Ornithine decarboxylase mutant and its use in producing 1,4-butanediamine

Abstract

This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to an ornithine decarboxylase mutant, an engineered bacterium, and its application in the production of 1,4-butanediamine. Compared to the protein with the amino acid sequence shown in SEQ ID NO: 1, this mutant contains a mutation at amino acid position 694. The mutant protein exhibits significantly enhanced catalytic activity compared to the parent enzyme and significantly improves the synthesis efficiency of 1,4-butanediamine, providing a new biosynthetic pathway and engineered bacterium for the production of 1,4-butanediamine.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to an ornithine decarboxylase mutant and its application in the production of 1,4-butanediamine. Background Technology

[0002] 1,4-Butanediamine (chemical formula: C4H) 12 1,4-Butanediamine (CAS No.: 110-60-1) is an important organic compound, also known as 1,4-diaminobutane, tetramethylenediamine, or putrescine. It has wide applications in industry, medicine, and biological research. Putrescine is also widely used in bio-agriculture, with its core value lying in enhancing plant stress resistance, improving crop quality and yield, and its unique role in animal gut health. Putrescine acts as a signaling molecule, helping organisms cope with environmental stress. Currently, the production of 1,4-butanediamine mainly relies on chemical synthesis. This involves reacting highly toxic styrene with hydrogen cyanide under expensive alkaline catalysts to obtain flammable succinate, which is then converted to 1,4-butanediamine through a harsh hydrogenation reaction. This entire chemical synthesis route poses significant risks to human health and the environment, and the chemical raw materials used in the reaction are derived from non-renewable petroleum resources. Given the shortage of petrochemical raw materials, finding a low-energy-consumption, environmentally friendly, sustainable, and efficient route for the production of 1,4-butanediamine is urgently needed. With the development of bioengineering, biosynthesis has gradually shown its application value in many fields such as bio-based chemical product production, bioenergy, biomedicine, and pollution control.

[0003] The biosynthetic pathway of 1,4-butanediamine mainly includes two pathways: ornithine decarboxylation and arginine decarboxylation. The core of the ornithine decarboxylation pathway is ornithine decarboxylase (ODC, EC4.1.1.17), which catalyzes the synthesis of 1,4-butanediamine from ornithine. Therefore, developing a highly active ornithine decarboxylase is crucial for achieving efficient biosynthesis of 1,4-butanediamine. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an ornithine decarboxylase mutant and its application in the production of 1,4-butanediamine. This mutant protein exhibits significantly enhanced catalytic activity compared to the parent enzyme and significantly improves the synthesis efficiency of 1,4-butanediamine, providing a novel biosynthetic pathway and engineered microorganism for the production of 1,4-butanediamine.

[0005] The technical solution of this invention is as follows: The present invention provides an ornithine decarboxylase mutant, which, compared with the protein shown in SEQ ID NO: 1, contains a mutation at amino acid position 694.

[0006] Preferably, the ornithine decarboxylase mutant, compared with the protein whose amino acid sequence is as shown in SEQ ID NO: 1, has a tyrosine mutation at position 694 to one of the following amino acids: glutamine, asparagine, cysteine, serine, or proline.

[0007] The present invention also provides the encoding gene of the above-mentioned ornithine decarboxylase mutant.

[0008] The present invention also provides vectors, recombinant vectors or expression vectors containing the above-mentioned coding genes.

[0009] The present invention also provides a host cell containing the above-described coding gene or recombinant vector.

[0010] The present invention also provides the application of the above-mentioned ornithine decarboxylase mutant, which uses the mutant protein to catalyze the conversion of L-ornithine or L-ornithine salt into 1,4-butanediamine (or putrescine).

[0011] The present invention also provides a method for preparing the above-mentioned ornithine decarboxylase mutant, which involves gene recombination and expression using the coding gene of the ornithine decarboxylase mutant or an expression vector containing the coding gene.

[0012] The present invention also provides a method for producing 1,4-butanediamine, the method comprising the steps of introducing the ornithine decarboxylase mutant into a microbial expression system and preparing 1,4-butanediamine with a suitable substrate under suitable conditions.

[0013] The present invention also provides a method for improving the activity of ornithine decarboxylase, wherein the method is by introducing one of the following substitutions into the parental ornithine decarboxylase: Y694Q, Y694N, Y694C, Y694S or Y694P; each position corresponds to the amino acid sequence of ornithine decarboxylase shown in SEQ ID NO: 1.

