxygenase CpuADO with an amino acid sequence as shown in SEQ ID NO:3 to catalyze the reaction of 4-vinylphenol, 4-vinylguaiacol, isoeugenol, sinigrin, or coniferyl alcohol, wherein the conditions for the catalytic reaction are 30°C to 45°C and pH 7.0 to 9.0.

[0043] In the following embodiments of the present invention, the enzyme activity and substrate conversion of the aromatic dioxygenase CpuADO and its mutants were determined as follows: The total volume of the reaction system was 1 mL, containing 50 mM NaH2PO4-Na2HPO4 buffer (pH 8.0), 10 mM substrate (the amount of DMSO added as a co-solvent did not exceed 5% of the total volume), and 10 μM purified CpuADO wild-type or the corresponding mutant. The reaction system was stirred in a 40°C water bath for 30 min. After the reaction was completed, 10 μL of 10 M HCl was added to the reaction solution to terminate the reaction, followed by the addition of 1 mL of ethyl acetate, extraction for 1 min, centrifugation at 12000 rpm for 10 min, and the upper organic phase was collected and injected into a gas chromatograph (GC-FID) for analysis. The concentrations of substrate and product were calculated using the external standard method. The substrate conversion rate was calculated as the percentage of the target product generated relative to the initial substrate amount (molar concentration). Specifically: Substrate conversion rate (%) = C t / C0×100%, where C t The molar amount of the target product measured by GC after the reaction is complete. In the reaction system of this invention, the molar ratio between the target product and the substrate is 1:1. Therefore, the above definition is equivalent to the substrate conversion rate characterized by the amount of target product generated. Those skilled in the art can choose the external standard or internal standard method to measure the C content according to actual needs. t Quantitative analysis is performed.

[0044] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Example 1: Sequence mining, gene synthesis, and protein expression of CpuADO

[0046] 1. Sequence Mining and Design

[0047] Using the reported amino acid sequence of the aromatic dioxygenase TthADO as the query sequence, a BLAST homology search was performed in the NCBI protein database. A putative protein, CpuADO, annotated as originating from *Coniochaeta pulveracea*, was obtained, with its amino acid sequence shown in SEQ ID NO:1. Based on this amino acid sequence and considering *E. coli* codon preferences, codon optimization was performed to obtain the nucleotide sequence for expressing CpuADO (as shown in SEQ ID NO:4).

[0048] SEQ ID NO:1>RKU43285.1 hypothetical protein DL546_006478 [Coniochaetapulveracea] (585 aa)

[0049] MAHIFDLAPEVSLPDEPIYKDGKLHRPNHVQFPQTPVFASMNKPSRFEGTILSLEHTGIIPPEINGTFFRVQPDHRFPPMFEDDIHFNGDGSVTAIRIFDGKVDFRQRYVHTERYKAETKARRSLFGRYRNPWTDNESVKGVIRTA SNTNVFFWRGMLLATKEDGPPYAMDPVTLETYGRYDFEGQILSPTFTAHPKFDPRTGMVCFAYETGGDGADCSREVMVWTLDKDGKKVSERWFEAPFAGMIHDCGLSENWLVLPLTPIKMDLERMKRGGNKFAWDPKEDQVYGLV PRRGDGEVKWFRGENAFHGHVAGCYENAQGHVVIDLTVADGNVFFWFPPDGEEQGQFAKRNKLSSPTHRWILDPSLPNNARITPALVWPTNGEFSRIDDRWTTRKYKHFWLAKVDPSRPYDFAKCGPPAGGLFNCLGHYTWDLDNE LATGQEDVYFAGPTCTFQEPTFIPKGDKEGEGWLIALVNHLDVLRNDVVILDAQNLAKGPVCTIHLPLKLKLGLHGNWVDWRDIEDWTKRRQEDGEVGPVQVATEMLPWQKAFWEKEKEKNGNGVEGPNINGTNGANGTNGVNGSSH

[0050] SEQ ID NO:4

[0051]

[0052] 2. Gene synthesis and vector construction

[0053] The optimized nucleotide sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and restriction endonuclease sites EcoRI and NotI, adapted to the pETDuet-1 vector (provided by Sangon Biotech (Shanghai) Co., Ltd.), were designed at both ends. The synthesized fragment was ligated to the linearized pETDuet-1 vector via enzyme digestion, transformed into TOP10, and positive clones were selected and sequenced to confirm their accuracy. The resulting plasmid, named pETDuet-CpuADO, was used for subsequent protein expression.

