Ornithine decarboxylase mutants, genes, recombinant plasmids, recombinant strains and their applications

Abstract

This application relates to the fields of genetic engineering and enzyme engineering, specifically to an ornithine decarboxylase mutant, gene, recombinant plasmid, recombinant strain, and its applications. The ornithine decarboxylase mutant is obtained by mutating the wild-type ornithine decarboxylase with the amino acid sequence shown in SEQ ID NO.1, and the mutation sites of the ornithine decarboxylase mutant include any one or more of Y104F, A264V, Q263E, R157K, S325C, and V265A. By performing single-point or multi-site combination mutations on the wild-type ornithine decarboxylase, a high-activity ornithine decarboxylase mutant is obtained, thereby significantly improving the substrate conversion rate of the ornithine decarboxylation reaction and the synthesis efficiency of butanediamine. This successfully overcomes the rate-limiting bottleneck of the ornithine decarboxylation reaction in the synthetic pathway from glucose to ornithine and then to butanediamine, significantly optimizing the overall efficiency of this metabolic pathway.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to an ornithine decarboxylase mutant, gene, recombinant plasmid, recombinant strain, and its applications. Background Technology

[0002] Heterologous expression of ornithine decarboxylase (ODC) to achieve efficient synthesis of butanediamine is an important research direction. Current techniques typically involve heterologous expression of ornithine decarboxylases from Escherichia coli, Enterobacter cloacae, etc., in Corynebacterium glutamicum. However, wild-type ODCs generally suffer from insufficient enzyme activity and low substrate conversion rates, which significantly limits the efficient synthesis of butanediamine.

[0003] Patent application CN202411655623.2 discloses an ornithine decarboxylase (ODC10) screened from Staphylococcus ludensii through single-point mutation, further enhancing its catalytic activity for efficient synthesis of 1,4-butanediamine. However, this mutant has several limitations in practical applications:

[0004] Firstly, during long-term use, the stability of this mutant will gradually decrease, which in turn leads to a decrease in its catalytic efficiency.

[0005] Secondly, the mutant has a narrow pH stability range, with better pH stability only in the pH range of 6.5-7.5. In addition, its temperature stability is also poor, making it difficult to meet the requirements of large-scale industrial production of 1,4-butanediamine.

[0006] Third, this mutant is suitable for producing 1,4-butanediamine using the whole-cell method. This method relies on the activity and stability of the recombinant strain. Although it works well in small and medium-sized laboratories, it is difficult to commercialize. In high-density culture and large-scale fermentation, these strains are easily inhibited or metabolically disordered, resulting in a significant reduction in production efficiency.

[0007] ODCs derived from *Citrobacter freundii* typically exhibit higher stability and wider adaptability in industrial applications. However, even after expression in *Corynebacterium glutamicum*, wild-type *Citrobacter freundii*-derived ODCs still suffer from low enzyme activity and limited catalytic conversion efficiency for L-ornithine, failing to meet the demands of industrial-scale 1,4-butanediamine production. Therefore, to improve the synthesis efficiency of 1,4-butanediamine, it is necessary to modify *Citrobacter freundii*-derived ODCs. Summary of the Invention

[0008] The purpose of this invention is to provide an ornithine decarboxylase mutant, which improves the activity of the ornithine decarboxylase mutant by performing single-point mutation or multi-site combination mutation on the ornithine decarboxylase, thereby improving the catalytic conversion efficiency of L-ornithine.

[0009] To achieve the above objectives, the present invention provides the following technical solution: an ornithine decarboxylase mutant, wherein the ornithine decarboxylase mutant is obtained by mutating the wild-type ornithine decarboxylase with the amino acid sequence shown in SEQ ID NO.1, and the mutation sites of the ornithine decarboxylase mutant include any one or more of Y104F, A264V, Q263E, R157K, S325C and V265A, wherein the wild-type ornithine decarboxylase is derived from Citrobacter freundii.

[0010] Furthermore, the mutation sites include any two to four of Y104F, A264V, Q263E, R157K, S325C, and V265A.

[0011] Furthermore, the mutation sites include Q263E and / or A264V.