[0014] The beneficial effects of this invention are as follows: 1. This invention modifies ornithine decarboxylase by means of directed evolution, selection of active sites, gene mutation, etc., and discovers the 694th active site of ornithine decarboxylase for the first time. High-activity mutants are screened and a new important target is provided for enzyme engineering modification.

[0015] 2. This invention utilizes ornithine decarboxylase with enhanced activity for the biosynthesis of 1,4-butanediamine, significantly improving the microbial fermentation level of 1,4-butanediamine.

[0016] 3. Taking the *E. coli* expression system as an example, this invention obtained a genetically engineered bacterium containing the ornithine decarboxylase mutant of this invention, and applied the genetically engineered bacterium to the production of 1,4-butanediamine. Compared with the unmutated engineered bacterium, the engineered bacterium containing the mutant of this invention exhibited high catalytic activity, with the highest concentration of 1,4-butanediamine reaching 1.4 times that of the control bacterium. The mutant of this invention effectively improves the synthesis efficiency of 1,4-butanediamine, providing a new biosynthetic pathway and genetically engineered bacterium for the production of 1,4-butanediamine. Attached Figure Description

[0017] Figure 1 The pET28a plasmid and recombinant vector pET28a-SpeC spectra in Example 1. Detailed Implementation

[0018] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0019] The following definitions are used in this invention: 1. Nomenclature of amino acids and DNA nucleic acid sequences Amino acid residues are named using the recognized IUPAC nomenclature, either in three-letter abbreviations or single-letter symbols. DNA nucleic acid sequences are named using the recognized IUPAC nomenclature.

[0020] 2. Identification of ornithine decarboxylase mutants The term "amino acid replaced at the original amino acid position" is used to represent the mutated amino acid in the ornithine decarboxylase mutant. For example, Y694Q indicates that the 694th amino acid residue in the sequence from the N-terminus to the C-terminus is replaced by glutamine (Q) instead of tyrosine (Y) from the parent ornithine decarboxylase. The numbering method of this invention is based on the sequence shown in SEQ ID NO: 1.

[0021] 3. The amino acid sequence of ornithine decarboxylase is shown in SEQ ID NO: 1: MKSMNIAASSELVSRLSSHRRVVALGDTDFTDVAAVVITAADSRSGILALLKRTGFHLPVFLYSEHAVELPAGVTAVINGNEQQWLELESAACQYEENLLPPFYDTLTQYVEMGNSTFACPGHQHGAFFKKHPAGRHFYDFFGENVFRADMCNADVKLGDLLIHEGSAKDAQKFAAK VFHADKTYFVLNGTSAANKVVTNALLTRGDLVLFDRNNHKSNHHGALIQAGATPVYLEASRNPFGFIGGIDAHCFNEEYLRQQIRDVAPEKADLPRPYRLAIIQLGTYDGTVYNARQVIDTVGHLCDYILFDSAWVGYEQFIPMMADSSPLLLELNENDPGIFVTQSVHKQQAGFSQT SQIHKKDNHIRGQARFCPHKRLNNAFMLHASTSPFYPLFAALDVNAKIHEGESGRRLWAECVEIGIEARKAILARCKLFRPFIPPVVDGKLWQDYPTSVLASDRRFFSFEPGAKWHGFEGYAADQYFVDPCKLLLTTPGIDAETGEYSDFGVPATILAHYLRENGIVPEKCDLNSILF LLTPAESHEKLAQLVAMLAQFEQHIEDDSPLVEVLPSVYNKYPVRYRDYTLRQLCQEMHDLYVSFDVKDLQKAMFRQQSFPSVVMNPQDAHSAYIRGDVELVRIRDAEGRIAAEGALPYPPGVLCVVPGEVWGGAVQRYFLALEEGVNLLPGFSPELQGVYSETDADGVKRLYGYVLK The present invention provides an ornithine decarboxylase mutant, which, compared with the protein shown in SEQ ID NO: 1, contains a mutation at amino acid position 694.

[0022] Preferably, the ornithine decarboxylase mutant, compared with the protein whose amino acid sequence is as shown in SEQ ID NO: 1, has a tyrosine mutation at position 694 to one of the following amino acids: glutamine, asparagine, cysteine, serine, or proline.

