Genetically engineered bacteria for producing butanediamine, preparation method and application thereof

Abstract

The application discloses a genetically engineered bacterium for producing butanediamine as well as a preparation method and application thereof, and belongs to the field of bioengineering. D494K / H729D The mutant AdiA D494K / H729D The specific enzyme activity of the mutant AdiA D494K / H729D at pH 7.0 reaches 9.3 times that of the wild type, and remains high activity in the whole alkaline range of pH 7.0-9.0. Further, the mutant AdiA D494K / H729D is expressed in E. coli, and through optimization of an expression vector, a genetically engineered bacterium with butanediamine yield reaching 155.2 g / L is constructed, thereby laying a foundation for efficiently synthesizing butanediamine.

Description

Technical Field

[0001] This invention relates to a genetically engineered bacterium for producing butanediamine, its preparation method and application, and belongs to the field of bioengineering. Background Technology

[0002] Homo oligomerization of proteins is a common phenomenon in natural evolution, with about half of all proteins existing in the form of homooligomers. These homooligomers have wide applications in physiological regulation, medicine, and chemical engineering, such as alkaline amino acid decarboxylases and tyrosine tRNA synthetases. Homooligomeric proteins are usually predominantly dimers or tetramers, while higher-order oligomers are relatively rare. Their oligomerization is crucial for structural stability and functional activity, and is maintained through covalent or non-covalent bonds at the monomer interfaces.

[0003] Arginine decarboxylase AdiA is a crucial member of the *E. coli* acid stress response system. It is a typical decameric high-order homologous oligomeric protein, consisting of five dimers forming a bilayer pentacyclic structure. This enzyme catalyzes the decarboxylation of L-arginine to produce guanidine, a key enzyme in the biosynthesis of putrescine (an important nylon monomer). However, the assembly of the AdiA decameric structure is highly pH-dependent, remaining stable only under acidic conditions (pH < 6.0). This is because its surface is rich in acidic amino acids, which protonate in acidic environments to neutralize the charge and promote oligomerization. Under neutral and alkaline conditions, the acidic amino acids deprotonate, generating electrostatic repulsion, causing the decameric to depolymerize into dimers or monomers, resulting in a significant loss of enzyme activity. This pH dependence severely limits the whole-cell catalytic application of AdiA in neutral or alkaline environments. Since the intracellular environment in industrial fermentation is typically neutral, and the putrescine product is alkaline, conventional AdiA cannot be efficiently used for the large-scale biosynthesis of putrescine.

[0004] Currently, various engineering strategies have been developed to promote protein oligomerization, such as regulating interfacial residue complementarity, introducing disulfide bonds or electrostatic attraction, and improving interfacial hydrophobicity. These strategies are easily implemented in dimer and tetramer proteins. However, for higher-order decadal proteins like AdiA, due to the complex interfacial interactions (involving both meridional and latitudinal interfaces) and the difficulty in molecular dynamics simulations, existing strategies struggle to effectively achieve oligomeric structural stability under moderately alkaline conditions. Furthermore, other acid-inducible decarboxylases, such as lysine decarboxylase CadA, also exhibit similar pH-regulating mechanisms, limiting the efficient biosynthesis of polyamine compounds.

[0005] Therefore, there is an urgent need to develop a new rational engineering strategy to precisely modify the interface of higher-order oligomeric decarboxylases, so as to shift the pH adaptability of the enzyme towards neutral and alkaline conditions, thereby improving the catalytic efficiency of the enzyme and promoting the green and large-scale production of polyamine compounds such as butanediamine. Summary of the Invention

[0006] The purpose of this invention is to first obtain an arginine decarboxylase mutant with optimal pH upregulation, and then, based on this arginine decarboxylase mutant with optimal pH upregulation, construct a genetically engineered bacterium capable of efficiently producing butanediamine, wherein the combined mutant AdiA D494K / H729D The enzyme activity at pH 7.0 was 9.3 times higher than that of AdiA, making the mutant AdiA the one with the best enzyme activity. D494K / H729D It was applied to the biosynthesis of butanediamine, resulting in a butanediamine yield of 155.2 g / L.

