Arginine decarboxylase AdiA mutant and application thereof in production of butanediamine

Abstract

This invention discloses an arginine decarboxylase mutant AdiA and its application in the production of butanediamine, belonging to the field of bioengineering. Five effective mutants were obtained by site-directed mutagenesis of the arginine decarboxylase encoding gene in *E. coli*, specifically by mutating aspartic acid at position 102 to histidine, arginine, or lysine, or by mutating glutamic acid at position 467 to histidine or lysine. The relative enzyme activity of mutant 467K at pH 7.0 increased from 2.6 U·g. ‑1 Increased to 8.3 U·g ‑1 The enzyme activity and stability were significantly improved within the neutral pH range. When the arginine decarboxylase mutant 467K was applied to the production of butanediamine from *E. coli*, the butanediamine yield reached 57.5 g / L, and the arginine molar conversion rate reached 0.95 mol·mol⁻¹. ‑1 Compared to the control strain, the efficiency was increased by 28%. This invention improves the conversion efficiency of arginine decarboxylase in a neutral environment and increases the synthesis rate of butanediamine in a neutral environment, laying the foundation for the efficient synthesis of butanediamine.

Description

Technical Field

[0001] This invention relates to an arginine decarboxylase AdiA mutant and its application in the production of butanediamine, belonging to the field of bioengineering. Background Technology

[0002] Butylene diamine (Putrescine), also known as putrescine, is a precursor for the synthesis of the high-performance plastic Nylon-46 and a raw material for the preparation of Nylon-6 and Nylon-66, possessing significant industrial value. As of 2006, the annual production of butylene diamine in Europe was approximately 10,000 tons, and this figure is expected to continue to grow in the future.

[0003] Currently, the large-scale production of butanediamine mainly relies on chemical synthesis. Although the technology is mature, the raw materials are non-renewable petroleum products, which does not meet the requirements of sustainable development. Furthermore, chemical reactions require expensive catalysts and relatively harsh reaction conditions. Therefore, there is a need to find a green and efficient method for synthesizing butanediamine using renewable resources. Biological methods have attracted widespread attention due to their advantages of being green and sustainable, having mild reaction conditions, and causing less environmental pollution. Currently, the main approach to producing butanediamine using biological methods, both domestically and internationally, involves modifying the metabolic pathways of engineered strains and combining fermentation optimization techniques to direct more substrate towards butanediamine production, thus promoting product accumulation. However, research on the biosynthesis of butanediamine is limited both domestically and internationally, and the yield of butanediamine is low, preventing its application in industrial production.

[0004] Arginine decarboxylase AdiA, a key enzyme in the synthesis of butanediamine, has an optimal pH of 5.4. Under neutral conditions (pH 6-8), AdiA typically exists in dimer or monomeric form. The dimer structure is relatively loose and cannot form a stable active site, resulting in virtually no activity of AdiA. However, the intracellular environment of *E. coli* is almost entirely neutral. Therefore, there is an urgent need to find arginine decarboxylases suitable for intracellular butanediamine production in *E. coli*. Summary of the Invention

[0005] The purpose of this invention is to solve the problem of inhibited enzyme activity of arginine decarboxylase (AdiA) under near-neutral conditions, and thus to solve the problem of low synthesis rate of butanediamine in near-neutral environments. Five arginine decarboxylase mutants with optimal pH upregulation are provided. Among them, mutant 467K has an enzyme activity 3.2 times higher than AdiA at pH 7.0. Applying mutant 467K with the best enzyme activity to the biosynthesis of butanediamine increases the accumulation of butanediamine in the fermentation broth.

[0006] 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 an arginine decarboxylase mutant obtained by mutating position 102 or 467 of the arginine decarboxylase as shown in SEQ ID NO. 1.

