A heme oxygenase mutant and use thereof

Abstract

The application discloses a heme oxygenase mutant and application thereof, and belongs to the technical field of biological catalysis and enzyme engineering. The application discloses a heme oxygenase mutant CtHO3m, and the amino acid sequence of the heme oxygenase mutant is shown as SEQ ID NO. 4. The application solves the problems of low catalytic efficiency, poor stability and weak substrate tolerance in the production of chlorin, and provides a new heme oxygenase mutant for the industrial production of chlorin, and has potential application value.

Description

Technical Field

[0001] This invention relates to the fields of biocatalysis and enzyme engineering technology, and more specifically to a heme oxygenase mutant CtHO3m designed based on enzyme engineering technology, and the use of this mutant for whole-cell expression in Escherichia coli and its application in the catalytic synthesis of biliverdin, comprising the enzyme mutant, the engineered bacteria expressing the enzyme, and the reaction route and application scheme. Background Technology

[0002] Biliverdin (BV), an important linear tetrapyrrole pigment, is a key precursor for the synthesis of bilirubin, the core component of the precious traditional Chinese medicine bezoar. It is widely used in over 100 kinds of traditional Chinese medicine preparations, with huge market demand. Natural biliverdin mainly comes from the bile of animals such as pigs and cattle, with a biliverdin content of only about 0.05%. Extracting 1 kg of biliverdin requires at least 5 tons of pig bile, resulting in high costs and unsustainable resources, limiting its application due to regulations and folk customs. Chemical methods can also be used to synthesize biliverdin, but they easily produce multiple isomers, resulting in low product purity and difficult downstream separation and purification, making it difficult to meet industrial application requirements and similarly limiting its application.

[0003] In recent years, enzymatic synthesis technology has become a research hotspot in biliverdin preparation due to its advantages such as mild reaction conditions, high optical purity of products, environmental friendliness, and wide range of applications. Heme oxygenase (HO) is the core catalyst in the enzymatic synthesis of biliverdin, catalyzing the oxidation of heme chloride to form biliverdin. However, natural heme oxygenase HO has significant performance defects: low catalytic efficiency, with the highest yield of biliverdin synthesized by existing biological methods only reaching 132 mg / L, a conversion rate of less than 20%; poor stability, easily and rapidly inactivated in aerobic environments or in the presence of trace amounts of hydrogen peroxide or organic cosolvents; and weak substrate and product tolerance, with heme tolerance concentration not exceeding 300 mg / L and biliverdin tolerance concentration not exceeding 30 mg / L, severely restricting industrial application. Patent CN114891711A reports a method that can express Clostridium tetani (… Clostridium tetani Recombinant Escherichia coli with heme oxygenase CtHO derived from 0.05% had an intracellular coenzyme cycle system and a membrane surface display system. When heme chloride was added to a final concentration of 100 mg / L and the conversion time was 20 h, the final biliverdin yield was only 77.6 mg / L, which is far from industrial application.

[0004] To significantly improve the efficiency of enzymatic bilirubin production, it is essential to substantially enhance the catalytic performance of heme oxygenase (HO). Previous research has largely focused on HO screening and traditional enzyme engineering. Traditional enzyme engineering primarily includes directed evolution and rational design. Directed evolution involves introducing mutations into the target protein and screening for variants with improved function to obtain enzyme variants with enhanced activity, stability, and other properties. Rational design, on the other hand, is based on known protein structure and function information, involving targeted modifications to the sequence or structure. Both methods require experimental screening of a vast number of mutants; a protein containing N amino acids may have 20... N The screening process for these mutations is expensive, time-consuming, and complex. Even with advanced high-throughput screening technologies, only a very small portion of the mutation space can be covered. This is especially true for low-activity enzymes like HO, where it is extremely difficult to significantly increase their activity using traditional enzyme engineering methods. This is a major reason why high-activity HO is currently difficult to obtain.

[0005] Therefore, providing a heme oxygenase mutant and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a heme oxygenase mutant that can be used for enzymatic preparation of biliverdin and its application, solving the problems of low efficiency, poor stability and weak substrate tolerance in current biliverdin production.

[0007] With the application of intelligent technologies such as deep learning in enzyme engineering, the efficiency of enzyme modification has been greatly improved. To effectively obtain a highly active HO mutant, this invention utilizes the deep learning tool LigandMPNN to extensively redesign and optimize CtHO derived from Clostridium tetani. Ultimately, by rewriting 57 non-conserved amino acids (accounting for 27.01% of the total sequence length), a mutant CtHO3m with high catalytic efficiency, strong stability, and good substrate tolerance was obtained, significantly improving the biliverdin synthesis rate: using 1500 mg / L heme chloride as a substrate, through whole-cell catalysis, after 8 h of reaction at 30 °C in the dark, the biliverdin yield reached 1245 mg / L, with a molar conversion rate of 92.88%. These indicators are far superior to those of CtHO (with a final concentration of 100 mg / L heme chloride, a conversion time of 20 h, and a final biliverdin yield of 77.6 mg / L, with a molar conversion rate of 86.5%). This provides an effective enzyme tool for the industrial-scale, efficient production of biliverdin and bilirubin.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] 1) Obtaining the heme oxygenase mutant CtHO3m The amino acid sequence of wild-type CtHO derived from Clostridium tetani used in this invention is shown in SEQ ID NO.1.