[0054] 3. Conditions of expression

[0055] Single clones were inoculated into 5 mL of LB medium and cultured at 37°C with shaking at 220 rpm for about 12 h. The culture was then transferred to 100 mL of LB medium and cultured at 37°C with shaking at 220 rpm for about 2 h until the OD600 reached approximately 0.6-0.8. The culture was then transferred to 500 mL of self-induction medium (each liter containing: 10 g peptone, 5 g yeast extract, 10 g NaCl, 5 g glycerol, 0.5 g glucose, 2 g lactose, 6.7 g Na2HPO4·7H2O, 3.4 g KH2PO4, 2.7 g NH4Cl, 0.7 g Na2SO4, 0.49 g MgSO4·7H2O). The culture was cultured at 37°C with shaking at 220 rpm until the OD600 reached approximately 0.6-0.8. The temperature was then lowered until 16°C, at which point 1 mM FeCl2 was added to continue inducing expression for another 20 h.

[0056] 4. Protein extraction and purification

[0057] Bacterial cells were collected (centrifuged at 4 ℃, 5000 rpm for 30 min), resuspended in Buffer A (25 mM Tris-HCl, pH 8.0, 300 mM NaCl), sonicated, and centrifuged to remove cell debris. The supernatant was purified by Ni-NTA affinity chromatography, and the target protein was eluted with elution buffer containing 200 mM imidazole. The eluent was then passed through a desalting column to remove small molecule salts, and finally concentrated by ultrafiltration.

[0058] 5. CpuADO SDS-PAGE analysis

[0059] The purified protein was analyzed by SDS-PAGE, such as... Figure 1 As shown, a single band can be observed at approximately 66.4 kDa, indicating good protein purity and that the subunit molecular weight is consistent with the theoretical value.

[0060] Example 2: Substrate Spectrum Study of CpuADO Wild-Type

[0061] The catalytic performance of wild-type CpuADO was investigated using a variety of structurally diverse aromatic olefins as substrates (including p-hydroxy aromatic olefins 4-vinylphenol and 4-isopropenylphenol; lignin monomer derivatives such as 4-vinylguaiacol, isoeugenol, sinigrin, and coniferyl alcohol; and biaromatic ring substrates such as resveratrol and pterostilbene). The reaction system consisted of a total volume of 1 mL, including 50 mM NaH2PO4-Na2HPO4 buffer (pH 8.0), 10 mM substrate (total system containing 2% v / v DMSO), and 10 μM enzyme. The reaction was carried out with stirring in a water bath at 40 °C for 24 h. After the reaction, an equal volume of ethyl acetate was added for extraction. The organic phase was used for GC analysis, and the conversion rate (%) of the substrate to the corresponding aromatic aldehyde / ketone product was quantitatively calculated using the external standard method (all experiments were performed in triplicate, and the data are expressed as mean ± standard deviation).

[0062] The substrate spectrum of wild-type CpuADO is as follows: Figure 2 As shown in Table 1, the substrates that cannot be catalyzed by the wild-type CpuADO are listed below.

[0063] Table 1

[0064]

[0065] Note: "-" indicates no product formation, and "Retention time" indicates the elution time of the compound on the gas chromatogram.