[0007] In one embodiment of the present invention, the arginine decarboxylase mutant is based on the arginine decarboxylase with the amino acid sequence shown in SEQ ID NO. 1, wherein the 102nd aspartic acid is mutated to histidine, and the amino acid sequence is shown in SEQ ID NO. 2, named 102H; or the 102nd aspartic acid is mutated to arginine, and the amino acid sequence is shown in SEQ ID NO. 3, named 102R; or the 102nd aspartic acid is mutated to lysine, and the amino acid sequence is shown in SEQ ID NO. 4, named 102K; or the 467th glutamic acid is mutated to histidine, and the amino acid sequence is shown in SEQ ID NO. 5, named 467H; or the 467th glutamic acid is mutated to lysine, and the amino acid sequence is shown in SEQ ID NO. 6, named 467K.

[0008] The present invention also provides a gene encoding the arginine decarboxylase mutant.

[0009] In one embodiment of the present invention, the nucleotide sequence of arginine decarboxylase mutant 102H is shown in SEQ ID NO. 7; the nucleotide sequence of arginine decarboxylase mutant 102R is shown in SEQ ID NO. 8; the nucleotide sequence of arginine decarboxylase mutant 102K is shown in SEQ ID NO. 9; the nucleotide sequence of arginine decarboxylase mutant 467H is shown in SEQ ID NO. 10; and the nucleotide sequence of arginine decarboxylase mutant 467K is shown in SEQ ID NO. 11.

[0010] The present invention also provides a recombinant vector carrying the said gene.

[0011] The present invention also provides genetically engineered bacteria carrying the gene or the recombinant vector.

[0012] In one embodiment of the present invention, the genetically engineered bacteria uses Escherichia coli as a host.

[0013] The present invention also provides a recombinant cell that simultaneously expresses the arginine decarboxylase mutant and the guanidine aminoase SpeB.

[0014] In one embodiment of the present invention, the recombinant cells also overexpress Escherichia coli. E. coli BL21(DE3) arginine decarboxylase SpeA.

[0015] In one embodiment of the present invention, the amino acid sequence of the guanidine amino acid enzyme SpeB is shown in SEQ ID NO.12, and the amino acid sequence of the arginine decarboxylase SpeA is shown in SEQ ID NO.13.

[0016] The present invention also provides a whole-cell catalyst containing the above-mentioned recombinant cells.

[0017] In one embodiment of the present invention, the whole-cell catalyst further contains a lyophilization protectant.

[0018] In one embodiment of the present invention, the freeze-drying protectant includes trehalose, sucrose, or mannitol.

[0019] The present invention also provides a method for producing butanediamine, wherein the method is any one of (a) to (c): (a) The recombinant cells were added to a reaction system containing arginine and glucose to prepare butanediamine; (b) The arginine decarboxylase mutant and the guanidine amino acid enzyme with the amino acid sequence shown in SEQ ID NO.12 were added to a reaction system containing arginine and glucose to prepare butanediamine; (c) The arginine decarboxylase mutant, the guanidine amino acid enzyme with the amino acid sequence shown in SEQ ID NO.12, and the arginine decarboxylase with the amino acid sequence shown in SEQ ID NO.13 were added to a reaction system containing arginine and glucose to prepare butanediamine.

[0020] The present invention also provides the use of the above-mentioned arginine decarboxylase mutant, or the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned genetically engineered bacteria, or the above-mentioned recombinant cells, or the above-mentioned whole-cell catalyst in the preparation of products containing butanediamine.

[0021] Beneficial effects: This invention modifies the arginine decarboxylase gene through semi-rational site-directed mutagenesis, mutating the aspartic acid D at position 102 or the glutamic acid E at position 467 of the arginine decarboxylase gene to a basic amino acid (histidine H, arginine R, or lysine K). The resulting glutamic acid decarboxylase mutants 102H, 102R, 102K, 467H, and 467K exhibit a relative enzyme activity at pH 7 that increases from the original 2.6 U·g. -1 Increased to 4.9 U·g -1 3.8 U·g -1 6.9 U·g -1 3.8 U·g -1 and 8.3 U·g -1The optimal pH of the arginine decarboxylase mutant obtained in this invention is upregulated, and the enzyme activity stability in the neutral pH range is improved, making it more suitable for the conditions required for industrial microbial fermentation and laying the foundation for the efficient synthesis of butanediamine. Attached Figure Description

[0022] Figure 1 Arginine is converted to guanidine by arginine decarboxylase; Figure 2 Enzyme activity characterization of AdiA and its mutants. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments. The embodiments described below are illustrative and not limiting, and should not be used to limit the scope of protection of the present invention.