[0010] MENTFLNEIRLNSSKLHDMAEHTGFIKRLIEGNANVTTYAEYIYNLYHIYNAIESNLEKNKGNKYIKDFALPEVYRAEAIMKDVKYLLKDKLDSMEPLISTKVFVNR INHIGEKNKELLIAHAYTRYLADLFGGRTIYQIVKENYKIDDKGLNYYIFHEINDLKNFVMGYHEKLNNIKFDETLKKDFINEISISYIYNISISNELEFDRFK; SEQ IDNO.1.

[0011] The mutant is based on Clostridium tetani ( Clostridium tetani CtHO (patent number: CN114891711A) was analyzed for amino acid conservation using the bioinformatics analysis software Consurf. The 57 amino acids were redesigned using the deep learning model LigandMPNN, resulting in three new CtHO mutants. The amino acid sequence of CtHO1m is shown in SEQ ID NO.2, the amino acid sequence of CtHO2m is shown in SEQ ID NO.3, and the amino acid sequence of CtHO3m is shown in SEQ ID NO.4.

[0012] CtHO1m mutant amino acid sequence: MANTFLNQIRDNSKALHDMAENTGFIKRLVAGEANVETYAEYIYNLYYIYKAIETNLEKNKDNPILKDFNLPEVYRAEAIKKDVEYLLGDKLKTAKPLESTKKFVER SEQ IDNO.2.

[0013] CtHO2m mutant amino acid sequence: MENTFLNQIRDNSKDLHDMAENTGFIKRLVDGKANVETYAEYIYNLYHIYKAIEDNLEKNKDNPIIKDFALPEVYRAEAIMKDVKALLGDKLKTAKPLESTKKFVERINKIGKENKELLIAHAYTRYLADLFGGRTIYKVVKEKYKIGPEGLNYYKFEKIKDMKAFVADYKKKLNNVPFNEEQRKKFIEEISISYQYNISISNELEEQRFK; SEQ ID NO.3。

[0014] Amino acid sequence of the CtHO3m mutant: M A NTFLN Q IR D NS KS LHDMAE N TGFIKRL VA G E A D V E TYAEYIYNLY Y IY E AIE T NLEKNK D N PLI KDFALPEVYRAEAIMKDVK A LL G DKL KEAK P IE STK K FV E RIN E IG KE NKELLIAHAY S RYLADLFGGRTIY KV VK DV YKIDD S GLNYY K F DK I K D M K A FV AD Y KK KLNN VP F N E EQ K EK FINEISISY N YNISISNELE EK RFK; SEQ ID NO.4。

[0015] Note: SEQ ID NO.4 contains 57 amino acid mutations compared to SEQ ID NO.1, involving catalytic domains, substrate binding pockets, and stability-related sites.

[0016] The synthesis was optimized according to the codon preference of *E. coli*. Subsequently, heterologous expression was performed in *E. coli* BL21(DE3) on the pET-28a vector. SDS-PAGE analysis showed that only the mutant enzyme CtHO3m could be solublely expressed in *E. coli*, and therefore it was selected for further research.

[0017] The optimized sequence of the CtHO gene is shown in SEQ ID NO.5.

[0018] ; SEQ ID NO.5.

[0019] The optimized sequence of the CtHO1m gene is shown in SEQ ID NO.6.

[0020] ATGGCTAACACCTTTCTGAACCAGATCCGTGACAACTCTAAAGCACTGCACGACATGGCAGAGAACACTGGTTTCATCAAACGTCTGGTTGCAGGTGAAGCGAACGTTGAAACCTACGCAGAATATATCTACAACCTGTACTACATCTACAAAGCGATCGAAACCAACCTGGAGAAGAACAAAGACAATCCGATTCTGAAAGACTTCAACCTGCCGGAAGTTTACCGTGCTGAAGCGATCAAGAAAGACGTTGAATACTTGCTGGGTGACAAACTGAAAACCGCGAAACCGTTGGAATCCACCAAGAAATTCGTGGAACGTATCAACAAGATCGGTGAAGAAGAGAAAGAACTGCTGATCGCGCACGCTTACACTCGTTACCTGGCTGATTTGTTCGGCGGTCGTACCATCTACGACATCGTGAAGAACGTGTACAAGATCGACGACTCTGGTCTGAACTACTACCGTTTCGATAAGATCAAAGACCTGAAAGCCTTCGTTAAAGACTACAAGAACAAACTGAACAACGTTCCGTTCAACGAAGAACAGCGTAAACGTTTCATCGAAGAAATCTCCATCAGCTACAACTACAACATCTCTATCTCCAACGAACTGGAAGAGAAACGTTTCAAATAG; SEQ ID NO.6。

[0021] The optimized sequence of the CtHO2m gene is shown in SEQ ID NO.7.