[0007] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: The first objective of this invention is to provide a genetically engineered bacterium for producing butanediamine, wherein the bacterium expresses an arginine decarboxylase mutant in a host. adiA D494K / H729D Arginine decarboxylase SpeA and guanidine amino acid enzyme SpeB, wherein the arginine decarboxylase mutant adiA D494K / H729D It is obtained by mutating aspartic acid at position 494 of the arginine decarboxylase, as shown in SEQ ID NO.1, to lysine, and mutating histidine at position 729 to aspartic acid.

[0008] In one embodiment of the present invention, in the genetically engineered bacteria, arginine decarboxylase SpeA and guanidine amino acid enzyme SpeB are expressed using an expression vector, and the arginine decarboxylase mutant... adiA D494K / H729D It uses another expression vehicle for expression.

[0009] In one embodiment of the present invention, the expression vector is a pET series expression vector or a pRSF-Duet-1 expression vector, wherein the pET series expression vector is preferably a pETDuet-1 expression vector.

[0010] In one embodiment of the present invention, the genetically engineered bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast.

[0011] In one embodiment of the present invention, the amino acid sequence of arginine decarboxylase SpeA is SEQ ID NO.3, and the amino acid sequence of guanidine amino acid enzyme SpeB is shown in SEQ ID NO.4.

[0012] The second objective of this invention is to provide an arginine decarboxylase mutant, which is obtained by mutating aspartic acid at position 494 of the arginine decarboxylase as shown in SEQ ID NO.1 to lysine, and mutating histidine at position 729 to aspartic acid.

[0013] A third object of the present invention is to provide a recombinant enzyme catalyst containing the said arginine decarboxylase mutant, which is any one of the following forms: (1) Culture recombinant expression transformants containing the arginine decarboxylase mutant and isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme; (2) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, and break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant arginine decarboxylase mutant enzyme to obtain lyophilized enzyme powder.

[0014] The fourth objective of this invention is to provide a method for synthesizing butanediamine, wherein the method uses arginine as a substrate and the genetically engineered bacteria or its lysate or fermentation broth as a catalyst to catalytically convert and synthesize butanediamine.

[0015] In one embodiment of the present invention, the amount of arginine added is 300~400g / L, the amount of genetically engineered bacteria added is 80~100 g wet weight / L, PLP is 0.08~0.15mmol / L, the temperature is 35~45℃, the rotation speed is 200~250r / min, and the initial pH is 7.0.

[0016] A fifth object of the present invention is to provide the use of the genetically engineered bacteria or the method described herein in the preparation of butanediamine or products containing butanediamine or in the preparation of nylon products.

[0017] Beneficial effects This invention yielded an arginine decarboxylase mutant, AdiA, with optimal pH upregulation. D494K / H729D The mutant AdiA D494K / H729D The specific enzyme activity at pH 7.0 reached 9.3 times that of the wild type, and maintained high activity throughout the entire neutral-alkaline range of pH 7.0-9.0. Furthermore, this invention uses the mutant AdiA... D494K / H729D The expression was performed in Escherichia coli, and through optimization of the expression vector, a genetically engineered bacterium with a yield of 155.2 g / L of butanediamine was constructed, laying the foundation for efficient butanediamine synthesis. Detailed Implementation

[0018] The culture media involved in the following examples: LB solid culture: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 2 g / L agar powder.

[0019] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0020] The detection methods involved in the following embodiments are as follows: The method for detecting arginine decarboxylase activity is as follows: Prepare a 200 µL enzyme activity reaction system according to Table 1, and determine the arginine decarboxylase activity. The reaction system was precisely reacted at 37°C for 10 min, and the reaction was terminated by adding 20 µL of 40% trichloroacetic acid. The reaction solution was then cooled in an ice-water bath. The reaction was then accelerated at 12000 r·min. -1 Centrifuge for 10 min, collect the supernatant, derivatize it, and use it for HPLC detection of butanediamine yield. Unit enzyme activity is defined as U / g = 1 mol butanediamine / min / g protein.

[0021] Table 1: Reaction system for arginine decarboxylase activity detection

[0022] The 15 mmol / L potassium sodium phosphate buffer solution is prepared as follows: the pH value of 15 mmol / L KH2PO4 is adjusted to the corresponding pH value using 15 mmol / L Na2HPO4.