[0066] from Figure 2 It can be seen that the CpuADO wild type has a high conversion rate for substrates with para-hydroxyl and methoxy substitutions (such as 4-vinylguaiacol (8) and isoeugenol (9)); it has a high conversion rate for biphenyl ring substrates (such as trans-4-hydroxystilbene (4), 4,4'-dihydroxystilbene (5), pterostilbene (6), resveratrol (7), and isopanthene (14)). However, when more hydroxyl groups or other substituents are introduced into the aromatic ring, the conversion rate of some substrates (such as leucine (12) and curcumin (15)) decreases; substrates without a para-hydroxystyrene skeleton are basically not converted (as shown in Table 1). It is evident that the CpuADO wild type has a clear preference for the electronic properties of the substrate and the position of the substituents, especially for lignin monomer-derived aromatic olefins with para-hydroxyl groups and electron donor substituents. This provides a theoretical basis for the high-value application of lignin monomer-derived aromatic compounds.

[0067] The gas chromatograms of the CpuADO-catalyzed formation of aromatic aldehydes from representative substrates such as sinaponicol, isoeugenol, and resveratrol are shown below. Figures 3-5 As shown.

[0068] Example 3: Crystal structure analysis and active center determination of CpuADO

[0069] 1. Crystallization conditions

[0070] The protein concentration was approximately 10 mg / mL, and the buffer solution was 25 mM Tris-HCl (pH 8.0) and 300 mM NaCl. An equal volume of protein solution was mixed with the stock solution (0.2 M NaCl, 0.1 M BIS-Tris (pH 5.5), 25% PEG 3350) and incubated at 16°C to obtain crystals suitable for diffraction.

[0071] 2. Data Collection and Structure Analysis

[0072] The crystals were rapidly transferred to a solution containing the original reservoir components and supplemented with 10% (v / v) glycerol for cryoprotection. Subsequently, the crystals were rapidly frozen in liquid nitrogen, and X-ray diffraction data were acquired at the Shanghai Synchrotron Radiation Facility (SSRF). The diffraction data were processed using XDS and Aimless programs. A molecular substitution method was employed, using the relevant ADO structure as a template for solution. The model was refined using Refmac5 and Coot, ultimately obtaining a CpuADO crystal structure with a resolution of approximately 2.2 Å (PDB ID: 9LP3, e.g., ...). Figure 6 (As shown); analysis of the crystal structure revealed that CpuADO exhibits a typical heptal β-helical folded structure with Fe coordinated by H195, H249, H312, and H513. 2+ Active site. A three-dimensional structural diagram of the substrate channel, catalytic active site, and structure-directed mutation site of CpuADO is shown below. Figure 7 As shown.

[0073] Comparison of CpuADO and the reported MapADO structure, as follows: Figure 8 As shown, the light blue represents the crystal structure of CpuADO, with mutated residues displayed as light blue rods; the light pink represents the crystal structure of MapADO, with mutated residues displayed as light pink rods; and the orange spheres represent Fe. 2+ .from Figure 8 It can be seen that the histidine residues in the active site are highly conserved, but there are differences in the channel regions F349, W338, R352, etc., which provides a structural basis for improving its catalytic efficiency through mutation.

[0074] Example 4 Construction and Catalytic Performance Study of CpuADO Mutant

[0075] 1. Construction of mutants

[0076] Using CpuADO as template DNA, primers were designed to introduce point mutations (mutation sites and primer sequences are shown in Table 2). The target fragment containing the mutation site was obtained using overlap extension PCR. The total system volume was 50 μl, containing 1 μL each of upstream and downstream primers, 1 μL of template DNA, 25 μL of 2×Prime STARMAX, and diluted to 50 μL with pure water. The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min; followed by 31 cycles (98℃ denaturation for 15 s, 57℃ annealing for 25 s, 72℃ extension for 3 min); and a final extension at 72℃ for 5 min. After the reaction, the product was incubated at 12℃. After amplification, the results were verified by 1% agarose gel electrophoresis (TAE). After verification, 1 μL of QuickCut DpnI restriction enzyme and 5 μL of 10×QuickCutbuffer were added to the PCR product, and the product was digested at 37℃ for 10 min to remove the template DNA. The product was then purified according to the PCR product purification kit instructions. After transformation into the TOP10 strain, plasmids were extracted using a plasmid extraction kit. Sequencing confirmed the extraction was successful, and the resulting plasmid containing the mutant was transformed into E. coli BL21(DE3) to obtain the mutant expression strain. Purification conditions were the same as in Example 1.