[0022] ATGGAGAACACCTTCTTGAACCAGATCCGTGACAACTCTAAAGACCTGCACGACATGGCAGAGAACACCGGTTTCATCAAACGTCTGGTTGACGGTAAAGCGAACGTTGAAACCTACGCGGAATACATCTACAACCTGTACCACATCTACAAAGCGATCGAAGATAACCTGGAGAAGAACAAAGACAATCCGATCATCAAAGACTTCGCGCTGCCGGAAGTTTACCGTGCTGAAGCGATCATGAAAGACGTTAAAGCGCTGCTGGGTGACAAACTGAAAACCGCGAAACCACTGGAATCTACCAAGAAATTCGTGGAACGTATCAACAAGATCGGCAAAGAGAACAAAGAACTGCTGATCGCACACGCGTACACTCGTTACCTGGCAGATCTGTTCGGTGGTCGTACCATCTACAAGGTTGTTAAAGAGAAGTACAAGATCGGTCCAGAAGGTCTGAACTACTACAAATTCGAGAAGATCAAAGACATGAAAGCGTTCGTAGCGGACTACAAGAAGAAACTGAACAACGTTCCGTTCAACGAAGAACAGCGTAAGAAATTCATCGAAGAAATCTCTATCAGCTACCAGTACAACATCTCCATCTCTAACGAACTGGAAGAGCAGCGTTTCAAGTAG; SEQ ID NO.7。

[0023] The optimized sequence of the CtHO3m gene is shown in SEQ ID NO.8.

[0024] ; SEQ ID NO.8.

[0025] 2) Characteristics of the heme oxygenase mutant CtHO3m The amino acid sequence of the CtHO3m mutant is shown in SEQ ID NO.4. Compared with the wild-type CtHO (SEQ ID NO.1), it has 57 amino acid mutations, and its gene sequence is shown in SEQ ID NO.5.

[0026] The in vitro crude enzyme activity of the CtHO3m mutant was 0.29 U / g, which was 1.23 times higher than that of wild-type CtHO (0.13 U / g). In terms of thermal stability, the half-life of CtHO3m at 30 °C was 11.5 h, which was 67% longer than that of wild-type CtHO (6.9 h). The substrate tolerance concentration was increased to 1000 mg / L, and it could achieve efficient conversion of high concentrations of heme chloride to biliverdin through whole-cell catalysis.

[0027] 3) E. coli whole-cell transformation process for preparing biliverdin To improve the expression efficiency of CtHO3m, codon-optimized CtHO3m was expressed in *E. coli* BL21(DE3) using pET-28a to construct the genetically engineered strain CtHO3m#. Since industrially engineered bacteria based on *E. coli* BL21(DE3) are highly sensitive to bacteriophages, they are frequently infected by phages during industrial applications, which is a common problem affecting their industrial application. To circumvent this problem, this invention uses the phage-resistant engineered strain BL21(DE3)-RMⅡ constructed in patent CN120554466 A (which integrates a type II restriction-modification (RM) system derived from *E. coli* Nissle 1917 on the basis of *E. coli* BL21(DE3)). Furthermore, the pET-28a-CtHO3m plasmid was introduced into BL21(DE3)-RMⅡ via thermal transformation, resulting in a highly efficient heterologous expression genetically engineered strain CtHO3m with superior phage resistance. Both strains were able to catalyze 1500 mg / L heme chloride using 100 g / L wet cells. After reacting at 30 °C in the dark for 8 h, the biliverdin yield reached >1200 mg / L, with a molar conversion rate exceeding 90%. The reaction system was 0.1 M citrate buffer (pH=7.0) containing 0.2% Triton-X 100 and 0.2 g / L ascorbic acid.

[0028] Furthermore, the encoding gene of the heme oxygenase mutant CtHO3m or the heme oxygenase mutant CtHO3m, or the recombinant expression vector, or the recombinant bacteria CtHO3m#, or the engineered bacteria CtHO3m Application in the catalytic synthesis of biliverdin.

[0029] Furthermore, using heme chloride as a substrate, the encoding gene of the heme oxygenase mutant CtHO3m or the heme oxygenase mutant CtHO3m, or the recombinant expression vector, or the recombinant bacteria CtHO3m#, or the engineered bacteria CtHO3m, can be used. As a catalyst to catalyze reactions.

[0030] The specific reaction conditions are as follows: cell wet weight concentration of 50-150 g / L (whole-cell catalysis) or enzyme concentration of 0.1-0.5 g / L (enzyme catalysis), substrate heme chloride concentration of 100-1500 mg / L, reaction system of 0.1 M citrate buffer containing 0.2% Triton-X 100 and 0.2 g / L ascorbic acid, pH of 6.5-7.5, and reaction temperature of 25-35 ℃.

[0031] Furthermore, the encoding gene of the heme oxygenase mutant CtHO3m or the heme oxygenase mutant CtHO3m, or the recombinant expression vector, or the recombinant bacteria CtHO3m#, or the engineered bacteria CtHO3m Application in increasing biliverdin production.

[0032] Furthermore, the encoding gene of the heme oxygenase mutant CtHO3m or the heme oxygenase mutant CtHO3m, or the recombinant expression vector, or the recombinant bacteria CtHO3m#, or the engineered bacteria CtHO3m Application in improving the conversion rate of heme chloride.