[0012] This application provides the gene encoding the above-mentioned ornithine decarboxylase mutant.

[0013] This application provides a recombinant plasmid loaded with the above-mentioned genes.

[0014] This application provides a recombinant strain expressing the above-mentioned ornithine decarboxylase mutant.

[0015] Furthermore, the recombinant plasmid was introduced into the CgP engineered strain for heterologous expression to construct the recombinant strain.

[0016] Furthermore, the CgP engineered strain was constructed using Corynebacterium glutamicum S3 as a matrix, by knocking out the argF, proB, speE, argR and poo genes, and by replacing the original gdh promoter with the strong promoter Ptuf.

[0017] This application provides the use of the above-mentioned ornithine decarboxylase mutant, the above-mentioned gene, the above-mentioned recombinant plasmid, or the above-mentioned recombinant strain in the preparation of 1,4-butanediamine.

[0018] The beneficial effects of this invention are as follows: This application obtains ornithine decarboxylase mutants with high enzyme activity and high catalytic performance by performing single-point mutations or multi-site combined mutations on wild-type ornithine decarboxylase, thereby significantly improving the substrate conversion rate of ornithine decarboxylation reaction and the synthesis efficiency of butanediamine. It successfully overcomes the rate-limiting bottleneck of ornithine decarboxylation reaction in the synthetic pathway from glucose to ornithine and then to butanediamine, significantly optimizing the overall efficiency of this metabolic pathway. By optimizing the activity and catalytic efficiency of ornithine decarboxylase, the accumulation of intracellular ornithine can be effectively avoided, promoting the continuous flow of carbon sources from glucose metabolism towards ornithine and butanediamine; at the same time, it can also reduce the feedback inhibition effect of intermediate product accumulation on strain growth, further improving the overall synthesis efficiency of butanediamine.

[0019] This application identifies several effective mutation sites that significantly enhance enzyme activity from numerous potential mutation sites, and further optimizes enzyme activity by superimposing these effective mutation sites, achieving a synergistic improvement in enzyme activity and butanediamine synthesis. This not only significantly reduces industrial production costs and improves production efficiency, but also provides a scientific and efficient approach for enzyme optimization and modification.

[0020] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0021] Figure 1 This is a liquid chromatogram of the synthesis of butanediamine catalyzed by the single point mutation ODC mutant with mutation site Q263E shown in Example 1 of the present invention. Detailed Implementation

[0022] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0023] It should be noted that the composition of each culture medium and reagent used in this application is as follows:

[0024] LB medium: 5 g•L -1 Yeast powder, 10g•L -1 Tryptone, 10 g / L -1 LB solid medium is prepared by adding sodium chloride, pH 7.0, and 1.8% agar powder.

[0025] BHI medium: 38.5 g•L -1 Brain and heart extract, with the addition of 1.8% agar powder, becomes BHI solid culture medium.

[0026] Secondary culture medium: BHI medium supplemented with 20 g•L -1 Glucose.

[0027] BHIS: BHI culture medium supplemented with 91 g•L -1 Sorbitol.

[0028] BHIS + 2% glycine: 91 g•L of BHI medium was added. -1 Sorbitol and 20 g•L -1 Glycine.

[0029] 10% (V / V) Tween 80: Filtration and sterilization.

[0030] 10% glycerol: sterilized at high temperature.

[0031] TG buffer: 10% glycerol + 1mM Tris, sterilized at high temperature.

[0032] The components of CGXII shake-flask fermentation medium (Corynebacterium glutamicum minimal medium XII) are shown in Table 1.

[0033]

[0034] A preferred embodiment of this application discloses a high-activity ornithine decarboxylase mutant, obtained by mutating the wild-type ornithine decarboxylase with the amino acid sequence shown in SEQ ID NO.1. The mutation sites of this ornithine decarboxylase mutant include any one or more of Y104F, A264V, Q263E, R157K, S325C, and V265A. The gene encoding the wild-type ornithine decarboxylase with the amino acid sequence shown in SEQ ID NO.1 has the sequence shown in SEQ ID NO.2.

