Genetically engineered bacteria for efficient production of butanediamine and application thereof

Abstract

The application discloses genetically engineered bacteria for efficiently producing butanediamine and application thereof, and belongs to the field of bioengineering. D606R / H730D The optimal mutant AdiA of the application maintains high activity in the whole alkaline range of pH 7.0-9.0. The optimal pH of the arginine decarboxylase mutant obtained by the application is up-regulated, the enzyme activity stability in the neutral pH range is improved, and the mutant is more suitable for the condition requirement of industrial microbial fermentation, thereby laying a foundation for efficient synthesis of butanediamine. D606R / H730D The recombinant whole cell catalyst of the optimal double mutant AdiA of the application realizes butanediamine yield of 149.7 g / L within 12 h.

Description

Technical Field

[0001] This invention relates to a genetically engineered bacterium for the efficient production of butanediamine and its applications, belonging to the field of bioengineering. Background Technology

[0002] 1,4-Butanediamine (Butanediamine for short), also known as putrescine or 1,4-diaminobutane, is a small-molecule aliphatic compound containing nitrogen and carrying a positive charge. It is the simplest of the biogenic amines (including putrescine, spermine, spermidine, and cadaverine). Butanediamine is of significant value in the production of engineering plastics, pharmaceuticals, agrochemicals, and surfactants. Its primary industrial application is in the production of the polyamide material Nylon 46, which possesses advantages such as high melting point, high crystallinity, high heat resistance, and high mechanical strength, finding wide application in textiles, machinery, chemicals, electronics, and automotive manufacturing. Therefore, butanediamine has a very broad market application scope.

[0003] Currently, the use of microbial engineered strains to construct a biosynthetic pathway for butylamine synthesis has been applied in factories and is being used for large-scale production. Arginine decarboxylase AdiA is an important member of the *E. coli* acid stress response system, a typical decadal high-order homologous oligomeric protein composed of five dimers forming a bilayer pentacyclic structure. This enzyme catalyzes the decarboxylation of L-arginine to generate guanidine, a key enzyme in the biosynthesis of butylamine. However, the assembly of the AdiA decadal 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 to neutralize the charge in an acidic environment, promoting oligomerization. Under neutral and alkaline conditions, the acidic amino acids deprotonate, generating electrostatic repulsion, causing the decadal 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. However, the intracellular environment in industrial fermentation is typically neutral, while the butylamine product is alkaline, making traditional AdiA inefficient for large-scale biosynthesis of butylamine.

[0004] Therefore, there is an urgent need to develop a butanediamine-producing bacterium suitable for industrial production, to increase butanediamine yield, and to promote the green and large-scale production of butanediamine and other polyamine compounds. Summary of the Invention

[0005] The purpose of this invention is to first obtain an arginine decarboxylase mutant with optimal pH upregulation, and further construct a genetically engineered bacterium capable of efficiently producing butanediamine based on the arginine decarboxylase mutant with optimal pH upregulation, wherein the combined mutant to A D606R / H730D It maintains high activity throughout the entire semi-alkaline pH range of 7.0-9.0. The mutant with optimal enzyme activity... to AD606R / H730D It was applied to the biosynthesis of butanediamine, resulting in a butanediamine yield of 149.7 g / L.

[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 a genetically engineered bacterium for the efficient production of butanediamine, wherein the genetically engineered bacterium expresses an arginine decarboxylase mutant in a host. to A D606R / H730D Arginine decarboxylase SpeA and guanidine amino acid enzyme SpeB, wherein the arginine decarboxylase mutant to A D606R / H730D It is obtained by mutating aspartic acid at position 606 of the arginine decarboxylase, as shown in SEQ ID NO.1, to arginine, and mutating histidine at position 730 to aspartic acid.

[0007] 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.

[0008] 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.