[0077] Table 2

[0078]

[0079]

[0080] 2. Investigation of catalytic performance

[0081] Using six lignin monomer-derived aromatic olefins as substrates (1,4-vinylphenol; 5,4,4'-dihydroxystilbene; 8,4-vinylguaiacol; 9, isoeugenol; 10, coniferyl alcohol; 11, sinigrin), the catalytic performance of CpuADO wild-type and several substrate channel-related mutants was investigated. The reaction system, reaction conditions, and methods for calculating substrate conversion were the same as in Example 2.

[0082] The amount of target product generated by each enzyme within a certain time period was converted into substrate conversion rate (%), and the results are shown in Table 3.

[0083] Table 3

[0084]

[0085] As shown in Table 3, most single-point mutants (such as M247R, F349V, F349L, N42A, R352K, W338F, etc.) showed similar conversion rates on some substrates to wild-type CpuADO, and even showed varying degrees of decrease. This indicates that simply changing the residues near the substrate channel cannot generally improve catalytic efficiency.

[0086] In comparison, mutant CpuADO-F349W (amino acid sequence as shown in SEQ ID NO:2, which replaces phenylalanine (F) with tryptophan (W) at residue 349 of SEQ ID NO:1, while the remaining amino acid sequence is identical to SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:5) and mutant CpuADO-W338D (amino acid sequence as shown in SEQ ID NO:3, which replaces tryptophan (W) with aspartic acid (D) at residue 338 of SEQ ID NO:1, while the remaining amino acid sequence is identical to SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:5) also exhibit similar characteristics. As shown in NO:6, the mutant CpuADO-F349W exhibited a representative activity enhancement advantage: the conversion rate of sinaponic acid was increased by 77.3%, and the conversion rates of 4-vinylphenol, 4-vinylguaiacol, isoeugenol, coniferol, and sinaponic acid by 53.8%, 32.3%, 22.2%, 35.8%, and 36.9%, respectively, with only a slight decrease in the conversion rate of 4,4'-dihydroxystilbene.

[0087] SEQ ID NO:2

[0088] MAHIFDLAPEVSLPDEPIYKDGKLHRPNHVQFPQTPVFASMNKPSRFEGTILSLEHTGIIPPEINGTFFRVQPDHRFPPMFEDDIHFNGDGSVTAIRIFDGKVDFRQRYVHTERYKAETKARRSLFGRYRNPWTDNESVKGVIRTASNTNVFFWRGMLLATKEDGPPYAMDPVTLETYGRYDFEGQILSPTFTAHPKFDPRTGEMVCFAYETGGDGADCSREVMVWTLDKDGKKVSERWFEAPFAGMIHDCGLSENWLVLPLTPIKMDLERMKRGGNKFAWDPKEDQVYGLVPRRGDGEVKWFRGENAFHGHVAGCYENAQGHVVIDLTVADGNVFFWFPPDGEEQGQWAKRNKLSSPTHRWILDPSLPNNARITPALVWPTNGEFSRIDDRWTTRKYKHFWLAKVDPSRPYDFAKCGPPAGGLFNCLGHYTWDLDNELATGQEDVYFAGPTCTFQEPTFIPKGDKEGEGWLIALVNHLDVLRNDVVILDAQNLAKGPVCTIHLPLKLKLGLHGNWVDWRDIEDWTKRRQEDGEVGPVQVATEMLPWQKAFWEKEKEKNGNGVEGPNINGTNGANGTNGVNGSSH

[0089] SEQ ID NO:5:

[0090]