[0023] HPLC detection of butanediamine: 1) Sample derivatization and extraction The pre-column derivatization procedure for dansyl chloride is as follows: After centrifuging the fermentation broth or reaction solution at 12000 r / min for 10 min, take 500 μL of the supernatant as the sample. Add 500 μL of saturated NaHCO3 solution and internal standard (5 μL of 10 g / L heptamethylenediamine), mix thoroughly, and adjust the pH to 10 with saturated NaOH solution. Then add 1 mL of the derivatization reagent dansyl chloride (5 g / L, soluble in acetone). Incubate the mixture in a light-protected water bath at 60°C for 30 min, add 2 mL of anhydrous diethyl ether, and extract for 10 min. Collect the upper organic phase. Repeat the extraction operation twice, mix the two organic phases, and dry them with nitrogen to remove the diethyl ether. Dissolve the derivatized compound in 500 μL of acetonitrile solution, filter through a 0.22 μm filter membrane, and use for HPLC detection.

[0024] 2) HPLC chromatographic determination Chromatographic conditions: High-performance liquid chromatography (HPLC) separation of diamine dansyl chloride derivatives was performed on a C18 column at a separation temperature of 30°C and a UV detection wavelength of 254 nm. The injection volume was 10 μL. Mobile phase A was ultrapure water, and mobile phase B was HPLC-grade acetonitrile. Both mobile phases were filtered through a 0.22 μm filter before use. The gradient elution program was set as follows: 0–4 min, 55%–70% B; 4–6 min, 70% B; 6–11 min, 70% B; 11–12 min, 95% B; 12–13 min, 95% B; 13–16 min, 55% B. The total flow rate was set to 0.7 mL / min.

[0025] Example 1: Determination of the AdiA mutation site in arginine decarboxylase The specific steps are as follows: Arginine decarboxylase AdiA catalyzes the synthesis of guanidinediamine from arginine and is a key enzyme in the butanediamine (ADC) synthesis pathway. Its catalytic reaction requires pyridoxal phosphate (PLP) to assist in decarboxylation and proton transfer, but does not consume PLP. The effective quaternary structure of AdiA is a decameric, but the decameric can only form in environments with pH < 6, resulting in an optimal reaction pH of slightly acidic (pH 5.4). Based on the crystal structure of the AdiA decameric (PDB ID: 2VYC), interface analysis was performed using PyMOL 2.5 software. The AdiA decameric consists of a bilayer pentacyclic structure composed of five dimers, exhibiting centrosymmetry. The meridional interface, located between the pentacyclic monomers, mainly involves acidic amino acid enrichment regions and exhibits significant electrostatic repulsion under moderately alkaline conditions. The latitudinal interface, located between the dimers, simultaneously forms substrate channels. The CAVER tool was used to predict these substrate channels, which directly affect the binding and transport of the substrate arginine. The surface of AdiA is rich in acidic amino acids, which results in the decadomer being stable only at pH < 6.0.

[0026] Based on the above research, D494 and H729 were identified as mutation sites for arginine decarboxylase AdiA.

[0027] Example 2: Preparation of Arginine Decarboxylase AdiA Mutant The specific steps are as follows: Through primers adiA -F and adiA -R from E. coli Amplification of arginine decarboxylase gene in the BL21(DE3) genome adiA ,use Nde I and Xho I performed double digestion of the PCR product and pETDuet-1 plasmid, and after purification, the product was ligated using T4 DNA ligase. adiA The ligation product was introduced into E. coli JM109 and screened by colony PCR and Sanger sequencing to finally obtain the recombinant plasmid pETDuet-adiA.

[0028] The amino acid sequence (SEQ ID NO.1) of the wild-type arginine decarboxylase is as follows: MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRT DMGIERTSLGSLLDHTGAFGESEKYAARVFGADRSWSVVVGTSGSNRTIMQACMTDNDVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPT VFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGLSLTQEVIDEAVDFRQAMARLYKEFTADGSWFFKPWNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRH GIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNAAYSGLPVAEVTPREAYNAIVDNNVELVSIENLPGRIAANSVIPYPPGIPMLLSGENFGDKNSPQVSYLRSLQSWDHHFPGFEHETEGTEIIDGIYHVMCVKA The nucleotide sequence (SEQ ID NO.2) encoding the arginine decarboxylase is as follows: Through primers adiA- D494K -F and adiA- D494K -R was used to perform full-plasmid PCR on pETDuet-adiA, and the PCR product was purified before use. Dpn The bacteria were digested with enzyme I, and the digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain adiA. D494K Mutant recombinant plasmid pETDuet-adiA D494K .