[0023] The present invention also provides a coding gene for the above-mentioned ornithine decarboxylase mutant. In some embodiments, the nucleotide sequence of the coding gene is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0024] The present invention also provides vectors, recombinant vectors or expression vectors containing the above-mentioned coding genes, such as a recombinant vector composed of vector plasmid pET28a and the coding gene described in the present invention.

[0025] The present invention also provides a host cell comprising the above-described coding gene or recombinant vector. The host cell can be any host suitable for producing the ornithine decarboxylase mutant of the present invention from the gene or vector of the present invention, such as microbial expression systems like *Escherichia coli*, *Bacillus subtilis*, *Corynebacterium glutamicum*, and *Saccharomyces cerevisiae*.

[0026] The present invention also provides the application of the above-mentioned ornithine decarboxylase mutant, which uses the mutant protein to catalyze the conversion of L-ornithine or L-ornithine salt into 1,4-butanediamine (or putrescine).

[0027] The ornithine decarboxylase mutant of the present invention has improved enzyme activity or catalytic activity compared with its parent ornithine decarboxylase, for example, more than 1.1 times in some embodiments, more than 1.2 times in others, more than 1.3 times in still others, and more than 1.4 times in yet others.

[0028] The present invention also provides a method for preparing the above-mentioned ornithine decarboxylase mutant, which involves gene recombination and expression using the coding gene of the ornithine decarboxylase mutant described in this invention or an expression vector containing the coding gene. Gene recombination methods and expression hosts known to those skilled in the art can be used, and suitable culture media and culture conditions for host expression can be selected. The method may further include a step of recovering the ornithine decarboxylase mutant, which may involve isolating or purifying the ornithine decarboxylase mutant from the host culture or expression product, and can be performed using any method known to those skilled in the art.

[0029] This invention also provides a method for producing 1,4-butanediamine, the method comprising the steps of introducing the ornithine decarboxylase mutant into a microbial expression system (e.g., an *E. coli* expression system) and preparing 1,4-butanediamine under suitable conditions with a suitable substrate (e.g., ornithine, ornithine salt). In some embodiments, the microbial expression system further includes other modifications or alterations, such as enhancing precursor supply, strengthening key enzyme catalytic reactions, blocking ornithine degradation pathways, and / or removing intracellular feedback repression and feedback inhibition to promote ornithine accumulation by introducing or knocking out genes.

[0030] In the above method for producing 1,4-butanediamine, the preferred culture medium composition is as follows: Glucose 20 g / L, yeast extract 1 g / L, peptone 2 g / L, K2HPO4 3 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 15 mg / L, MnSO4·7H2O 15 mg / L, V B1 V B3 V B5 V B12 V H Each 1 mg / L, L-arginine 2 g / L, the remainder being water, pH 7.0.

[0031] In other embodiments, the preferred culture medium composition is: 5-10 g / L peptone, 2-5 g / L yeast extract, 5-10 g / L NaCl, with the remainder being water, and the pH being natural.

[0032] The present invention also provides a method for improving the activity of ornithine decarboxylase, wherein the method is by introducing one of the following substitutions into the parental ornithine decarboxylase: Y694Q, Y694N, Y694C, Y694S or Y694P; each position corresponds to the amino acid sequence of ornithine decarboxylase shown in SEQ ID NO: 1.

[0033] The present invention will be described in more detail below through specific embodiments. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] Example 1: This embodiment illustrates the screening method for ornithine decarboxylase mutant libraries and highly active ornithine decarboxylases.

[0035] Recombinant strains E.coli Construction of BL21-pET28a-SpeC: First, pET28a plasmid ( Figure 1 Using pET28a as a template, a linearized vector was obtained by reverse PCR with primer pET28a-S / A. The ornithine decarboxylase encoding gene was then obtained by PCR using primer speC-S / A. speC Gene fragment (nucleotide sequence as shown in SEQ ID NO:2, encoding amino acid sequence as shown in SEQ ID NO:1), pET28a linearized vector and speC The gene fragment was ligated using the ClonExpress® II One Step Cloning Kit series of homologous recombinases (purchased from Nanjing Novizan Biotechnology Co., Ltd.) to obtain the recombinant vector pET28a-SpeC. Figure 1 ).

[0036] The PCR reaction system consisted of: a total volume of 50 μL, 19 μL of sterile water, 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of template, and 25 μL of Primestar DNA Polymerase. The PCR reaction program was: 98℃ for 30 s, 98℃ for 10 s, 57℃ for 30 s, 72℃ for 5 s / kb, 72℃ for 10 min, 16℃ ± ∞, with 30 cycles. The homologous recombination reaction conditions were: 37℃ for 10 min. The reaction system consisted of: a total volume of 10 μL, 2 μL of 5×CE II Buffer, 1 μL of linearized cloning vector, 1 μL of insert fragment, 1 μL of Exnase® II, and 5 μL of ddH2O.