[0033] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a heme oxygenase mutant and its application. The CtHO3m mutant is derived from Clostridium tetani (… Clostridium tetani Heme monooxygenase CtHO, derived from [source missing], was modified. Through redesign and screening of 57 non-conserved amino acids in CtHO, a mutant CtHO3m with high catalytic efficiency, strong stability, and good substrate tolerance was obtained. Using 1500 mg / L heme chloride as a substrate, the genetically engineered bacteria CtHO3m [missing information]. Whole-cell catalysis, after 8 h of reaction at 30 °C in the dark, yielded 1245 mg / L of biliverdin, with a molar conversion rate of 92.88%. This invention solves the problems of low catalytic efficiency, poor stability, and weak substrate tolerance in existing biliverdin production processes, providing a novel heme oxygenase mutant for the industrial production of biliverdin, with potential application value. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0035] Figure 1 The analysis of the conservation of wild-type CtHO amino acid sequences is presented in the figure. The figure shows the conservation distribution of CtHO sequences, where Conserved indicates highly conserved regions, Variable indicates variable regions, and Average indicates the average level of conservation. This figure supports the rationality of screening non-conserved amino acid sites in CtHO.

[0036] Figure 2 The design results for three candidate mutant sequences (CtHO1m-CtHO3m).

[0037] Figure 3The pET-28a-CtHO3m recombinant expression vector map is shown; the vector contains a 6×His tag, promoter (lacI), terminator (T7 terminator), kanamycin resistance gene (KanR), and target gene insertion site, clarifying the core elements of vector construction.

[0038] Figure 4 This is an electrophoretic verification image of the supernatant proteins from recombinant bacterial cell lysates. The image includes electrophoretic analysis of heme oxygenase (CtHO) and its mutants (CtHO1m, CtHO2m, CtHO3m) expressed in *E. coli*, showing the target protein band (approximately 25 kDa). The left side shows the protein molecular weight standard (kDa). Lane 1: CtHO1m whole cell solution; Lane 2: CtHO1m supernatant; Lane 3: CtHO2m whole cell solution; Lane 4: CtHO2m supernatant; Lane 5: CtHO3m whole cell solution; Lane 6: CtHO3m supernatant; Lane 7: CtHO whole cell solution; Lane 8: CtHO supernatant; Lane 9: Control group whole cell solution; Lane 10: Control group supernatant.

[0039] Figure 5 This is an HPLC standard chromatogram of biliverdin.

[0040] Figure 6 This is an HPLC sample image of CtHO3m catalyzing the formation of biliverdin from heme.

[0041] Figure 7 The comparison shows the crude enzyme activity of wild-type CtHO and CtHO3m mutant; the vertical axis represents enzyme activity (U / g wet cell weight). The enzyme activity of CtHO3m is significantly higher than that of wild-type CtHO, confirming the improved catalytic efficiency of CtHO3m.

[0042] Figure 8 The half-life of wild-type CtHO and CtHO3m mutant at 30 °C is compared; the horizontal axis represents incubation time (h), the vertical axis represents residual enzyme activity (%), and the curve shows the fitted half-life decay trend, proving that CtHO3m has better stability than wild-type.

[0043] Figure 9 The graph shows the kinetic reaction curve of the CtHO3m mutant enzyme; where the x-axis represents the concentration of heme chloride (μM) and the y-axis represents the reaction rate (μmol). min -1 mL -1 The curve represents the fitting result of the Michaelis-Menten equation, providing data support for the analysis of enzyme catalytic characteristics.

[0044] Figure 10The graph shows the kinetic reaction curve of wild-type CtHO enzyme; where the x-axis represents the concentration of heme chloride (μM) and the y-axis represents the reaction rate (μmol). min -1 mL -1 The curve represents the fitting result of the Michaelis-Menten equation, and is compared with CtHO3m.

[0045] Figure 11 The substrate tolerance of wild-type CtHO and CtHO3m mutant is compared; the horizontal axis represents the concentration of heme chloride (mg / L), the left vertical axis represents the concentration of biliverdin (mg / L), and the right vertical axis represents the conversion rate (%), reflecting the stronger substrate tolerance of CtHO3m.

[0046] Figure 12 The graphs show the fermentation growth and IPTG induction curves of recombinant strain CtHO3m#; the horizontal axis represents fermentation time (h), and the left vertical axis represents OD. 600 The right vertical axis represents glucose concentration (g / L), and the arrows indicate the IPTG induction time points, providing a reference for optimizing the fermentation process.

[0047] Figure 13 The performance of whole-cell catalysis for biliverdin production at a scale of 1L CtHO3m# was demonstrated; the x-axis represents reaction time (h), the y-axis represents biliverdin concentration (mg / L), and the curves show the fitting trend of the detection results at different time points, verifying the feasibility of scale-up reaction.

[0048] Figure 14 The engineered bacteria CtHO3m Performance against CtHO3m# antiphage.

[0049] Figure 15 The engineered bacteria CtHO3m Fermentation growth and IPTG induction curves; where the horizontal axis represents fermentation time (h) and the left vertical axis represents OD. 600 The right vertical axis represents glucose concentration (g / L), and the arrows indicate the IPTG induction time points, providing a reference for optimizing the fermentation process.

