Efficient butanediamine biosynthesis method

Abstract

The invention discloses an efficient butanediamine biosynthesis method, and belongs to the field of bioengineering. The arginine decarboxylase mutant provided by the invention has the advantages that the specific enzyme activities of the arginine decarboxylase mutant under the alkaline condition can reach 15.6 U / g, 17.8 U / g and 23.4 U / g respectively and are increased by about 4.2 times, 4.9 times and 6.8 times respectively compared with wild type enzymes, the catalytic efficiency of the enzymes is obviously enhanced, and an efficient enzyme catalyst is provided for biosynthesis of butanediamine. In addition, CO2 is introduced in the fermentation process, and the yield of butanediamine is further increased. And the CO2 throughput is controlled within the range of 0.5-2 vvm, so that the catalytic efficiency can be ensured, the resource waste can be avoided, and the optimization of the production process is realized.

Description

Technical Field

[0001] This invention relates to an efficient method for the biosynthesis of butanediamine, belonging to the field of bioengineering. Background Technology

[0002] Butylene diamine (Putrescine), also known as putrescine, is an amino acid derivative. 1,4-Butylene diamine is not only an important molecule in life processes, regulating cellular metabolism, but also holds significant value in the production of engineering plastics, pharmaceuticals, agrochemicals, and surfactants. Its primary application is in the preparation of polyamide materials, including PA46, PA410, PA4T, and other products, which have wide applications in textiles, machinery, chemicals, electronics, and automotive manufacturing.

[0003] Currently, traditional chemical synthesis remains the primary production method. However, the raw materials used in synthesis are toxic, and the production process suffers from stringent reaction conditions, demanding equipment, expensive catalysts, high hydrogen pressure, and high energy consumption, resulting in high technological barriers for butanediamine synthesis. Therefore, utilizing synthetic biology techniques to directly convert biomass raw materials into butanediamine through microbial cells or enzyme catalysis not only effectively circumvents the technical bottlenecks of traditional processes but also demonstrates significant environmental friendliness and economic advantages, providing an important solution for the sustainable production of butanediamine. Summary of the Invention

[0004] [Technical Solution] The first objective of this invention is to provide an arginine decarboxylase mutant, which is obtained by simultaneously mutating glutamic acid at position 95 to histidine, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid, based on the amino acid sequence shown in SEQ ID NO.1. Alternatively, based on the amino acid sequence shown in SEQ ID NO.1, the glutamic acid at position 95 is mutated to histidine, and the glutamic acid at position 467 is mutated to lysine. Alternatively, it can be obtained by simultaneously mutating glutamic acid at position 95 to histidine and histidine at position 736 to glutamic acid, based on the amino acid sequence shown in SEQ ID NO.1.

[0005] A second object of the present invention is to provide a gene encoding the arginine decarboxylase mutant or a vector carrying the gene.

[0006] A third object of the present invention is to provide recombinant cells expressing the arginine decarboxylase mutant or the vector.

[0007] Preferably, the recombinant cells use bacteria or fungi as host cells.

[0008] A fourth objective of this invention is to provide a recombinant strain expressing the arginine decarboxylase mutant, arginine decarboxylase SpeA, and guanidine aminoase SpeB.

[0009] In one embodiment of the present invention, the amino acid sequence of the guanidine amino acid enzyme SpeB is shown in SEQ ID NO.3, and the amino acid sequence of the arginine decarboxylase SpeA is shown in SEQ ID NO.4.

[0010] A fifth object of the present invention is to provide a whole-cell catalyst containing the recombinant strain.

[0011] The sixth objective of this invention is to provide a method for producing butanediamine, wherein butanediamine is prepared by using arginine as a substrate and catalyzing a reaction with the recombinant strain, the whole-cell catalyst, or the mutant, while maintaining a CO2 atmosphere or an inert atmosphere throughout the reaction.