[0024] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0025] The Escherichia coli of the present invention E. coli BL21(DE3), plasmid pETDuet-1, molecular manipulation techniques, microbial culture techniques, and the detection of butanediamine are all well-known to those skilled in the art.

[0026] The culture media involved in the following examples: LB solid culture: 10 g·L -1 Tryptone, 5 g·L -1 Yeast extract, 10 g·L -1 Sodium chloride and 2 g·L -1 Agar powder.

[0027] LB liquid medium: 10 g·L -1 Tryptone, 5 g·L -1 Yeast extract and 10 g·L -1 Sodium chloride.

[0028] SOB medium: 5 g·L -1 Yeast extract, 20 g·L -1 Peptone, 0.5 g·L -1 Sodium chloride, 0.95 g·L -1 MgCl2 and 0.186 g·L -1 KCl.

[0029] Table 1 Primer sequences involved in the examples

[0030] The detection methods involved in the following embodiments: (1) The method for detecting arginine decarboxylase activity is as follows: Prepare a 200 µL enzyme activity reaction system according to Table 2, and determine the arginine decarboxylase activity. The reaction system was incubated at 37°C for exactly 10 min, then 20 µL of 40% trichloroacetic acid was added to terminate the reaction, and the reaction solution was cooled in an ice-water bath. The reaction was then accelerated at 12000 r·min. -1 Centrifuge for 10 min, collect the supernatant, derivatize, and use for HPLC detection of butanediamine yield. Unit enzyme activity is defined as U·g. -1 = 1 mole butanediamine·min -1 ·g -1 protein.

[0031] The 15 mmol·L -1 Potassium sodium phosphate buffer is prepared as follows: using 15 mmol·L⁻¹ -1 Adjust the Na2HPO4 concentration by 15 mmol·L -1 Adjust the pH of KH2PO4 to prepare a buffer solution with the corresponding pH.

[0032] Table 2. Reaction system for arginine decarboxylase activity detection

[0033] (2) HPLC detection of butanediamine 1) Sample derivatization and extraction The pre-column derivatization procedure for dansyl chloride is as follows: the fermentation broth is subjected to 12000 r·min -1 After centrifugation for 10 min, take 500 μL of the supernatant as the sample, add 500 μL of saturated NaHCO3 solution and internal standard (5 μL with a concentration of 10 g·L⁻¹) -1 After mixing thoroughly with heptanediamine, the pH was adjusted to 10 with saturated NaOH solution, followed by the addition of 1 mL of the derivatization reagent dansyl chloride (5 g·L⁻¹). -1 (Soluble in acetone). The mixture was incubated in a light-protected water bath at 60°C for 30 min, then extracted with 2 mL of anhydrous diethyl ether for 10 min. The upper organic phase was collected, and the extraction was repeated twice. The two organic phases were mixed and dried using a nitrogen evaporator to remove the diethyl ether. The derivatized compound was dissolved in 500 μL of acetonitrile solution, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC.

[0034] 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. -1 .

[0035] Example 1: Determination of Arginine Mutation Site Arginine decarboxylase AdiA catalyzes the synthesis of guanidine from arginine and is a key enzyme in the butanediamine ADC synthesis pathway. Figure 1 Its catalytic reaction requires the participation of 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). The decameric of AdiA consists of 5 dimers, which can also be viewed as a bilayer ring composed of 5 monomers. The catalytic active center of AdiA is located at the interface between the two dimers. Because there are too many acidic amino acids on the surface of AdiA, under neutral conditions, AdiA usually exists in the form of dimers or monomers. The dimer structure is relatively loose and cannot form a stable active center, resulting in AdiA having virtually no activity.