[0035] Ornithine decarboxylases from different sources often exhibit differences in their gene sequences, encoding different amino acid sequences and three-dimensional structures. This significantly impacts their enzymatic properties, such as catalytic activity, substrate affinity, thermal stability, and pH stability. In this and other embodiments, the wild-type ornithine decarboxylase used is derived from *Citrobacter freundii*. A mutant of this wild-type ornithine decarboxylase was obtained through molecular modification. Ornithine decarboxylase from *Citrobacter freundii* maintains high stability over a relatively wide pH range and possesses strong thermostability, making it better suited to the harsh conditions of industrial production. Furthermore, optimizing and regulating its expression level and activity through genetic engineering can further enhance its production efficiency, making it more suitable for industrial-scale production applications.

[0036] In one embodiment, the mutation sites include any two to four of Y104F, A264V, Q263E, R157K, S325C, and V265A. Combining any two to four of these mutation sites results in a synergistic effect, further optimizing enzyme activity and significantly improving catalytic efficiency. In other embodiments, to ensure the ornithine decarboxylase mutant activity reaches the ideal level, the mutation sites preferably include Q263E and / or A264V. Experiments have shown that the Q263E and A264V mutation sites have a crucial impact on the ornithine decarboxylase mutant activity; incorporating them into the mutation combination helps to stably and efficiently enhance the ornithine decarboxylase mutant activity.

[0037] In one embodiment, a gene encoding the ornithine decarboxylase mutant described above is provided. In this and other embodiments, a recombinant plasmid loaded with the above gene is provided. The construction of the recombinant plasmid includes designing and synthesizing a DNA sequence containing the gene encoding the ornithine decarboxylase mutant described above, cloning the DNA sequence into an expression vector to form the recombinant plasmid. The expression vector may be pEC-ODC. cf .

[0038] In one embodiment, a recombinant strain expressing the above-mentioned ornithine decarboxylase mutant is provided. In this embodiment and other embodiments, the recombinant strain is constructed by introducing a recombinant plasmid carrying the gene encoding the above-mentioned ornithine decarboxylase mutant into a CgP engineered strain and performing heterologous expression. In some embodiments, the CgP engineered strain is constructed using Corynebacterium glutamicum S3 as a matrix, knocking out the argF, proB, speE, argR, and poo genes, and replacing the original gdh promoter with the strong promoter Ptuf.

[0039] In one embodiment, the use of the above-mentioned ornithine decarboxylase mutant, the above-mentioned gene, the above-mentioned recombinant plasmid, or the above-mentioned recombinant strain in the preparation of 1,4-butanediamine is also provided.

[0040] Example 1

[0041] Based on the amino acid sequence of wild-type ornithine decarboxylase (ODC) from *Citrobacter freundii*, a three-dimensional structural model of ODC was constructed using homology modeling, and its active pocket region was located. Next, molecular docking technology was used to simulate the binding mode between the substrate L-ornithine and the enzyme's active site, and the key interactions between them were analyzed. Then, the RosettaDesign algorithm was used to predict 15 high-scoring mutation sites, as shown in Table 2. These mutation sites scored between 1.98 and 4.39 and covered both the active pocket (e.g., positions 265 and 104) and the substrate-binding channel (e.g., position 157).

[0042] Based on the amino acid sequence of the wild-type ornithine decarboxylase shown in SEQ ID NO.1, a whole-gene synthesis was performed to obtain a plasmid including the gene fragment encoding the wild-type ornithine decarboxylase shown in SEQ ID NO.2. Based on this gene fragment encoding the wild-type ornithine decarboxylase, the primer pair pEC-ODC was designed and synthesized. cf -F and pEC-ODC cf -R. Using the plasmid containing the above-mentioned fragment encoding the wild-type ornithine decarboxylase gene as a template, pEC-ODC cf -F and pEC-ODC cf -R represents the primers. PCR amplification was performed using a Taq DNA polymerase PCR amplification kit purchased from Agilent Technologies (China) Co., Ltd. The amplification reaction mixture consisted of: 10 μL of 2-fold Taq DNA polymerase premix, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), and template (2.5–50 ng), with the volume brought to 20 μL using double-distilled water. The amplification program was: 95℃ for 5 min; 95℃ for 15 s and 50–60℃ for 15 sec, for 30–35 cycles; 72℃ for 1 min·kb. -1 Incubate at 72℃ for 5-10 minutes. The amplified PCR product was purified by agarose gel electrophoresis to obtain a recombinant plasmid carrying the gene encoding wild-type ODC, named pEC-ODC. cf -WT.