[0012] In one embodiment of the present invention, the CO2 atmosphere is continuously introduced at a flow rate of 0.5~2 vvm.

[0013] In one embodiment of the present invention, the inert atmosphere includes at least one of argon and nitrogen.

[0014] The seventh objective of this invention is to provide a method for improving the activity of arginine decarboxylase, wherein the method involves mutating glutamic acid at position 95 of the arginine decarboxylase as shown in SEQ ID NO.1 to histidine, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid. Alternatively, based on the amino acid sequence shown in SEQ ID NO.1, the glutamic acid at position 95 is mutated to histidine, and the glutamic acid at position 467 is mutated to lysine. Alternatively, it can be obtained by simultaneously mutating glutamic acid at position 95 to histidine and histidine at position 736 to glutamic acid, based on the amino acid sequence shown in SEQ ID NO.1.

[0015] An eighth object of the present invention is to provide a method for increasing the yield of butanediamine, wherein CO2 is introduced during the preparation of butanediamine using the mutant or the whole-cell catalyst.

[0016] In one embodiment of the present invention, CO2 is continuously introduced at a ventilation rate of 0.5~2 vvm.

[0017] A ninth object of the present invention is to provide the use of the arginine decarboxylase mutant, or the vector, or the recombinant cell, or the recombinant strain, or the whole-cell catalyst in the preparation of butanediamine or products containing butanediamine or in the preparation of nylon products.

[0018] Preferably, the nylon products include, but are not limited to, PA46, PA410, and PA4T.

[0019] [Beneficial Effects] (1) The present invention provides an arginine decarboxylase mutant with specific enzyme activities of 15.6 U / g, 17.8 U / g and 23.4 U / g under alkaline conditions (pH 7.0), which are about 4.2 times, 4.9 times and 6.8 times higher than the wild-type enzyme (3 U / g), respectively, significantly improving the enzyme's catalytic efficiency and providing a highly efficient enzyme catalyst for the biosynthesis of butanediamine.

[0020] (2) The present invention further improves the yield of butanediamine by introducing CO2, and the CO2 gas flow rate is controlled within the range of 0.5~2vvm, which not only ensures catalytic efficiency but also avoids waste of resources and achieves optimization of the production process. Detailed Implementation

[0021] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0022] 1. The culture medium 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.

[0023] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0024] SOB medium: 5 g / L yeast extract, 20 g / L peptone, 0.5 g / L sodium chloride, 0.95 g / L MgCl2 and 0.186 g / L KCl.

[0025] 2. The biomaterials involved in the following examples Plasmid pETDuet-adiA: published in the literature L. Wang, B. Ding, X. Hu, G. Li, Y. Deng, Rationally Engineering pH Adaptation of Acid-Induced Arginine Decarboxylase from Escherichia coli to Alkaline Environments to Efficiently BiosynthesizePutrescine. Adv. Sci. 2024, 11, 2307779. https: / / doi.org / 10.1002 / advs.202307779 .

[0026] Plasmid pETDuet-speA-speB: disclosed in Chinese patent application document with publication number CN117535274A.

[0027] 3. The detection methods involved in the following embodiments (1) Method for detecting arginine decarboxylase activity: 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. After centrifugation at 12000 r / min for 10 min, the supernatant was collected, derivatized, and used for HPLC analysis to determine the butanediamine yield. The unit enzyme activity was defined as U / g = 1 mole of butanediamine / min / g protein.

[0028] The 15 mmol / L potassium sodium phosphate buffer solution was prepared as follows: the pH of 15 mmol / L KH2PO4 was adjusted to 7.0 using 15 mmol / L Na2HPO4.