[0009] The second objective of this invention is to provide an arginine decarboxylase mutant, which is obtained by mutating aspartic acid at position 606 of the arginine decarboxylase amino acid sequence as shown in SEQ ID NO.1 to arginine, and mutating histidine at position 730 to aspartic acid.

[0010] A third objective of this invention is to provide a gene encoding the arginine decarboxylase mutant.

[0011] A fourth objective of this invention is to provide a recombinant vector carrying the said gene.

[0012] A fifth 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.

[0013] The sixth 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.

[0014] 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.

[0015] A seventh 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.

[0016] Beneficial effects The optimal mutant AdiA for dual-interface synergistic modification according to the present invention D606R / H730D It maintains high activity throughout the entire alkaline pH range of 7.0-9.0. 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. The optimal double mutant AdiA obtained in this invention is utilized. D606R / H730D The recombinant whole-cell catalyst achieved a butanediamine yield of 149.7 g / L within 12 h. Detailed Implementation

[0017] 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.

[0018] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0019] 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.

[0020] Table 1: Reaction system for arginine decarboxylase activity detection

[0021] 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.

[0022] The HPLC method for detecting butanediamine is as follows: 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.

[0023] 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.

[0024] 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.

[0025] Based on the above research, D606 and H730 were identified as mutation sites for arginine decarboxylase AdiA.

[0026] Example 2: Preparation of Arginine Decarboxylase AdiA Mutant The specific steps are as follows: Through primers to A -F and to A -R from E. coli Amplification of arginine decarboxylase gene in the BL21(DE3) genome to A ,use Yes 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. to A 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.

[0027] The amino acid sequence (SEQ ID NO.1) of the wild-type arginine decarboxylase is as follows: MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGIERTSLGSLLDHTGAFGESEKYAARVFGADRSWSVVVGTSGSNRTIMQACMTDNDVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGLSLTQEVIDEAVDFRQAMARLYKEFTADGSWFFKPWNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNAAYSGLPVAEVTPREAYNAIVDNNVELVSIENLPGRIAANSVIPYPPGIPMLLSGENFGDKNSPQVSYLRSLQSWDHHFPGFEHETEGTEIIDGIYHVMCVKA The nucleotide sequence encoding said arginine decarboxylase is as follows: Through primers to A -F1 and to A -R1 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. D606R Mutant recombinant plasmid pETDuet-adiA D606R .

[0028] Through primers to A -F2 and to A -R2 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. D606K Mutant recombinant plasmid pETDuet-adiA D606K .

[0029] Through primers to A -F3 and to A -R3 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 digestion product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain AdiA. D606H The mutant recombinant plasmid pETDuet-AdiA D606H .

[0030] Through primers to A -F4 and to A -R4 was used to perform full-plasmid PCR on pETDuet-adiA. The PCR product was purified and then used. Dpn The bacteria were digested with enzyme I, and the digestion product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain AdiA. H730D The mutant recombinant plasmid pETDuet-AdiA H730D Then through primers to A -F1 and to A -R1 for pETDuet-adiA H730D Perform whole-plasmid PCR, and use the PCR products after purification. DpnThe bacteria were digested with enzyme I, and the digestion product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain AdiA. D606R / H730D The mutant recombinant plasmid pETDuet-AdiA D606R / H730D .

[0031] Table 2: Primer Sequence Listing

[0032] 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, and the recombinant plasmid expressing the guanidine aminotransferase SpeB gene, prepared in Example 1, 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.

[0033] The method for preparing the recombinant vector expressing the guanidine aminotransferase SpeB gene is as follows: First, the guanidine amino acid enzyme SpeB overexpression plasmid pETDuet-SpeB was constructed using a double enzyme digestion and ligation method; 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.

[0034] 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 aminotransferase SpeB pure enzyme 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.

[0035] The enzyme activity detection results of each mutant by HPLC are shown in Table 3 below. The results show that the mutant AdiA D606R / H730D It maintains high activity within the pH range of 7.0–9.0.