[0037] The recombinant vector pET28a-SpeC ligation system was transformed into Escherichia coli via CaCl2 chemical transformation. E.coli After DH5α, the bacteria were plated on LB agar plates containing 100 mg / L kanamycin sulfate and incubated overnight at 37°C. Once single colonies appeared on the plates, colony PCR was performed using primers JD-pET28a-S / A to identify the recombinant strain. E.coli DH5α-pET28a-SpeC.

[0038] Using the OMEGA DNA Extraction Kit (brand: Omega, catalog number: D3350-01), DNA was extracted from recombinant strains. E.coli Plasmid pET28a-SpeC was extracted from DH5α-pET28a-SpeC and chemically transformed into... E.coli After single colonies of BL21 competent cells grew on plates, colony PCR was performed using primers JD-pET28a-S / A to identify the recombinant expression strain. E.coli BL21-pET28a-SpeC was used as a control strain.

[0039] Construction of an ornithine decarboxylase mutant library: The original SpeC fragment was randomly mutated using a dye-based real-time error-prone PCR kit (brand name: Tianjingsha, catalog number: CAT#:230712) to obtain a SpeC protein mutant library.

[0040] Error-prone PCR reaction system: total volume 30 μL, sterile water 15 μL, forward primer 1 μL, reverse primer 1 μL, template 1 μL, error-prone PCR enhancer 3 μL, dNTP 3 μL, MnCl2 3 μL, 10× error-prone PCR Mix (containing enzyme) 3 μL; PCR reaction program: 94℃ 3 min, 94℃ 1 min, 60℃ 1 min, 72℃ 3-10 min; 60 cycles. After verification and recovery of the PCR products by agarose gel electrophoresis, they were ligated into the pET28a linearized vector and transformed into...E.coli DH5α competent cells were used to obtain recombinant mutant strains after verification with primers JD-pET28a-S / A. Plasmid libraries carrying the SpeC mutation were obtained using the OMEGA DNA extraction kit (Omega, catalog number D3350-01).

[0041] The obtained plasmid was chemically transformed to... E.coli After single colonies of BL21(DE3) competent cells grew on plates, colony PCR was performed using primers JD-pET28a-S / A to identify the mutant strain library.

[0042] Screening methods for highly active SpeC mutant proteins: The obtained mutant strains were cultured and fermented as follows.

[0043] Plate culture: Recombinant bacterial strains stored at -80℃ were streaked onto activated plates and incubated overnight at 37℃ to obtain slant seed culture; the solid culture medium consisted of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar, and the remainder was water, with natural pH.

[0044] 96-well plate seed culture: The bacterial cells were scraped with a toothpick and inoculated into a 96-well plate containing 1 mL of LB medium. The culture was shaken overnight at 37℃ and 220 rpm to obtain the primary seed culture. The seed culture medium consisted of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and the remainder was water, with the pH set to natural.

[0045] 96-well plate fermentation culture: The primary seed culture was inoculated into 96-well plates containing 0.8 mL of fermentation medium at an inoculation rate of 10% (v / v) (final volume 1 mL). After shaking culture at 37℃ and 220 rpm for 4 h, lactose was added to a final concentration of 2%, and expression was induced at 20℃ and 160 rpm for 20 h. The fermentation medium consisted of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and the remainder was water, with natural pH.

[0046] After fermentation, the fermentation broth was used for a catalytic reaction at 37℃ for 8 hours. The catalytic reaction system consisted of a total volume of 50 mL and an OD of the fermentation broth. 600 =2, L-ornithine hydrochloride 2 g / L, 1 mM PLP, 50 mM sodium citrate to make up the difference.

[0047] The liquid chromatography method for detecting 1,4-butanediamine in the fermentation broth was as follows: a reversed-phase column (Shim-pack GIST-HP-C18 column, 2.1 x 100 mm, particle size 3 μm) was used, with a detection wavelength of 284 nm. The mobile phase system consisted of 100% acetonitrile (A); 25 mM sodium acetate aqueous buffer (pH 4.8) (B). The flow rate was 0.5 mL / min. The gradient elution program was as follows: 0–2 min: 80%–75% B; 2–27 min: 75%–37.5% B; 27.01–37 min: 80% B. The flow rate was 0.5 mL / min, the column temperature was 30 °C, the UV detection wavelength was 284 nm, and the retention time was 40 min.