[0043] Based on the above predictions, 15 mutation sites were obtained, and the amplified recombinant plasmid pEC-ODC was also obtained. cf-WT, design and synthesize the corresponding site-directed mutagenesis primer pairs, as detailed in Table 3. Using the corresponding primer pairs as primers, the above recombinant plasmid pEC-ODC was used... cf Using WT as a template, PCR amplification was performed using a Taq DNA polymerase PCR amplification kit purchased from Agilent Technologies (China) Co., Ltd., following the amplification reaction procedure described above, to introduce a single mutation site. The amplified PCR products were purified by agarose gel electrophoresis, ultimately yielding 15 recombinant plasmids, each carrying a gene fragment encoding the aforementioned single-point mutation in the ODC mutant. These recombinant plasmids were named and labeled according to the mutation type they carried; for example, the recombinant plasmid carrying the ODC mutant gene encoding the V265A mutation site was named pEC-ODC. cf -V265A.

[0044] Using electroporation, the 16 recombinant plasmids were introduced into CgP engineered strains for heterologous expression, constructing one group of wild-type ODC recombinant strains and 15 groups of ODC mutant recombinant strains. These strains were named and labeled according to their corresponding mutation types, such as CgP / pEC-ODC. cf -WT and CgP / pEC-ODC cf -V265A, etc. Among them, the CgP engineered strain was constructed based on Corynebacterium glutamicum S3 (CgS3). Corynebacterium glutamicum S3 is deposited at the China General Microbiological Culture Collection Center (CGMCC No. 26921, deposit date: March 28, 2023, deposit address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing). In constructing the CgP engineered strain, gene editing technology was used to knock out five genes in Corynebacterium glutamicum S3: argF, proB, speE, argR, and poo, to optimize the metabolic pathway, reduce the generation of unnecessary metabolic byproducts, and improve the synthesis efficiency of the target product. Simultaneously, the original promoter of gdh was replaced with the strong promoter Ptuf to increase the metabolic flux from glucose to ornithine precursor glutamate. This CgP engineered strain is labeled: CgS3,ΔargFΔproBΔspeEΔargRΔpuo,Pgdh::Ptuf.

[0045] The electroconversion process specifically includes:

[0046] (1) Preparation of competent cells: Take an appropriate amount of CgP engineered strain bacterial suspension, inoculate it into a test tube containing 5 mL of BHI medium, and incubate overnight at 30℃ to activate the bacterial suspension. Then, transfer the activated bacterial suspension to BHIS medium containing 0.1% Tween 80 + 2% glycine, and adjust the initial OD. 600Reduce to approximately 0.2. Incubate at 30℃ for 3-4 hours until OD... 600 Stop culturing when the culture reaches 0.9. Then, transfer the bacterial culture to a centrifuge tube and centrifuge at 4°C and 4500 rpm for 5 min. Discard the supernatant and resuspend the cells in 25 mL of pre-chilled TG buffer (10% glycerol + 1 mM Tris, autoclaved). Gently mix and centrifuge again at 4°C and 4500 rpm for 5 min, discarding the supernatant. Place the centrifuge tube on ice and gently resuspend the cells with the remaining liquid to a final volume of approximately 500 μL. If the volume is insufficient, add pre-chilled 10% glycerol to obtain competent cells.