[0091] SEQ ID NO:3

[0092] MAHIFDLAPEVSLPDEPIYKDGKLHRPNHVQFPQTPVFASMNKPSRFEGTILSLEHTGIIPPEINGTFFRVQPDHRFPPMFEDDIHFNGDGSVTAIRIFDGKVDFRQRYVHTERYKAETKARRSLFGRYRNPWTDNESVKGVIRTASNTNVFFWRGMLLATKEDGPPYAMDPVTLETYGRYDFEGQILSPTFTAHPKFDPRTGEMVCFAYETGGDGADCSREVMVWTLDKDGKKVSERWFEAPFAGMIHDCGLSENWLVLPLTPIKMDLERMKRGGNKFAWDPKEDQVYGLVPRRGDGEVKWFRGENAFHGHVAGCYENAQGHVVIDLTVADGNVFFDFPPDGEEQGQFAKRNKLSSPTHRWILDPSLPNNARITPALVWPTNGEFSRIDDRWTTRKYKHFWLAKVDPSRPYDFAKCGPPAGGLFNCLGHYTWDLDNELATGQEDVYFAGPTCTFQEPTFIPKGDKEGEGWLIALVNHLDVLRNDVVILDAQNLAKGPVCTIHLPLKLKLGLHGNWVDWRDIEDWTKRRQEDGEVGPVQVATEMLPWQKAFWEKEKEKNGNGVEGPNINGTNGANGTNGVNGSSH

[0093] SEQ ID NO:6:

[0094]

[0095] 3. Steady-state dynamics research

[0096] The steady-state dynamics results of several representative mutants are shown in Table 4.

[0097] Table 4

[0098]

[0099] Note: Reaction conditions: 1 mL reaction system, substrate concentrations of 0.05, 1, 2, 3, 5, 7, 10, 15, 20, 30 mM, 2 μM pure enzyme, 50 mM phosphate buffer (pH 8.0), 40 ℃, 500 rpm for 10 min, each experiment was repeated 3 times.

[0100] The steady-state kinetics studies in Table 4 further reveal the molecular basis of the activity alterations. With sinapoxetine (11) as the substrate, the k values ​​of each mutant... cat Slightly increased, but accompanied by K M A slight increase in the value leads to a decrease in its overall catalytic efficiency (k). cat / K M The results were comparable to those of the wild-type parent. For substrate (8), the k-value of the mutant CpuADO-W338D was... cat It increased 10-fold, while for isoeugenol (9), the k value of the mutant CpuADO-W338D was significantly higher. cat It increased by more than 2 times, K M The value also decreased, ultimately leading to an overall catalytic efficiency increase of 12 times, demonstrating optimal catalytic performance.

[0101] The results of this embodiment show that the mutant CpuADO-W338D (SEQ ID NO:3) exhibits high catalytic activity for a variety of lignin monomer-derived aromatic olefin substrates and has good broad-spectrum substrate conversion ability; while the mutant CpuADO-F349W (SEQ ID NO:2) mainly improves the catalytic efficiency for sinapoxetine.

[0102] Example 5 Molecular dynamics simulation analysis of mutants

[0103] To elucidate the effects of the mutation on substrate binding and catalysis, MD simulations were performed on the complex of the mutant and sinapoxetine (11), primarily monitoring the iron ion (Fe) at the catalytic center. 2+ The distance between the double bond (C=C) and the substrate is shown in the figure. Figure 9 As shown. In the last frame of the CpuADO-F349W-sinopeptidyl complex, Fe 2+ The distance from the C=C double bond is approximately 5.5 Å. Figure 9In the CpuADO-W338D-sinopeptidyl complex, Fe 2+ The distance to the C=C double bond is the shortest among all the tested systems, approximately 4.7 Å. Figure 9 (B in the middle).

[0104] The active sites and structural dynamics analysis results of CpuADO WT-sinopeptide complex, CpuADO-F349W-sinopeptide complex, and CpuADO-W338D-sinopeptide complex in 100 ns molecular dynamics simulations are as follows: Figure 10 As shown. Fe 2+ The trajectory of the -C=C distance over time shows that both mutants converge rapidly and maintain the distance within a narrow range (approximately 0.45-0.55 nm) with minimal fluctuations. This suggests that the substrate's C=C double bond maintains a more persistent precatalytic conformation near the catalytic metal center. Figure 10 (A in the original text). Furthermore, the root mean square deviation (RMSD) curve of the complex backbone atoms showed that it remained at a low level (<0.25 nm) after 100 ns of simulated equilibrium, indicating that the protein-substrate complex was generally stable. Figure 10 (B in the middle).