[0050] Figure 16 1L of CtHO3m The performance of whole-cell catalytic production of biliverdin. Detailed Implementation

[0051] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] (1) Strains and vectors Host strains: Escherichia coli BL21(DE3) and Escherichia coli BL21(DE3)-RMⅡ, which integrates the heterologous expression of the Type II RM system derived from Escherichia coli Nissle 1917 and has a strong phage resistance.

[0053] Expression vector: pET-28a vector.

[0054] (2) Culture medium formulation LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and deionized water to a final volume of 1 L. Sterilize at 121 °C for 20 min.

[0055] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder, and deionized water to a final volume of 1 L. Sterilize at 121 °C for 20 min.

[0056] Fermentation medium: 10 g / L glycerol, 10 g / L peptone, 5 g / L yeast extract, 3 g / L disodium hydrogen phosphate, 0.7 g / L sodium sulfate, 3.4 g / L potassium dihydrogen phosphate, 0.25 g / L magnesium sulfate, 2.7 g / L ammonium chloride, 1 mL / L nonionic compound defoamer, and deionized water to a final volume of 1 L. Adjust the pH to 7.0 and sterilize at 121 °C for 20 min.

[0057] Feed 1: Dissolve 500 g / L glucose, 20 g / L magnesium sulfate, and 15 g / L ammonium chloride in deionized water, bring the volume to 1 L, and sterilize at 121 °C for 20 min. Feed 2: Ammonia water. Feed 3: Defoamer.

[0058] (3) Buffer solution and reagent formulation 0.1 M Citrate Buffer 1: Citrate monohydrate 21.01 g / L, dissolved in deionized water, brought to a final volume of 1 L, adjusted pH to 7.0, and stored at 4 °C.

[0059] 0.1 M Citrate Buffer 2: Citrate monohydrate 21.01 g / L, 0.2% Triton-X 100 (V / V), ascorbic acid 0.2 g / L, dissolved in deionized water, brought to a final volume of 1 L, adjusted pH to 7.0, and stored at 4 °C.

[0060] 0.5 M IPTG stock solution: Take 2.38 g of IPTG dry powder, dissolve it in 20 mL of deionized water, filter it through a 0.22 μm filter membrane for sterilization, and store it at -20 ℃.

[0061] 25 g / L heme chloride stock solution: Weigh 1 g of heme chloride and dissolve it in an appropriate amount of 0.1 M NaOH aqueous solution, adjust the pH to 7.2, make up to 40 mL, and store at -20 ℃ in the dark.

[0062] 50 mg / mL kanamycin stock solution: Dissolve 1 g of kanamycin powder in 20 mL of deionized water, filter through a 0.22 μm filter membrane for sterilization, and store at -20 ℃.

[0063] SM buffer: 5.8 g sodium chloride, 2 g magnesium sulfate heptahydrate, 50 mL Tris-HCl (1M, pH=7.5), add water to a final volume of 1 L, store at room temperature.

[0064] (4) Detection method Enzyme activity assay: The reaction system contained 100 mg / L heme chloride, 1 mM NADPH, and 1 mL of crude enzyme supernatant (wet weight 20 g / L). The reaction volume was 1 mL. After reacting at 30 ℃ for 1 h, 10 μL HCl was added to terminate the reaction. The amount of biliverdin produced was determined by HPLC. The enzyme activity unit is defined as the amount of enzyme (U / g) required to produce 1 μmol of biliverdin per hour.

[0065] HPLC detection method: UV detector, SBAQ column (4.6 mm × 250 mm), mobile phase: methanol:2% acetic acid aqueous solution = 70:30, flow rate 0.7 mL / min, detection wavelength 376 nm, column temperature 30 ℃, injection volume 10 μL.

[0066] Glucose concentration determination: Refer to the glucose kit (glucose oxidase method) from Nanjing Jiancheng Bioengineering Institute.

[0067] Example 1: Redesign of heme oxygenase CtHO and construction of heterologous expression engineered bacteria Clostridium tetani ( Clostridium tetaniThe heme oxygenase CtHO (NCBI No.: WP_035111656.1) derived from [source missing] has the function of catalyzing the oxidation of heme chloride to biliverdin. To improve the feasibility of this enzyme's industrial application and enhance its catalytic activity towards heme, its protein was redesigned. The specific process is as follows: (1) Sequence conservation analysis: The amino acid sequence of CtHO (as shown in SEQ ID NO. 1) was analyzed for conservation using the bioinformatics analysis software Consurf. Figure 1 As shown in the figure, highly and moderately conserved regions closely related to catalytic function were retained, and 57 non-conserved amino acids with conserve values ​​of 1-6 were selected as hotspot residues for redesign.

[0068] (2) Protein design for CtHO: The 57 non-conserved amino acids were redesigned using the deep learning model LigandMPNN, generating three candidate mutant sequences (CtHO1m-CtHO3m, amino acid sequences as shown in SEQ ID NO.2-4, results are shown in [see table]). Figure 2 Gene synthesis was commissioned to Beijing Qingke Biotechnology Co., Ltd., and codon optimization was performed to target the codon bias of *E. coli*. The optimized gene (sequence shown in SEQ ID NO. 5-8) was cloned into the EcoRI and HindIII sites of the pET-28a vector. Plasmids carrying the target gene (pET-28a-CtHO1m, pET-28a-CtHO2m, pET-28a-CtHO3m, pET-28a-CtHO) were transformed into *E. coli* BL21(DE3) via heat shock to construct strains CtHO1m#, CtHO2m#, CtHO3#, and CtHO#, respectively, for subsequent protein expression and evaluation. The pET-28a-CtHO3m recombinant expression vector map is shown below. Figure 3 .