[0047] (2) Electroporation: Take 100 μL of competent cells, add 10 μL of the above recombinant plasmid, mix gently, and transfer to a pre-cooled electroporation cuvette (2 mm in diameter). Set the electroporation parameters to 2.5 kV and 25 μF, and perform electroporation. After electroporation, immediately add 4 mL of BHIS medium preheated to 46 °C to the electroporation cuvette, mix gently, and perform heat shock at 46 °C for 6 min. Then, insert the electroporation cuvette into ice, and after the temperature of the mixture drops to room temperature, transfer it to a 30 °C incubator for 60 min. After incubation, transfer the bacterial culture to a centrifuge tube, centrifuge at 4 °C and 4500 rpm for 5 min, discard part of the supernatant, and spread the remaining bacterial culture onto BHI plates containing the corresponding antibiotics (the concentration of kanamycin is approximately 25 μg / mL, and the concentration of chloramphenicol is approximately 7.5 μg / mL). Place the plate in a 30 °C incubator for 2 days.

[0048] Single colonies grown from the above plates were picked and streaked onto BHI plates containing the corresponding antibiotics, and incubated overnight at 30°C. The resulting colonies were dissolved in 100 μL of sterile water and then streaked onto BHI+10% sucrose plates, one region per single colony, and incubated at 30°C for 48-72 hours. Five to ten single colonies were picked from the sparsely grown streaked regions and streaked onto BHI+K and BHI+10% sucrose plates, respectively. Clones that grew on BHI+10% sucrose plates but not on BHI+K plates were selected and sent to Shanghai Sangon Biotech Co., Ltd. for colony PCR identification. Ultimately, 15 groups of recombinant ODC mutant strains with single point mutations and 1 group of wild-type ODC recombinant strains with correct sequencing were obtained.

[0049] Fifteen groups of recombinant ODC mutant strains with single point mutations and one group of wild-type recombinant ODC strains were selected and inoculated into test tubes containing 5 mL of BHI medium. The cultures were incubated overnight at 30°C and 200 rpm in a shaker to obtain primary seed culture. The primary seed culture was then inoculated into shake flasks containing 50 mL of CGXII fermentation medium, and the initial OD was adjusted. 600 The initial sugar concentration of the fermentation medium was 80 g / L, and the concentration was adjusted to approximately 0.3. The shake flasks were incubated at 30°C and 200 rpm for 96 h to allow the strain to reach maximum growth under suitable temperature and shaking conditions. Afterward, the cells were collected, washed twice with physiological saline, and then sonicated at 300 W with 3 / 5 s pulses for 10 min. The resulting supernatant was collected by centrifugation, yielding 16 groups of crude enzyme solutions.

[0050] The enzyme activities of the above-mentioned wild-type ornithine decarboxylases and their mutants were determined. Enzyme activity was defined as the amount of enzyme required to catalyze the production of 1 μmol of 1,4-butanediamine (putrescine) per minute at 30°C using L-ornithine as a substrate; this was defined as one unit of activity (U). The specific enzyme activity of each ornithine decarboxylase was obtained by dividing the measured enzyme activity by the enzyme mass; the unit of specific enzyme activity was U / g. Total enzyme activity was defined as the enzyme activity units (U / mL) required to catalyze the production of butanediamine per unit volume of crude enzyme solution.

[0051] The activity assay was performed as follows: 50 µL of the crude enzyme solution was added to a reaction system comprising 10 mM L-ornithine, 0.04 mM pyridoxal-5-phosphate monohydrate (PLP), 1.67 mM dithiothreitol, 100 mM L-glutamine, and 100 mM HEPES / NaOH buffer (pH 7.25). The reaction was carried out at 30 °C for 10 minutes. Then, 100 µL of the reaction solution was mixed with 900 µL of 100 mM HEPES / NaOH buffer (pH 7.25) preheated at 85 °C to terminate the enzymatic reaction. The mixture was then centrifuged at 10,000 r / min for 5 min, and the supernatant was collected for derivatization. The concentrations of 1,4-butanediamine and the remaining L-ornithine in the mixture were determined by high-performance liquid chromatography (HPLC) (mobile phase: methanol / water = 20:80, flow rate 1 mL / min, detection wavelength 210 nm). The results are shown in Table 4. Figure 1 To ensure the accuracy and reliability of the experimental results and to minimize the impact of random errors on the data, three parallel experiments were set up for all data.