[0105] MD simulations were also performed on the complex of the mutant CpuADO-W338D with 4-vinylguaiacol (8), isoeugenol (9), and coniferyl alcohol (10), with the Fe in the last frame... 2+ The -C=C distance, the active site and structural dynamics analysis results in the 100 ns molecular dynamics simulation are as follows: Figure 11 and Figure 12 As shown. The results show that CpuADO-W338D can also support the binding of 4-vinylguaiacol (8), isoeugenol (9), and coniferyl alcohol (10). In these complexes, Fe 2+ The distances between -C and C stabilize at approximately 4.7 Å, 6.2 Å, and 5.2 Å, respectively (as shown in Figure 1). Figure 11 (A, B, and C in the text), and the fluctuation within 100 ns is limited (0.55-0.6 nm). Figure 12 The root mean square deviation (RMSD) curves of the backbone atoms of the complex (A, C, and E) show that the RMSD values ​​remain at a low level (approximately 0.2-0.3 nm). Figure 12 The presence of B, D, and F in the data indicates that CpuADO-W338D can form stable complexes with a variety of monoaryl substrates.

[0106] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0107] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A mutant of aromatic dioxygenase CpuADO, characterized in that, The amino acid sequence of which is shown as SEQ ID NO: 2 or SEQ ID NO:

3.

2. A gene encoding a mutant of aromatic dioxygenase CpuADO, characterized in that, The nucleotide sequence of which is shown as SEQ ID NO: 5 or SEQ ID NO: 6, or the amino acid sequence encoded by the nucleotide sequence is shown as SEQ ID NO: 2 or SEQ ID NO:

3.

3. A recombinant expression vector into which the coding gene of the mutant of the aromatic dioxygenase CpuADO according to claim 2 is inserted.

4. The recombinant expression vector of claim 3, wherein, The expression vector is pET.

5. A recombinant engineering bacterium transformed with the recombinant expression vector according to claim 3 or 4.

6. The recombineering bacteria of claim 5, wherein, The engineering bacterium is E. coli.

7. Use of the mutant of the aromatic dioxygenase CpuADO according to claim 1, the coding gene of the mutant of the aromatic dioxygenase CpuADO according to claim 2, the recombinant expression vector according to claim 3 or 4, or the recombinant engineering bacterium according to claim 5 or 6 in catalyzing a lignin monomer-derived aromatic olefin to generate an aromatic aldehyde or an aromatic ketone; the lignin monomer-derived aromatic olefin is 4-vinylphenol, 4-isopropenylphenol, 4-vinylguaiacol, isoeugenol, brassinol, coniferyl alcohol, 4,4'-dihydroxy stilbene, trans-4-hydroxystilbene, piceatannol, isodamnacanthal, oxy-piceatannol, and / or pterostilbene.

8. A process for the preparation of an aromatic aldehyde, characterized in that, comprising the step of: using the mutant of the aromatic dioxygenase CpuADO to catalyze brassinol, under the conditions of 30-45℃ and pH 7.0-9.0, and the amino acid sequence of the mutant of the aromatic dioxygenase CpuADO is shown as SEQ ID NO:

2.

9. A process for the preparation of an aromatic aldehyde or an aromatic ketone, characterized in that, comprising the step of: using the mutant of the aromatic dioxygenase CpuADO to catalyze 4-vinylphenol, 4-vinylguaiacol, isoeugenol, brassinol, or coniferyl alcohol, under the conditions of 30-45℃ and pH 7.0-9.0, and the amino acid sequence of the mutant of the aromatic dioxygenase CpuADO is shown as SEQ ID NO:

3. comprising the step of: using the mutant of the aromatic dioxygenase CpuADO to catalyze 4-vinylphenol, 4-vinylguaiacol, isoeugenol, brassinol, or coniferyl alcohol, under the conditions of 30-45℃ and pH 7.0-9.0, and the amino acid sequence of the mutant of the aromatic dioxygenase CpuADO is shown as SEQ ID NO:

3. ​

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