[0069] Example 2 Screening of mutants and their engineered bacteria (1) Strains culture and protein induction ① Strawberries CtHO1m#, CtHO2m#, CtHO3m#, and the control strain CtHO# were streaked onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37 ℃ to obtain single colonies. A single colony was picked and inoculated into a 5 mL LB tube containing 50 μg / mL kanamycin and incubated at 37 ℃ and 220 rpm for no more than 16 h. Then, a 2% inoculum was transferred to a 50 mL LB shake flask containing 50 μg / mL kanamycin and incubated at 37 ℃ and 220 rpm until OD (dose elapsed). 600The concentration was 0.6, and IPTG was added to a final concentration of 0.3 mM. The mixture was induced overnight at 20°C and 220 rpm to obtain a well-soluble expressed protein. A strain containing only the pET-28a blank vector was used as a blank control strain.

[0070] ② After induction, collect the bacterial cells by centrifugation at 4 ℃ and 8000 rpm for 10 min. Wash the bacterial cells once with 0.1 M citrate buffer 1, weigh the wet weight, and then resuspend the cells in 0.1 M citrate buffer 2 until the wet weight concentration of the bacterial cells is 100 g / L.

[0071] (2) Screening of mutant engineered bacteria After induction of expression, the cells were sonicated to obtain whole-cell fluid, and the supernatant was collected. The soluble expression of the target mutant was verified by SDS-PAGE protein electrophoresis. Figure 4 The results showed that CtHO3m had the highest soluble expression level, therefore CtHO3m was selected as the best mutant.

[0072] Example 3: Determination and Comparison of Enzymatic Properties of CtHO3m and CtHO (1) Enzyme activity assay Following the method in Example 2, CtHO3m# and CtHO# were induced to express. Cells with a wet weight of 20 g / L were ultrasonically disrupted, and 1 mL of crude enzyme supernatant was used as the enzyme source. The reaction system contained 1 mL of crude enzyme supernatant, 100 mg / L heme chloride, and 1 mM NADPH. After reacting at 30 °C for 1 h, 10 μL of HCl was added to terminate the reaction. HPLC was used under the following conditions: SBAQ column (4.6 × 250 mm), mobile phase methanol:2% acetic acid aqueous solution = 70:30, flow rate 0.7 mL / min, detection wavelength 376 nm. The amount of biliverdin produced was detected to calculate enzyme activity. The HPLC standard chromatogram of biliverdin is shown below. Figure 5 The image of the biliverdin sample obtained after the reaction is shown below. Figure 6 The results showed that the enzyme activity of CtHO3m was 0.29 U / g wet cell weight, and the enzyme activity of CtHO was 0.13 U / g wet cell weight. The wet cell enzyme activity of CtHO3m was 1.23 times higher than that of the wild type. Figure 7 ).

[0073] (2) Half-life determination The supernatant of the crude enzyme solution prepared above was incubated at 30 °C for 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, and 20 h, respectively. Samples were taken at each time point, and 100 mg / L heme chloride and 1 mM NADPH were added. The reaction was carried out at 30 °C for 1 h, and the residual enzyme activity was measured. The enzyme activity at 0 h of incubation was taken as 100%, and a curve showing the change in residual enzyme activity over time was plotted. Figure 8 The results showed that the half-life of variant CtHO3m was 11.5 h, while that of CtHO was 6.9 h. That is, the half-life of CtHO3m was 67% longer than that of the wild type.

[0074] (3) Determination of enzyme kinetic properties Take 1 mL of the supernatant of the crude enzyme solution prepared above (wet weight 20 g / L), and set different heme chloride concentration gradients: 5 μM-480 μM for the CtHO3m group; 5 μM-160 μM for the CtHO group. Add 1 mM NADPH to each system, react at 30 ℃ for 1 h, and measure the reaction rate. Fit the kinetic parameters using the Michaelis-Menten equation (M). Figure 9 , Figure 10 The results showed that the maximum reaction rate V of CtHO3m was... max =0.709 μmol min -1 mL -1 Michaelis constant K m =29.1 μM, catalytic constant K cat =8.15 s -1 Catalytic efficiency K cat / K m =2.8 × 10 5 M -1 s -1 ;CtHO's V max =0.402 μmol min -1 mL -1 K m =21.8 μM, K cat =4.15 s -1 K cat / K m =1.9×10 5 M -1 s -1 These data indicate that the catalytic performance of the mutant CtHO3m obtained in this invention is significantly improved compared to its wild type, with the catalytic efficiency constant K... cat / K m It increased by 47%.