[0052]

[0053] Table 4 shows that among the 15 mutation sites, Y104F, A264V, Q263E, R157K, S325C, and V265A were effective mutation sites. The corresponding ODC mutants significantly increased enzyme activity and butanediamine production, with enzyme activity increases ranging from 10.5% to 28.1% and butanediamine production ranging from 27.84 mM to 38.96 mM, representing increases of 24.5%-74.2% compared to the wild-type ODC's 22.36 mM. The consumption of the substrate L-ornithine was positively correlated with butanediamine production. The reaction system catalyzed by the ODC mutant with mutation site Q263E exhibited the highest substrate conversion rate. The remaining nine mutation sites showed decreased or no significant change in enzyme activity, and were therefore considered ineffective mutation sites.

[0054] In this embodiment, six ODC mutants with high enzyme activity were successfully obtained. Among them, the ODC mutant with the Q263E mutation site showed the best performance, with an enzyme activity increase of 28.1% and a butanediamine yield of 38.96 mM, which is 74.2% higher than that of the wild type. It can be seen that by optimizing the key residues in the active pocket (such as Y104 and Q263), the substrate binding and catalytic efficiency can be significantly improved, providing a high-quality enzyme candidate for industrial-grade butanediamine biosynthesis.

[0055] To further enhance ODC enzyme activity, based on the effective mutation sites screened in Example 1, multiple groups of ODC mutants containing two or more mutation sites were constructed by progressively stacking mutation sites. Their enzyme activity and other properties were measured to explore the relationship between the number of mutation sites and the enhancement of enzyme activity and butanediamine synthesis, thus providing a more efficient enzyme preparation for industrial-grade butanediamine biosynthesis. The test results are shown in Table 5.

[0056] Example 2

[0057] The recombinant plasmid pEC-ODC obtained from the above amplification... cf -Q263E, designed and synthesized the corresponding mutant primer pair pEC-ODC cf -Q263E+A264V-F and pEC-ODC cf -Q263E+A264V-R, see Table 3 for details. Use this primer pair to recombinant plasmid pEc-ODC. cf Using WT as a template, the corresponding mutant primer pair pEC-ODC was designed and synthesized. cf -A264V-F and pEC-ODC cf -A264V-R, see Table 3 for details. Use this primer pair to recombinant plasmid pEC-ODC. cfUsing Q263E as a template, PCR amplification was performed using the Phanta® EVO HS Super-Fidelity DNA Polymerase kit purchased from Nanjing Novizan Biotechnology Co., Ltd. The amplification reaction system consisted of: 5×EVO buffer (containing 10 mM MgCl2, 10 μL), a mixture of deoxyribonucleoside triphosphates (10 mM each, 1 μL), forward primer (10 μM, 2 μL), reverse primer (10 μM, 2 μL), template (100-300 ng), high-fidelity DNA polymerase (1 μL), and double-distilled water to a final volume of 50 μL. The amplification reaction program was as follows: 95℃, 30 s or 3 min; 95℃, 15 s, 60℃, 15 s, and 72℃, 15-30 s·kb⁻¹, for 30-35 cycles; 72℃, 5 min. The amplified PCR product was purified by agarose gel electrophoresis to obtain a recombinant plasmid carrying the gene encoding the ODC mutant, which was named and labeled pEC-ODC. cf -Q263E+A264V.

[0058] Using the same method as in Example 1, the recombinant plasmid was introduced into a CgP engineered strain for heterologous expression to construct a recombinant ODC mutant strain with two site mutations, which was named and labeled CgP / pEC-ODC. cf -Q263E+A264V. The ODC mutant recombinant strain with this two-site mutation was fermented to obtain crude enzyme solution, and its specific enzyme activity, butanediamine yield, and other enzyme activity indicators were measured.

[0059] Example 3

[0060] The recombinant plasmid pEC-ODC obtained from the above amplification... cf -Q263E+A264V, design and synthesize the corresponding mutant primer pair pEC-ODC cf -R157K-F and pEC-ODC cf -R157K-R, see Table 3 for details. Use this primer pair to recombinant plasmid pEC-ODC. cf Using Q263E+A264V as a template, PCR amplification was performed using the same amplification reaction system and procedure as in Example 2. The amplified PCR products were purified by agarose gel electrophoresis to obtain the recombinant plasmid, which was named and labeled pEC-ODC. cf -Q263E+A264V+R157K.