[0048] The fermentation results are shown in Table 1. The yield of 1,4-butanediamine in the reaction solution catalyzed by the E. coli BL21-pET28a-SpeC-Y694N mutant strain was 0.655 g / L; compared with the control strain... E.coli Compared to BL21-pET28a-SpeC, this mutant strain exhibited a 1.3-fold increase in the production of 1,4-butanediamine (from 0.505 g / L to 0.655 g / L). The nucleotide sequence of the gene encoding the SpeCY694N mutant carried in this mutant strain is shown in SEQ ID NO:3.

[0049] Table 1. Effect of the SpeC Y694N mutant on the synthesis efficiency of 1,4-butanediamine.

[0050] Example 2: This example illustrates the construction of a saturated mutant at the SpeC Y694 site and its application in 1,4-butanediamine-producing strains.

[0051] Based on the codon bias of *E. coli*, the recombinant expression vector used the pET28a plasmid as a backbone to verify the potential highly active SpeC mutant protein. Two complementary primers were designed to replace the tyrosine residue at position 694 with the remaining 19 amino acids (taking Y694Q as an example, whose coding gene sequence is shown in SEQ ID NO:4, and the others are similar; Y694Q-S: 5'-GGTGTTCAGAGCGAAACCG-3'; Y694Q-A: 5'-TTCGCTCTGAACACCTTGCAG-3'), constructing a single-point mutant. The primers are shown in Table 7.

[0052] According to the method described in Example 1, plasmid pET28a-SpeC was extracted using an OMEGA DNA extraction kit. Using pET28a-SpeC as a template, a saturation mutation was performed at position 694 of the amino acid sequence of the ornithine decarboxylase SpeC by whole-plasmid PCR to obtain a recombinant expression vector containing the ornithine decarboxylase mutant. After digestion, purification, and recovery, the PCR product was transformed into Escherichia coli by chemical transformation. E.coli In BL21(DE3), after single colonies grew on the plate, colony PCR was performed to identify recombinant strains containing nucleotide sequences encoding ornithine decarboxylase mutants.

[0053] The obtained mutants were subjected to fermentation expression, catalytic reaction and high performance liquid chromatography detection as described in Example 1. The concentration of 1,4-butanediamine in the supernatant is shown in Table 2.

[0054] Table 2 shows that the mutant strain E.coli BL21-pET28a-SpeC-Y694Q exhibited high activity, increasing the 1,4-butanediamine concentration from 0.505 g / L to 0.702 g / L, which was lower than the control strain. E.coli 1.4 times that of BL21-pET28a-SpeC; secondly, E.coli BL21-pET28a-SpeC-Y694C E.coli BL21-pET28a-SpeC-Y694P E.coli BL21-pET28a-SpeC-Y694S also showed varying degrees of increased activity, with its 1,4-butanediamine concentrations being 1.2, 1.1, and 1.2 times higher than those of the control strain, respectively. This indicates that mutants at position 694 of ornithine decarboxylase, such as Y694Q, Y694N, Y694C, Y694S, or Y694P, significantly enhance the catalytic activity of this enzyme, thereby improving the 1,4-butanediamine production performance of the strain.

[0055] Table 2 Catalytic results of the SpeC Y694 saturated mutant strain

[0056] Example 3: This embodiment illustrates the application of ornithine decarboxylase mutants in 1,4-butanediamine-producing strains. The mutants can be Y694N or Y694Q, specifically involving the construction of genetically engineered bacteria HCBPUT1, HCBPUT2, or HCBPUT3.

[0057] 1. Methods of gene editing The gene editing method used in this embodiment refers to the literature (A modified pCas / pTargetF system for CRISPR-Cas9 assisted genome editing in...). Escherichia coli (DOI: 10.1093 / abbs / gmab036). The pEcCas9 plasmid carries the pEcgRNA elimination system, pSC101 replicon, SacB sucrose self-elimination system, Red recombination system, and Cas9 protein expression system. It also includes kanamycin resistance (working concentration: 50 mg / L) and is cultured at 37°C. The pEcgRNA includes the promoter J23119 (SpeI), gRNA-Cas9 binding region sequence and terminator sequence, CcdB toxicity marker, and zizomycin resistance (working concentration: 50 mg / L). It is cultured at 37°C.