[0029] Through primers adiA- H729D -F and adiA- H729D -R was used to perform full-plasmid PCR on pETDuet-adiA, and the PCR product was purified before use. Dpn The bacteria were digested with enzyme I, and the digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain adiA. H729D Mutant recombinant plasmid pETDuet-adiA H729D Further, using primers... adiA- D494K -F and adiA- D494K -R for pETDuet-adiA H729D Perform whole-plasmid PCR, and use the PCR products after purification. Dpn The bacteria were digested with enzyme I, and the digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain adiA. D494K / H729D Mutant recombinant plasmid pETDuet-adiA D494K / H729D .

[0030] Table 2: Primer Sequence Listing

[0031] Example 3: Expression of mutant arginine decarboxylase and purification of AdiA The specific steps are as follows: The recombinant plasmids expressing the AdiA gene and its mutants, prepared in Example 1, and the recombinant plasmid expressing the guanidine aminotransferase SpeB gene, were expressed in *E. coli* BL21 with an N-terminal 6-His tag. All expression strains were cultured in 500 mL SOB medium at 37°C for 3 hours, followed by overnight culture with 0.1 mmol / L IPTG at 30°C to induce protein expression. The overnight cultured cells were collected by refrigerated centrifugation at 8000 rpm for 5 min, resuspended in 50 mL Tris-HCl buffer, and then sonicated. The supernatant was purified by affinity chromatography using a Ni-NTA Superflow resin column.

[0032] The method for preparing the recombinant vector expressing the guanidine aminotransferase SpeB gene is as follows: First, it is digested with double enzymes (the enzyme sites are: Nde I, Xho I) The ligation method was used to construct the guanidine amino acid enzyme SpeB overexpression plasmid pETDuet-SpeB; the amino acid sequence of SpeB (SEQ ID NO.3) is as follows: MSTLGHQYDNSLVSNAFGFLRLPMNFQPYDSDSDWVITGVPFDMATSGRAGGRHGPAAIRQVSTNLAWEHNRFPWNFDMRERLNVVDCGDLVYAFGDAREMSEKLQAHAEKLLAAGKRMLSFGGDHFVTLPLLRAHAKHFGKMALVHFDAHTD TYANGCEFDHGTMFYTAPKEGLIDPNHSVQIGIRTEFDKDNGFTVLDACQVNDRSVDDVIAQVKQIVGDMPVYLTFDIDCLDPAFAPGTGTPVIGGLTSDRAIKLVRGLKDLNIVGMDVVEVAPAYDQSEITALAAATLALEMLYIQAAKKGE* Pure enzyme solutions of wild-type AdiA, AdiA mutant, and guanidine amino acid enzyme SpeB were prepared.

[0033] Example 4: Enzyme activity assay of wild-type and mutant enzymes at different pH values The specific steps are as follows: Following the reaction system in Table 1, i.e., adding arginine decarboxylase AdiA or mutant pure enzyme solution to a potassium sodium phosphate buffer containing dithiothreitol, EDTA, pyridoxal phosphate (PLP), MgSO4, arginine, and guanidine amino acid enzyme pure solution, to achieve the final concentration shown in Table 1, the reaction system was incubated at 37°C for 10 min. The reaction was terminated by adding 20 µL of 40% trichloroacetic acid, and the reaction solution was cooled in an ice-water bath. After centrifugation at 12000 r / min for 10 min, the supernatant was derivatized and used for HPLC detection of butanediamine yield. The unit enzyme activity was defined as U / g = 1 mole of butanediamine / min / g protein.

[0034] HPLC analysis revealed the enzyme activity of each mutant, as shown in Table 3. The results indicate that mutant AdiA... D494K / H729D At pH 7.0, the enzyme activity was 9.3 times that of the wild type, and it maintained high activity in the pH range of 7.0–9.0.