[0036] Example 2 Preparation of Arginine Decarboxylase AdiA Mutant Through primers adiA -F and adiA -R Amplification of the arginine decarboxylase gene fragment from the genome of Escherichia coli BL21(DE3) adiA (Amino acid sequence as shown in SEQ ID NO.1), and used Nde I and Xho I pair of gene fragments adiA Perform double enzyme digestion, ligation insertion into... Nde I and XhoFrom the pETDuet-1 expression plasmid digested with enzyme I, a recombinant plasmid pETDuet-adiA was constructed with an N-terminus adiA gene fragment (nucleotide sequence shown in SEQ ID NO.14). Using the corresponding primers in Table 1, full-plasmid PCR was performed on the pETDuet-adiA plasmid to construct recombinant expression plasmids with 102H, 102R, 102K, 467H, and 467K mutants.

[0037] Example 3 Expression of mutant arginine decarboxylase Through primers SpeB -his-F and SpeB -his-R amplifies a guanidine amino acid enzyme gene fragment from the genome of Escherichia coli BL21(DE3). SpeB and use Nde I and Xho I pair of gene fragments SpeB Perform double enzyme digestion, ligation insertion into... Nde I and Xho The recombinant plasmid pETDuet-SpeB with a 6×His tag at the N-terminus of the SpeB gene was constructed from the pETDuet-1 expression plasmid digested with I double enzyme.

[0038] The recombinant expression plasmid of AdiA or its mutant constructed in Example 2, or the recombinant plasmid pETDuet-SpeB, was transformed into *E. coli* BL21(DE3) to obtain recombinant bacteria carrying guanidine amino acid enzyme SpeB, arginine decarboxylase AdiA, or arginine decarboxylase AdiA mutant, respectively. All recombinant bacteria were first inoculated into 20 mL of LB medium at 50 μL and incubated at 37°C and 250 r·min. -1 The culture was incubated overnight on a shaker as a seed culture; then, at an inoculum rate of 1% (v / v), it was incubated in 500 mL of SOB medium at 37°C for 3 hours, followed by inoculation with 0.1 mmol·L⁻¹. -1 IPTG was incubated overnight at 30°C to induce protein expression. The overnight cultured cells were then subjected to 8000 rpm·min... -1 Collect the protein by freezing and centrifuging for 5 min, then resuspend it in 50 mL of Tris-HCl buffer, followed by sonication. The supernatant was then purified by affinity chromatography using a Ni-NTA Superflow resin column.

[0039] The results showed that the enzyme activity detection results of each mutant were as follows: Figure 2 As shown, the 467K mutant exhibited the highest enzyme activity at pH 7.0, reaching 8.3 U·g. -1Compared to the wild-type enzyme, the activity was increased by 3.2 times, effectively increasing the stability of AdiA enzyme in a neutral pH environment. The relative enzyme activities of mutants 102H, 102R, 102K, and 467H at pH 7 increased from 2.6 U·g⁻¹ to [missing value]. -1 Increased to 4.9 U·g -1 3.8 U·g -1 6.9 U·g -1 3.8 U·g -1 It is evident that the optimal pH of the arginine decarboxylase mutant obtained in this invention is upregulated, and the enzyme activity stability in the neutral pH range is improved, making it more suitable for the conditions required for industrial microbial fermentation and laying the foundation for the efficient synthesis of butanediamine.

[0040] Example 4: Production of Butanediamine by Recombinant Bacteria Containing Mutant Arginine Decarboxylase 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 Ligation was performed on 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. Primers were then used... speB -F and speB -R amplifies the gene from the genome of Escherichia coli BL21(DE3). speB .use EcoR I and Hind III. The PCR product and pETDuet-speA plasmid were double-digested, and the purified product was then ligated using T4 DNA ligase. speB Ligate to 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. (The text then abruptly shifts to a different topic:) ...using primers... adiA -F and adiA -R amplifies the mutant gene adiA467K from pETduet-adiA467K, using 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. adiA467K 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-adiA467K-speB. The recombinant plasmid pETDuet-speA-adiA467K-speB was then transformed into... E. coli BL21(DE3) was used to obtain the recombinant butanediamine-synthesizing strain PUT1. In the same manner, wild-type... adiA Gene replacement adiA The 467K gene was used to construct the pETDuet-speA-adiA-speB recombinant plasmid, which was then transformed into... E. coli BL21(DE3) was used to obtain the butanediamine-synthesizing recombinant strain PUT0 as a control strain.