[0058] The specific steps of this method are as follows: 1.1 Construction of pEcgRNA plasmid The purpose of constructing plasmid pEcgRNA is to transcribe the corresponding gRNA, thereby forming a complex with Cas9 protein, and to achieve double-strand breaks in the target DNA by recognizing the target gene site through base pairing and PAM.

[0059] 1.1.1 Target Sequence Design The target sequence (PAM:5'-NGG-3') was designed using CRISPR RGEN Tools (rgenome.net).

[0060] 1.1.2 Preparation of plasmids containing target sequences The base sequence of the site to be knocked out or integrated is input into CRISPR RGEN Tools. Primers are designed according to the target sequence provided by CRISPR RGEN Tools. Using the empty pEcgRNA plasmid as a template, the appropriate N20 sequence is integrated into the pEcgRNA plasmid through whole plasmid PCR amplification.

[0061] 1.1.3 Plasmid Transformation Slowly add the plasmid PCR product to E. coli In DH5α competent cells, after gently mixing by pipetting, incubate on ice for 20 min, incubate in water at 42℃ for 1 min, then immediately incubate on ice for 90 s, add 900 μL of recovery solution, and recover for 1 h. Take 200 μL of the recovered bacterial culture and spread it evenly on LB agar plates containing zirconia-resistant bacteria. Incubate upside down at 37℃ for 12-15 h until single bacteria grow on the plates. Select positive recombinants by colony PCR identification.

[0062] 1.1.4 Cloning Identification PCR-positive colonies were inoculated into LB medium containing 100 mg / L zizomycin and cultured overnight for preservation before plasmid extraction.

[0063] 1.2 Preparation of Recombinant DNA Fragments The target gene base sequence was located in KEGG or NCBI, and primers were designed using Primer Premier 5.0 software. The PCR system and methods are shown in Table 3.

[0064] Table 3

[0065] The system for overlap PCR is shown in Table 4.

[0066] Table 4

[0067] Note: The template consists of equimolar amounts of amplified fragments from upstream and downstream homologous arms and the target gene, and the total amount does not exceed 10 ng.

[0068] PCR reaction conditions (Takara Bio PrimeSTAR HS enzyme): Pre-denaturation (95℃) for 5 min; then 30 cycles: denaturation (98℃) for 10 s, annealing ((Tm-3 / 5)℃) for 15 s, extension at 72℃ (this enzyme activity extends by about 1 kb per min); continue extension at 72℃ for 10 min; maintenance (22℃).

[0069] 1.3 Electroconversion of plasmids and recombinant fragments 1.3.1 Conversion of pEcCas9 The pEcCas9 plasmid was electroporated into the electrocompetent cells of the starting strain. After cell resuscitation and culture, the cells were plated on LB agar plates containing kanamycin and incubated overnight at 37°C. Single colonies growing on the antibiotic-resistant plates were subjected to colony PCR using identification primers to screen for positive recombinants.

[0070] 1.3.2 Preparation of electrotransformation competent cells of the target strain containing pEcCas9 Pipette 1 mL of bacterial culture into a 2×YT shake flask, add 100 µL of kanamycin and 1 mL of arabinose (final concentration 10 mM), and incubate at 37°C with shaking at 220 r / min for approximately 2-3 h (OD). 600 (When the value is around 0.6). The culture medium and preparation process for competent cells should follow standard operating procedures.

[0071] 1.3.3 Electroporation of pEcgRNA and Recombinant Fragments pEcgRNA and donor DNA recombinant fragments were simultaneously electroporated into electrocompetent cells containing pEcCas9. The revived cells after electroporation were plated on LB agar plates containing kanamycin and zizomycin and incubated overnight at 37°C. Colony PCR was performed using specially designed identification primers to verify the colonies, screen for positive recombinants, and maintain the cells.

[0072] 1.4 Plasmid Elimination 1.4.1 Elimination of pEcgRNA 10 μL of positive transformant bacterial culture was inoculated into a shaker tube, and 80 μL of rhamnose (final concentration 10 mM) was added to induce the loss of the pEcgRNA plasmid. After culturing for 20 h, the cultured bacterial culture was streaked on a kanamycin-resistant plate to separate single colonies. Strains that eliminated the pEcgRNA plasmid were screened by cross-validation using zizomycin-resistant and kanamycin-resistant plates.