[0075] (4) Substrate tolerance test CtHO3m# and CtHO# were induced to express using the method described above. The bacterial cells were resuspended twice in 0.1 M citrate buffer to a wet weight of 100 g / L. One mL of the whole-cell reaction solution was taken, and heme chloride was added to final concentrations of 100 mg / L, 250 mg / L, 500 mg / L, 750 mg / L, and 1000 mg / L, respectively. 1 mM NADPH was added, and the reaction was carried out at 30 ℃ and 220 rpm in the dark. The generation of biliverdin and the conversion rate of heme were detected by HPLC. Figure 11 The results showed that CtHO3m maintained high catalytic activity at a substrate concentration of 1000 mg / L, while the conversion rate of CtHO decreased significantly after the substrate concentration exceeded 100 mg / L, indicating that CtHO3m has stronger substrate tolerance.

[0076] Example 4: Reaction system and process for the oxidation of heme chloride to biliverdin (1) Scale-up of CtHO3m# fermentation process CtHO3m# glycerol bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37 ℃ for no more than 16 h. A small amount of bacterial culture was streaked onto LB solid medium containing 50 μg / mL kanamycin and incubated at 37 ℃ for 18 h. A single colony was picked and transferred to 100 mL of LB shake flask containing 50 μg / mL kanamycin and incubated at 37 ℃ and 220 rpm for 12 h until OD was reached. 600 =2.5, used as seed solution.

[0077] The fermenter temperature was set to 37 ℃, the stirring speed to 300 rpm, and the air flow rate to 3 L / min. After the parameters stabilized, 100 mL of seed culture was inoculated into a fermenter containing 3 L of fermentation medium (glycerol 10 g / L, peptone 10 g / L, yeast extract 5 g / L, disodium hydrogen phosphate 3 g / L, sodium sulfate 0.7 g / L, potassium dihydrogen phosphate 3.4 g / L, magnesium sulfate 0.25 g / L, ammonium chloride 2.7 g / L, and nonionic compound defoamer 1 mL / L) under flame protection. During fermentation, when the dissolved oxygen dropped below 30%, the dissolved oxygen was controlled at 30% by adjusting the stirring speed. The aeration rate was increased by 1 L / min every hour until the maximum value was reached. Ammonia was added to maintain the pH at around 7.0. Samples were taken at regular intervals after fermentation began to measure OD. 600 and residual glucose concentration; after 5 h of fermentation, carbon and nitrogen sources (containing 500 g / L glucose, 20 g / L magnesium sulfate, and 15 g / L ammonium chloride) were added at a rate of 35 mL / h; OD at 18 h of fermentation600 =80, adjust the fermentation temperature to 25 ℃, reduce the feeding rate to 5 mL / h, and add IPTG to a final concentration of 0.5 mM to induce expression; at 27 h of fermentation, add antifoaming agent at a rate of 0.3 mL / h; after 22 h of induction, stop fermentation and collect 492 g of resting cells by centrifugation. Figure 12 ).

[0078] (2) 1L whole-cell catalytic reaction of heme chloride to biliverdin Take 100 g of the prepared wet-weight cells, add 0.1 M citrate buffer 2 (pH=7.0) to a final volume of 1 L, and magnetically stir until the cells are completely resuspended. Add heme chloride to a final concentration of 1500 mg / L, and add 20 mM NADPH to a final concentration. Initiate the reaction in a 5 L fermenter at 30 ℃ and an aeration rate of 1 L / min. Samples are taken every 1 h, and the amount of biliverdin produced is determined by HPLC. Figure 13 Using 1500 mg / L heme chloride as a substrate, biliverdin was produced at 1209.67 mg / L after whole-cell catalysis and reaction at 30 °C in the dark for 8 h, with a molar conversion rate of 90.24%.

[0079] Example 5: Construction of engineered bacteria with super-strong resistance to bacteriophages Given that engineered Escherichia coli BL21(DE3) as the chassis is highly sensitive to phage contamination, the engineered chassis bacterium BL21(DE3)-RMⅡ in patent CN120554466 A was used. The pET-28a-CtHO3m plasmid was introduced into BL21(DE3)-RMⅡ via thermal transformation, resulting in a highly efficient heterologous expression gene engineered bacterium CtHO3m with superior phage resistance. .

[0080] Using 24 industrially derived bacteriophages and four conventional bacteriophages (T1, T4, T5, and T7), the genetically engineered bacteriophage CtHO3m was studied. The phage defense capability of CtHO3m# was tested and evaluated. Twenty-eight phages previously stored in our laboratory were reactivated and subjected to 10x concentration tests using SM buffer. 0 -10 -6 Serial dilutions were performed, with 500 μL of bacterial suspension mixed with LB solid medium at 45℃ to a total volume of 30 mL. After thorough mixing, the mixture was quickly poured into sterile LB culture dishes and allowed to cool completely for 20 min. 2 μL of the pre-diluted phage solution was then evenly spotted onto the solid medium. The culture dishes were then inverted and incubated at 37℃ for 12 h. Results ( Figure 14 This indicates that CtHO3m# is highly sensitive to 25 of the bacteriophages; while CtHO3m It exhibits significant resistance to all 27 bacteriophages. Except for plaque formation against T7, HH3, S9, S6, and P005 at high concentrations, it demonstrates complete resistance to the remaining 22 bacteriophages. This indicates that CtHO3m... It has superior phage resistance and better industrial applicability compared to CtHO3m#.