[0061] Using the same method as in Example 1, the recombinant plasmid was introduced into a CgP engineered strain for heterologous expression to construct a recombinant ODC mutant strain with three point mutations, which was named and labeled CgP / pEC-ODC. cf-Q263E+A264V+R157K. The ODC mutant recombinant strain with this three-point mutation was fermented to obtain crude enzyme solution, and its specific enzyme activity, butanediamine yield, and other enzyme activity indicators were measured.

[0062] Example 4

[0063] The recombinant plasmid pEC-ODC obtained from the above amplification... cf -Q263E+A264V+R157K, designed and synthesized the corresponding mutant primer pair pEC-ODC cf -Y104F-F and pEC-ODC cf -Y104F-R, see Table 3 for details. Use this primer pair to recombinant plasmid pEC-ODC. cf Using Q263E+A264V+R157K as a template, PCR amplification was performed using the same amplification reaction system and procedure as in Example 2. The amplified PCR products were purified by agarose gel electrophoresis to obtain the recombinant plasmid, which was named and labeled pEC-ODC. cf -Q263E+A264V+R157K+Y104F.

[0064] Using the same method as in Example 1, the recombinant plasmid was introduced into a CgP engineered strain for heterologous expression to construct a recombinant strain with a four-site mutation in the ODC mutant, which was named and labeled CgP / pEC-ODC. cf -Q263E+A264V+R157K+Y104F. The recombinant ODC mutant strain with this four-site mutation was fermented to obtain crude enzyme solution, and its specific enzyme activity, butanediamine yield, and other enzyme activity indicators were measured.

[0065] Example 5

[0066] The recombinant plasmid pEC-ODC obtained from the above amplification... cf -Q263E+A264V+R157K+Y104F, designed and synthesized the corresponding mutant primer pair pEC-ODC cf -S325C-F and pEC-ODC cf -S325C-R, see Table 3 for details. Use this primer pair to recombinant plasmid pEC-ODC. cf Using Q263E+A264V+R157K+Y104F as a template, PCR amplification was performed using the same amplification reaction system and procedure as in Example 2. The amplified PCR products were purified by agarose gel electrophoresis to obtain the recombinant plasmid, which was named and labeled pEC-ODC. cf -Q263E+A264V+R157K+Y104F+S325C.

[0067] Using the same method as in Example 1, the recombinant plasmid was introduced into a CgP engineered strain for heterologous expression to construct a five-site mutated ODC mutant recombinant strain, which was named and labeled CgP / pEC-ODC. cf -Q263E+A264V+R157K+Y104F+S325C. The recombinant ODC mutant strain with this five-site mutation was fermented to obtain crude enzyme solution, and its specific enzyme activity, butanediamine yield, and other enzyme activity indicators were measured.

[0068] Example 6

[0069] The recombinant plasmid pEC-ODC obtained from the above amplification... cf -Q263E+A264V+R157K+Y104F+S325C, designed and synthesized the corresponding mutant primer pair pEC-ODC cf -V265A-F and pEC-ODC cf -V265A-R, see Table 3 for details. Use this primer pair to recombinant plasmid pEC-ODC. cf Using Q263E+A264V+R157K+Y104F+S325C as a template, PCR amplification was performed using the same amplification reaction system and procedure as in Example 2. The amplified PCR products were purified by agarose gel electrophoresis to obtain the recombinant plasmid, which was named and labeled pEC-ODC. cf -Q263E+A264V+R157K+Y104F+S325C+V265A.

[0070] Using the same method as in Example 1, the recombinant plasmid was introduced into a CgP engineered strain for heterologous expression to construct a recombinant strain with a six-site mutation in the ODC mutant, which was named and labeled CgP / pEC-ODC. cf -Q263E+A264V+R157K+Y104F+S325C+V265A. The recombinant ODC mutant strain with this six-site mutation was fermented to obtain crude enzyme solution, and its specific enzyme activity, butanediamine yield, and other enzyme activity indicators were measured.