[0073] 1.4.2 Elimination of pEcCas9 plasmid The selected strains with pEcgRNA plasmid removed were transferred to 5 mL antibiotic-free LB agar shakers and cultured for 18 h. Single colonies were then separated by streaking three zones on sucrose-resistant plates. Strains with pEcCas9 plasmid removed were screened by spotting on kanamycin-resistant and antibiotic-free plates; finally, antibiotic-free strains were obtained.

[0074] 2. Construction of genetically engineered bacteria HCBPUT1, HCBPUT2, or HCBPUT3 In detail, ycjV Site integration speC Genes, original genes, speC Y694N and speC Y694Q mutant enzyme gene. ycjV ::P trc - speC Reconstructed fragments: First, based on E. coli In the MG1655 genome speC Using the gene (NCBI-Gene ID: 947457) as a template, upstream and downstream primers were designed. speC -S-trc、 speC -A-trc, where P trc - speC The fragment is E. coli MG1655 was obtained from template amplification, P trc - speC Y694N The fragment was amplified using the pET28a-SpeC-Y694N mutant plasmid constructed in this invention as a template. trc - speC Y694QThe fragment was amplified using the pET28a-SpeC-Y694Q mutant plasmid constructed in this invention as a template. According to... E. coli In the MG1655 genome ycjV Based on the nucleotide sequence of the gene (NCBI-GeneID: 945890), primers were designed for amplification of the upstream homologous arm. ycjV -UPS, ycjV Primers required for UP-A and downstream homologous arm amplification ycjV -DN-S、 ycjV -DN-A. Among them, the upstream and downstream homologous arms and... speC The target gene is P trc The promoter sequence was used as the overlapping region, and the above primers were used to obtain the overlapping PCR according to method 1.2. ycjV ::P trc - speC , ycjV ::P trc - speC Y694N and ycjV ::P trc - speC Y694Q Recombinant fragments. gRNA- ycjV :according to E. coli In the MG1655 genome ycjV Sequences were used to design and construct plasmid gRNA using the online tool CRISPR RGEN Tools. ycjV Required primers ycjV -gRNA-S and ycjV -gRNA-A was constructed and obtained by circular PCR according to method 1.1. ycjV Plasmid.

[0075] The recombined fragments were processed according to method 1.3. ycjV ::P trc -speC , ycjV ::P trc - speC Y694N and ycjV ::P trc - speC Y694Q With plasmid gRNA- ycjV The cells were co-electroporated into HCBPUT0 strain electrotransformation competent cells containing the pEcCas9 plasmid using primers. ycjV -UPS, ycjV -DN-A was used as a primer for colony PCR identification to obtain strains HCBPUT1, HCBPUT2, or HCBPUT3. Among them, the engineered strain HCBPUT0 was a wild-type strain. E.coliStarting with MG1655, referencing strategies already reported in the industry (e.g., Metabolic Engineering of Escherichia coli For the Production of Putrescine: A FourCarbon Diamine. DOI: 10.1002 / bit.22502), the enzyme encoding glutamate-butanediamine ligase was knocked out. puuA Gene (NCBI-GeneID: 946202), encoding spermidine N-acetyltransferase speG Gene (NCBI-GeneID: 946117), encoding succinyldiamine aminotransferase patA Gene (NCBI-GeneID: 947120), encoding spermidine synthase. speE Gene (NCBI-GeneID: 947726) and the gene encoding the repression factor argR Gene (NCBI-GeneID: 947861), source of introduction C. glutamicum ATCC 13032 argC (NCBI-ProteinID:CAF21405)- argJ (NCBI-ProteinID: CAF21406) - argB (NCBI-ProteinID:CAF21407)- argD (NCBI-ProteinID: CAF21408) L-ornithine synthesis operon gene, and knockout of the gene encoding ornithine carbamoyltransferase. argF (NCBI-GeneID: 944844) and argI The engineered strain HCBPUT0 was constructed from a recombinant strain of gene (NCBI-GeneID: 948774).

[0076] The sources and descriptions of the genetically engineered bacteria in this embodiment are summarized in Table 5.

[0077] Table 5. Sources and descriptions of genetically engineered bacteria

[0078] Example 4: This embodiment illustrates a method for producing 1,4-butanediamine using a genetically engineered strain. The genetically engineered strain may be HCBPUT1, HCBPUT2, or HCBPUT3.