[0041] After streaking the butylene diamine recombinant strains PUT0 and PUT1, preserved in glycerol tubes, onto a plate, a single colony was picked and inoculated into 20 mL of LB medium and incubated at 37°C and 200 r·min. -1 The culture was incubated overnight on a shaker to activate the bacterial strain. The activated strain was then transferred to 50 mL of LB liquid medium at a 2% (v / v) inoculation rate and incubated at 37°C and 200 rpm. -1 The culture was incubated on a shaker for 12 h to prepare the seed culture. The seed culture was then transferred to a medium containing 100 μg / mL... -1 Ampicillin and 8 g·L -1 Glucose was fermented in 2 L SOB medium in a top-fermentation tank at 37°C and 250 r·min. -1 After culturing for 3 hours under the specified conditions, a final concentration of 0.1 mmol·L⁻¹ was added. -1 IPTG and 40 g·L -1 Arginine, continued at 250 r·min in a 30°C environment. -1 The culture was carried out, and 40 g·L⁻¹ was added at two time points, 12 h and 24 h, respectively. -1 Arginine was depleted, and the yield of butanediamine was determined by HPLC using fermentation broth collected every 6 hours until the arginine in the fermentation broth was exhausted. The final butanediamine yield of strain PUT0 reached 45 g / L. -1 The molar conversion rate of arginine reached 0.74 mol·mol⁻¹. -1 The PUT1 strain achieved a butanediamine yield of 57.5 g·L⁻¹. -1 The molar conversion rate of arginine reached 0.95 mol·mol⁻¹. -1 It increased by 28% compared to the control strain.

[0042] 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. An arginine decarboxylase mutant, characterized in that, The arginine decarboxylase mutant is based on the arginine decarboxylase shown in SEQ ID NO.1, with the aspartic acid at position 102 mutated to histidine, the amino acid sequence of which is shown in SEQ ID NO.2 and named 102H; or with the aspartic acid at position 102 mutated to arginine, the amino acid sequence of which is shown in SEQ ID NO.3 and named 102R; or with the aspartic acid at position 102 mutated to lysine, the amino acid sequence of which is shown in SEQ ID NO.4 and named 102K.

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

3. A recombinant vector carrying the gene of claim 2.

4. Genetically engineered bacteria carrying the gene of claim 2 or the recombinant vector of claim 3.

5. A recombinant cell, characterized in that, The recombinant cells simultaneously expressed the arginine decarboxylase mutant of claim 1, guanidine amino acid enzyme SpeB, and arginine decarboxylase SpeA.

6. The recombinant cell according to claim 5, characterized in that, The amino acid sequence of the guanidine amino acid enzyme SpeB is shown in SEQ ID NO.12, and the amino acid sequence of the arginine decarboxylase SpeA is shown in SEQ ID NO.

13.

7. A whole-cell catalyst, characterized in that, The whole-cell catalyst contains the recombinant cells as described in claim 5 or 6.

8. A method for producing butanediamine, characterized in that, The method is any one of (a) to (c): (a) The recombinant cells of claim 5 or 6 are added to a reaction system containing arginine and glucose to prepare butanediamine; (b) The arginine decarboxylase mutant of claim 1 and the guanidine amino acid enzyme with the amino acid sequence shown in SEQ ID NO. 12 were added to a reaction system containing arginine and glucose to prepare butanediamine; (c) The arginine decarboxylase mutant of claim 1, the guanidine amino acid enzyme with the amino acid sequence shown in SEQ ID NO.12, and the arginine decarboxylase with the amino acid sequence shown in SEQ ID NO.13 are added to a reaction system containing arginine and glucose to prepare butanediamine.

9. The use of the recombinant cell of claim 5 or 6 or the whole-cell catalyst of claim 7 in the preparation of butanediamine.

Images