[0036] Table 3:

[0037] 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 hope -F and hope -R amplifies the gene from the genome of Escherichia coli BL21(DE3). hope .use No 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. hope The ligation product was ligated into pETDuet-1. The ligation product was introduced into E. coli JM109, and the plasmid pETDuet-speA was finally constructed by screening by colony PCR and Sanger sequencing.

[0038] Through primers hope -F and hope -R amplifies the gene from the genome of Escherichia coli BL21(DE3). hope .

[0039] use EcoR I and HindIII. The PCR product and pETDuet-speA plasmid were double-digested, and the purified products were then ligated using T4 DNA ligase. hope 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... to A -F and to A -R from pETduet-adiA D606R / H730D amplified mutant gene adiA D606R / H730D ,use Yes 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 to ligate adiA. D606R / H730D 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. D606R / H730D -speB. The recombinant plasmid pETDuet-speA-adiA... D606R / H730D -speB conversion to E. coli BL2(DE3) was used to obtain the butanediamine-synthesizing recombinant strain PUT1.

[0040] In the same way, using wild type to A Gene replacement adiA D606R / H730D Gene, construct pETDuet-speA-adiA-speB recombinant plasmid, and transform it into E. coli BL2(DE3) was used to obtain the butanediamine-synthesizing recombinant strain PUT0 as a control strain.

[0041] Table 4: Primer Sequence Listing

[0042] The amino acid sequence (SEQ ID NO.4) of the arginine decarboxylase SpeA is as follows: * 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.

[0043] (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 OD. 600 The temperature was lowered to 30 °C at ≈15-20 °C, and 0.1 mmol / L IPTG was added to induce expression for 16 h to prepare the fermentation broth.

[0044] (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.

[0045] 3. Whole-cell catalytic preparation of butanediamine Whole-cell catalysis was conducted in a 5 L bioreactor with a working volume of 3 L. The reaction system composition was as follows: total L-arginine concentration of 348.4 g / L (added in batches), and wet cell concentration equivalent to OD0.05. 600 ≈80 (approximately 80 g wet weight / L), PLP 0.1 mmol / L, temperature 42 ℃, rotation speed 300 r / min, initial pH 7.0, closed reaction, reaction time 12 h.

[0046] 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.

[0047] The results are shown in Table 5: Table 5:

[0048] Finally, the optimal double mutant AdiA of this invention was utilized. D606R / H730D The recombinant whole-cell catalyst achieved a butanediamine yield of 149.7 g / L within 12 h.

[0049] 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 efficient production of butanediamine, characterized in that, The genetically engineered bacteria expressed the arginine decarboxylase mutant adiA in the host. D606R / H730D Genetically engineered bacteria containing arginine decarboxylase SpeA and guanidine aminoase SpeB, wherein the arginine decarboxylase mutant adiA D606R / H730D It is obtained by mutating aspartic acid at position 606 of the arginine decarboxylase, as shown in SEQ ID NO.1, to arginine, and mutating histidine at position 730 to aspartic acid. 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; The host was Escherichia coli JM109.

2. An arginine decarboxylase mutant, characterized in that, The arginine decarboxylase mutant is obtained by mutating aspartic acid at position 606 of the arginine decarboxylase amino acid sequence as shown in SEQ ID NO.1 to arginine, and mutating histidine at position 730 to aspartic acid.

3. A gene encoding the arginine decarboxylase mutant of claim 2.

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

5. A method for synthesizing butanediamine, characterized in that, The method involves using arginine as a substrate and the genetically engineered bacteria described in claim 1 as a catalyst to catalyze the conversion into butanediamine.

6. The method according to claim 5, characterized in that, The amount of arginine added is 300~400g / L, the amount of genetically engineered bacteria added is 80~100g 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.

7. The genetically engineered bacteria of claim 1 or the method of claim 5 or 6 is used in the preparation of butanediamine.

Images