[0081] Example 6: Scale-up preparation of biliverdin (1) Genetically engineered bacteria CtHO3m Fermentation amplification CtHO3m Glycerol-containing bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37 °C for no more than 16 h. A small amount of bacterial culture was streaked onto LB solid medium containing 50 μg / mL kanamycin and incubated at 37 °C for 18 h. A single colony was picked and transferred to 100 mL of LB shake flask containing 50 μg / mL kanamycin and incubated at 37 °C and 220 rpm for 12 h until OD (distillation time) was reached. 600 =2.5, used as seed solution.

[0082] The fermenter temperature was set to 37 ℃, the stirring speed to 300 rpm, and the aeration rate to 3 L / min. After the parameters stabilized, 100 mL of seed culture was inoculated into a fermenter containing 3 L of fermentation medium (glycerol 10 g / L, peptone 10 g / L, yeast extract 5 g / L, disodium hydrogen phosphate 3 g / L, sodium sulfate 0.7 g / L, potassium dihydrogen phosphate 3.4 g / L, magnesium sulfate 0.25 g / L, ammonium chloride 2.7 g / L, and nonionic compound defoamer 0.1 mL / L) under flame protection. During fermentation, when the dissolved oxygen dropped below 30%, the dissolved oxygen was controlled at 30% by adjusting the stirring speed. The aeration rate was increased by 1 L / min every hour until the maximum value was reached. Ammonia was added to maintain the pH at around 7.0. Samples were taken at regular intervals after fermentation began to measure the OD. 600 And residual glucose concentration; after 5 h of fermentation, carbon and nitrogen sources (containing 500 g / L glucose, 20 g / L magnesium sulfate, and 15 g / L ammonium chloride) were added at a rate of 35 mL / h; OD at 19 h 600 =72, the fermentation broth temperature was adjusted to 25 ℃, the feeding rate was reduced to 5 mL / h, and IPTG was added to a final concentration of 0.5 mM to induce expression; fermentation was carried out for 28 h, and antifoaming agent was added at a rate of 0.3 mL / h; after induction for 21 h, fermentation was stopped, and 405 g of resting cells were collected by centrifugation. Figure 15 ).

[0083] (2) Scale-up of biliverdin reaction at the 5 L bioreactor level Take 100 g of the prepared wet weight cells, add 0.1 M citrate buffer 2 (pH=7.0) to a final volume of 1 L, and magnetically stir until the cells are completely resuspended. Add heme chloride to a final concentration of 1500 mg / L, and add 20 mM NADPH to a final concentration. Initiate the reaction in a 5 L fermenter at 30℃ and an aeration rate of 1 L / min. Samples are taken every 1 h, and the amount of biliverdin produced is determined by HPLC. Figure 16 Using 1500 mg / L heme chloride as a substrate, biliverdin yield reached 1245 mg / L after whole-cell catalysis and reaction at 30 °C in the dark for 8 h, with a molar conversion rate of 92.88%.

[0084] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A heme oxygenase mutant CtHO3m, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

4.

2. The encoding gene of the heme oxygenase mutant CtHO3m according to claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

8.

3. A recombinant expression vector, characterized in that, It contains the encoding gene of the heme oxygenase mutant CtHO3m as described in claim 2.

4. A recombinant bacterium CtHO3m#, using BL21(DE3) as the host bacterium, expressing the encoding gene of the heme oxygenase mutant CtHO3m as described in claim 2.

5. An engineered bacterium CtHO3m Using BL21(DE3)-RMⅡ as the host bacterium, the gene encoding the heme oxygenase mutant CtHO3m described in claim 2 is expressed.

6. The encoding gene of the heme oxygenase mutant CtHO3m as described in claim 1 or the heme oxygenase mutant CtHO3m as described in claim 2, or the recombinant expression vector as described in claim 3, or the recombinant bacteria CtHO3m# as described in claim 4, or the engineered bacteria CtHO3m as described in claim 5. Application in the catalytic synthesis of biliverdin.

7. The application according to claim 6, characterized in that, Using heme chloride as a substrate, the encoding gene of the heme oxygenase mutant CtHO3m as described in claim 1 or the heme oxygenase mutant CtHO3m as described in claim 2, or the recombinant expression vector as described in claim 3, or the recombinant bacteria CtHO3m# as described in claim 4, or the engineered bacteria CtHO3m as described in claim 5. As a catalyst to catalyze reactions.

8. The encoding gene of the heme oxygenase mutant CtHO3m as described in claim 1 or the heme oxygenase mutant CtHO3m as described in claim 2, or the recombinant expression vector as described in claim 3, or the recombinant bacteria CtHO3m# as described in claim 4, or the engineered bacteria CtHO3m as described in claim 5. Application in increasing biliverdin production.

9. The encoding gene of the heme oxygenase mutant CtHO3m according to claim 1 or the heme oxygenase mutant CtHO3m according to claim 2, or the recombinant expression vector according to claim 3, or the recombinant bacteria CtHO3m# according to claim 4, or the engineered bacteria CtHO3m according to claim 5. Application in improving the conversion rate of heme chloride.

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