[0035] Table 3

[0036] Example 5: Construction of genetically engineered bacteria containing mutant arginine decarboxylase and its whole-cell catalytic production of butanediamine The specific steps are as follows: 1. Construction of genetically engineered bacteria Through primers speA -F and speA -R amplifies the gene from the genome of Escherichia coli BL21(DE3). speA .use Nco I and EcoR I performed double digestion of the PCR product and pETDuet-1 plasmid, and after purification, the product was ligated using T4 DNA ligase. speA The ligation product was ligated into pETDuet-1. The ligation product was introduced into E. coli JM109, and screened by colony PCR and Sanger sequencing to finally construct the plasmid pETDuet-speA.

[0037] Through primers speB -F and speB -R amplifies the gene from the genome of Escherichia coli BL21(DE3). speB .

[0038] use EcoR I and Hind III. The PCR product and pETDuet-speA plasmid were double-digested, and the purified products were then ligated using T4 DNA ligase. speB Ligation was performed on pETDuet-speA. The ligation product was introduced into *E. coli* JM109, and screened by colony PCR and Sanger sequencing to finally construct the plasmid pETDuet-speA-speB. Primers were then used to... adiA -F and adiA -R (shown in Table 2) from pETduet- adiA D494K / H729D amplified mutant gene adiA D494K / H729D ,use Nde I and Xho I performed double digestion of the PCR product and the pETDuet-speA-speB plasmid, and after purification, the product was ligated using T4 DNA ligase. adiA D494K / H729D The plasmid pETDuet-speA-speB was ligated into pETDuet-speA-speB, and the ligation product was introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to obtain the recombinant plasmid pETDuet-speA-. adiA D494K / H729D -speB. The recombinant plasmid pETDuet-speA- adiA D494K / H729D -speB conversion to E. coli BL2(DE3) was used to obtain the butanediamine-synthesizing genetically engineered bacterium PUT1.

[0039] In the same way, using wild type adiA Gene replacement adiA D494K / H729D Gene, construct pETDuet-speA-adiA-speB recombinant plasmid, and transform it into E. coli BL2(DE3) was used to obtain the butanediamine-synthesizing genetically engineered bacterium PUT0 as a control strain.

[0040] The amino acid sequence (SEQ ID NO.4) of the arginine decarboxylase SpeA is as follows: * The primer sequences involved are as follows: Table 4

[0041] 2. Preparation of wet mycelium (1) After the recombinant butanediamine strains PUT0 and PUT1 stored in glycerol tubes were streaked on LB solid plates containing 50 μg / mL kanamycin, single colonies were picked and inoculated into 50 mL LB liquid medium and cultured in a shaker at 37 ℃ and 250 r / min for 12 h to prepare seed culture.

[0042] (2) The prepared seed culture was transferred to 1 L LB medium containing the same antibiotic at an inoculation rate of 2% (v / v), and cultured at 37 °C until OD600≈15-20. The temperature was then reduced to 30 °C, and 0.1 mmol / L IPTG was added to induce expression for 16 h to prepare the fermentation broth.

[0043] (3) After induction, the prepared fermentation broth was centrifuged at 4 ℃ and 8000 r· / min for 20 min to collect the cells. The cells were washed twice with 50 mmol / L phosphate buffer at pH 7.0 and then resuspended to obtain wet cells.

[0044] 3. Whole-cell catalytic preparation of butanediamine Whole-cell catalysis was carried out in a 5 L bioreactor with a working volume of 3 L. The reaction system consisted of: a total L-arginine concentration of 348.4 g / L (added in batches), a wet cell concentration equivalent to OD600≈80, PLP of 0.1 mmol / L, a temperature of 42 ℃, a rotation speed of 300 r / min, an initial pH of 7.0, a closed reaction, and a reaction time of 12 h.

[0045] Samples were taken every 2 hours. After centrifugation at 12,000 r / min, the supernatant was derivatized with dansyl chloride, and the concentration of butanediamine was detected by HPLC.

[0046] The results are shown in Table 5: Table 5

[0047] Finally, the optimal double mutant AdiA of this invention was utilized. D494K / H729D The recombinant whole-cell catalyst achieved a butanediamine yield of 146.6 g / L within 12 h.