[0071]

[0072] As shown in Table 5, compared with the single-site mutation ODC mutant with mutation site Q263E provided in Example 1, the enzyme activity of the dual-site mutation ODC mutant with mutation sites Q263E and A264V combined increased by approximately 36.40%; compared with the dual-site mutation ODC mutant provided in Example 2, the enzyme activity of the three-site mutation ODC mutant with mutation sites Q263E+A264V+R157K combined provided in Example 3 increased by approximately 32.7%; similarly, compared with the three-site mutation ODC mutant provided in Example 3, the enzyme activity of the four-site mutation ODC mutant with mutation sites Q263E+A264V+R157K+Y104F combined provided in Example 4 increased by approximately 21.9%. This indicates a significant synergistic effect among multiple mutation sites, which can significantly enhance the enzyme activity of ODC mutants. Starting with a single mutation site, one mutation site was added sequentially. When adding mutations to the four-site ODC mutant, the enzyme activity of each resulting ODC mutant showed a significant synergistic increase, with an increase rate exceeding 20%, and the amount of butanediamine synthesized also increased significantly. However, when a five-site mutation was added to the four-site ODC mutant to form a five-site mutation, the enzyme activity increased by only 0.2% compared to the four-site ODC mutant, and the amount of butanediamine synthesized did not change significantly. Adding another site to form a six-site mutation ODC mutant resulted in a slight decrease in enzyme activity compared to the five-site ODC mutant, and the amount of butanediamine synthesized did not increase. This indicates that more mutation sites are not necessarily better. Within a certain range, increasing the number of mutation sites can significantly improve the enzyme activity and the amount of butanediamine synthesized in the ODC mutant; however, once this range is exceeded, further additions of mutation sites not only fail to further increase enzyme activity but may also have adverse effects on enzyme activity.

[0073] The crude enzyme solution containing the four-site mutation ODC mutant obtained in Example 4 was compared with the crude enzyme solution containing wild-type ODC and the crude enzyme solution containing the single-site mutation ODC mutant with mutation site Q263E obtained in Example 1. The results are shown in Table 6.

[0074]

[0075] As shown in Table 6, compared with wild-type ODC, the single-point mutation ODC mutant exhibited a significant increase in enzyme activity, with a 28.10% increase in specific enzyme activity, a substrate conversion rate of 47.80%, and a 74.20% increase in butanediamine synthesis concentration. In contrast, the four-site mutation ODC mutant showed an even more significant increase in enzyme activity. The four-site mutation ODC mutant exhibited a 182.8% increase in specific enzyme activity compared to wild-type ODC, a substrate conversion rate as high as 84.7%, and a butanediamine synthesis concentration of 68.92 mM, a 208.2% increase compared to the wild type. This clearly demonstrates that the synergistic effect between these mutation sites can achieve a significant increase in enzyme activity, significantly promote butanediamine synthesis, and effectively solve the problems of low enzyme activity and limited catalytic conversion efficiency of L-ornithine in existing ODCs derived from *Citrobacter freundii* expressed in *Corynebacterium glutamicum*.

[0076] 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.

[0077] 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 ornithine decarboxylase, characterized in that, The ornithine decarboxylase mutant was obtained by mutating the wild-type ornithine decarboxylase with the amino acid sequence shown in SEQ ID NO.1, and the mutation sites of the ornithine decarboxylase mutant are Q263E, Q263E+A264V, Q263E+A264V+R157K, Q263E+A264V+R157K+Y104F, Q263E+A264V+R157K+Y104F+S325C or Q263E+A264V+R157K+Y104F+S325C+V265A. The wild-type ornithine decarboxylase is derived from Citrobacter freundii.

2. The gene encoding the ornithine decarboxylase mutant of claim 1.

3. A recombinant plasmid loaded with the gene of claim 2.

4. A recombinant strain expressing the ornithine decarboxylase mutant of claim 1.

5. The use of the ornithine decarboxylase mutant of claim 1, the gene of claim 2, the recombinant plasmid of claim 3, or the recombinant strain of claim 4 in the preparation of 1,4-butanediamine.

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