[0079] Slant culture: Streaking the -80℃ preserved strain onto the activated slant, incubating at 37℃ for 12 h, and subculturing once.

[0080] Shake-flask seed culture: Use an inoculation loop to scrape a loop of slanted seeds and inoculate them into a 500mL Erlenmeyer flask containing 30mL of seed culture medium. Seal the flask with nine layers of gauze and incubate at 37℃ and 200rpm for 8 hours.

[0081] Shake-flask fermentation: Inoculate 10% of the seed culture volume into a 500 mL Erlenmeyer flask containing fermentation medium (final volume 30 mL), seal with nine layers of gauze, and incubate at 37℃ with shaking at 200 r / min. During fermentation, maintain the pH at 7.0-7.2 by adding ammonia; add 60% ( m / v Fermentation was maintained using glucose solution; the fermentation cycle was 20 hours.

[0082] The slant culture medium consisted of: 1 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 1 g / L NaCl, 25 g / L agar, and the remainder was water, with a pH of 7.0.

[0083] The seed culture medium consisted of: glucose 20 g / L, yeast extract 5 g / L, peptone 2 g / L, K₂HPO₄ 1 g / L, MgSO₄·7H₂O 1 g / L, FeSO₄·7H₂O 15 mg / L, MnSO₄·7H₂O 15 mg / L, and V B1 V B3 V B5 V B12 V H Each 1 mg / L, L-arginine 2 g / L, the remainder is water, pH 7.0.

[0084] The fermentation medium consisted of: glucose 20 g / L, yeast extract 1 g / L, peptone 2 g / L, K₂HPO₄ 3 g / L, MgSO₄·7H₂O 1 g / L, FeSO₄·7H₂O 15 mg / L, MnSO₄·7H₂O 15 mg / L, V B1 V B3 V B5 V B12 V H Each 1 mg / L, L-arginine 2 g / L, the remainder is water, pH 7.0.

[0085] The above method was used to treat the genetically engineered bacterium HCBPUT1. 、 HCBPUT2 or HCBPUT3 were used for shake-flask fermentation, and the fermentation results are shown in Table 6.

[0086] Table 6HCBPUT1 、 Shake-flask fermentation results of HCBPUT2 and HCBPUT3 genetically engineered bacteria

[0087] Table 6 shows that the 1,4-butanediamine yields in the genetically engineered strains HCBPUT2 or HCBPUT3, which integrate the SpeC Y694N or SpeC Y694Q mutants, were 7.76 g / L and 7.95 g / L, respectively, which were 21.06% and 24% higher than those in strain HCBPUT1, which integrates the original ornithine decarboxylase SpeC. The mutants of this invention have the beneficial effect of significantly increasing the 1,4-butanediamine yield of genetically engineered bacteria.

[0088] The primer sequences used in the embodiments of this invention are shown in Table 7.

[0089] Table 7 Primer sequence list

[0090] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. An ornithine decarboxylase mutant, wherein, compared with the protein whose amino acid sequence is as shown in SEQ ID NO: 1, the tyrosine at position 694 is mutated to one of the following amino acids: glutamine, asparagine, cysteine, serine, or proline.

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

3. The encoding gene as described in claim 2, wherein the nucleotide sequence is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

4. A recombinant vector, characterized in that, It includes the encoding gene as described in claim 2.

5. A host cell, characterized in that, It contains the coding gene of claim 2 or the recombinant vector of claim 4.

6. The application of the ornithine decarboxylase mutant according to claim 1, characterized in that, The ornithine decarboxylase mutant is used to catalyze the conversion of L-ornithine or L-ornithine salt to 1,4-butanediamine.

7. A method for preparing the ornithine decarboxylase mutant of claim 1, wherein the method involves gene recombination and expression using the coding gene of the ornithine decarboxylase mutant or an expression vector containing the coding gene.

8. A method for producing 1,4-butanediamine, the method comprising the step of introducing the ornithine decarboxylase mutant of claim 1 into a microbial expression system and preparing 1,4-butanediamine with a suitable substrate under suitable conditions.

9. The method as described in claim 8, characterized in that, The microbial expression system is Escherichia coli.

10. A method for increasing the activity of ornithine decarboxylase, the method comprising introducing one of the following substitutions into the parental ornithine decarboxylase: Y694Q, Y694N, Y694C, Y694S or Y694P; each position corresponding to the amino acid sequence of the parental ornithine decarboxylase shown in SEQ ID NO: 1.

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