[0048] Example 6: Optimization of expression vectors for genetically engineered bacteria The specific steps are as follows: Through primers speA -F and speA -R amplifies the gene from the genome of Escherichia coli BL21(DE3). speA .use Nco I and EcoR I performed double digestion of the PCR product and pETDuet-1 plasmid, and after purification, the product was ligated using T4 DNA ligase. speA The ligation product was ligated into pETDuet-1. The ligation product was introduced into E. coli JM109, and screened by colony PCR and Sanger sequencing to finally construct the plasmid pETDuet-speA.

[0049] Through primers speB -F and speB -R amplifies the gene from the genome of Escherichia coli BL21(DE3). speB .

[0050] use EcoR I and Hind III. The PCR product and pETDuet-speA plasmid were double-digested, and the purified products were then ligated using T4 DNA ligase. speB The ligation product was ligated into pETDuet-speA. The ligation product was then introduced into *E. coli* JM109, and screened by colony PCR and Sanger sequencing to finally construct the plasmid pETDuet-speA-speB.

[0051] Through primers adiA -F and adiA -R from pETduet- adiA D494K / H729D amplified mutant gene adiA D494K / H729D ,use Nco I and BamH I performed double digestion of the PCR product and pRSF-Duet-1 plasmid, and after purification, the product was ligated using T4 DNA ligase. adiA D494K / H729D The recombinant plasmid pRSF-Duet-1 was obtained by ligating it into pRSF-Duet-1. adiA D494K / H729D .

[0052] The recombinant plasmid pRSF-Duet-1- adiA D494K / H729D Transformed with recombinant plasmid pETDuet-speA-speB into E. coli BL2(DE3) was used to obtain the butanediamine-synthesizing genetically engineered bacterium PUT2.

[0053] Following the method in Example 5, wet bacterial cell preparation and whole-cell transformation were performed to prepare butanediamine, and the results showed that the butanediamine yield reached 155.2 g / L.

[0054] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A genetically engineered bacterium for producing butanediamine, characterized in that, The genetically engineered bacteria expressed an arginine decarboxylase mutant in the host. adiA D494K / H729D Arginine decarboxylase SpeA and guanidine amino acid enzyme SpeB, wherein the arginine decarboxylase mutant adiA D494K / H729D It is obtained by mutating aspartic acid at position 494 of the arginine decarboxylase, as shown in SEQ ID NO.1, to lysine, and mutating histidine at position 729 to aspartic acid. The genetically engineered bacteria is Escherichia coli. The amino acid sequence of the arginine decarboxylase SpeA is SEQ ID NO.3, and the amino acid sequence of the guanidine amino acid enzyme SpeB is shown in SEQ ID NO.

4.

2. The genetically engineered bacterium according to claim 1, characterized in that, In the genetically engineered bacteria, arginine decarboxylase SpeA and guanidine amino acid enzyme SpeB are expressed using a pETDuet-1 expression vector, and the arginine decarboxylase mutant... adiA D494K / H729D Another pRSF-Duet-1 expression vector was used for expression.

3. An arginine decarboxylase mutant, characterized in that, The arginine decarboxylase mutant is obtained by mutating aspartic acid at position 494 to lysine and histidine at position 729 to aspartic acid in the arginine decarboxylase amino acid sequence shown in SEQ ID NO.

1.

4. A recombinant enzyme catalyst containing the arginine decarboxylase mutant of claim 3, characterized in that, It is any of the following forms: (1) Culture recombinant expression transformants containing the arginine decarboxylase mutant and isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme; (2) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, and break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing the arginine decarboxylase mutant, isolate transformant cells containing the recombinant arginine decarboxylase mutant enzyme, break the transformant cells containing the recombinant arginine decarboxylase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant arginine decarboxylase mutant enzyme to obtain lyophilized enzyme powder.

5. A method for synthesizing butanediamine, characterized in that, The method involves using arginine as a substrate and the fermentation broth of the genetically engineered bacteria described in any one of claims 1 to 2 as a catalyst to catalytically convert and synthesize butanediamine.

6. The genetically engineered bacteria according to any one of claims 1 to 2 or the method according to claim 5, used in the preparation of butanediamine or products containing butanediamine.

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