[0029] Table 1. Reaction system for detecting arginine decarboxylase activity

[0030] (2) HPLC detection of butanediamine 1) Sample derivatization and extraction The pre-column derivatization procedure for dansyl chloride was as follows: After centrifuging the fermentation broth at 12000 r / min for 10 min, 500 μL of the supernatant was collected as a sample. 500 μL of saturated NaHCO3 solution and an internal standard (5 μL of 10 g / L heptamethylenediamine) were added and mixed thoroughly. 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, soluble in acetone). The mixture was incubated in a light-protected water bath at 60°C for 30 min. Extraction was performed for 10 min with 2 mL of anhydrous diethyl ether, and the upper organic phase was collected. This 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.

[0031] 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.

[0032] Example 1 Preparation of Arginine Decarboxylase AdiA Mutant The amino acid sequence of the wild-type arginine decarboxylase AdiA is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. Using the recombinant plasmid pETDuet-adiA as a template, primers containing the mutation site were designed (Table 2), and full-plasmid PCR was performed on the pETDuet-adiA plasmid using the corresponding primers in Table 2 to construct the mutant recombinant expression plasmid. pETDuet-adiA E95H Using recombinant plasmid pETDuet-adiA as a template, primers were used... adiA The E95H-F / R mutant was subjected to full-plasmid PCR, and the PCR product was transformed into E. coli JM109. After overnight culture, colonies were picked for PCR and sequencing verification, finally yielding the E95H mutant recombinant plasmid pETDuet-adiA. E95H .

[0033] pETDuet-adiA E95H-H736EUsing the single mutant plasmid pETDuet-adiA E95H Using a template, through primers adiA Full plasmid PCR was performed using the -H736E-F / R strain. The PCR product was transformed into E. coli JM109, cultured overnight, and colonies were picked for PCR and sequencing verification. The E95H and H736E mutant recombinant plasmid pETDuet-adiA was finally obtained. E95H-H736E .

[0034] pETDuet-adiA E95H-E467K : Using the double mutant plasmid pETDuet-adiA E95H Using a template, through primers adiA Full plasmid PCR was performed using E467K-F / R, and the PCR product was transformed into E. coli JM109. After overnight culture, colonies were picked for PCR and sequencing verification, finally yielding the E467K, H736E mutant recombinant plasmid pETDuet-adiA. E95H-E467K .

[0035] pETDuet-adiA E95H-E467K-H736E : Using the double mutant plasmid pETDuet-adiA E95H-E467K Using a template, through primers adiA Full plasmid PCR was performed using the -H736E-F / R strain. The PCR product was transformed into E. coli JM109, cultured overnight, and colonies were picked for PCR and sequencing verification. The resulting recombinant plasmid pETDuet-adiA, containing E95H, E467K, and H736E mutants, was finally obtained. E95H -E467K-H736E .

[0036] Table 2 Primer sequences involved in the examples

[0037] Example 2 Expression of mutant arginine decarboxylase pETDuet-adiA, pETDuet-SpeB, pETDuet-speA-speB, and the mutant plasmid constructed in Example 1 were transformed into Escherichia coli BL21(DE3) to obtain recombinant bacteria carrying guanidine amino acid enzyme SpeB, arginine decarboxylase AdiA, or arginine decarboxylase AdiA mutants, respectively.

[0038] All recombinant bacteria were first inoculated into 20 mL LB liquid medium at 50 μL and cultured overnight at 37°C with shaking at 250 r / min as seed culture. Then, the seed culture was inoculated at 1% (v / v) in 500 mL SOB medium and cultured at 37°C for 3 h. IPTG was then added to a final concentration of 0.1 mmol / L, and the culture was incubated overnight 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.

[0039] Pure enzyme solutions of wild-type AdiA, guanidine amino acid enzyme SpeB, and various mutants were prepared.

[0040] According to the reaction system in Table 1, that is, to the reaction system containing dithiothreitol, EDTA, pyridoxal phosphate (PLP), MgSO4, arginine, and guanidine amino acid enzyme SpeB pure enzyme solution in potassium sodium phosphate buffer, add arginine decarboxylase AdiA or mutant pure enzyme solution, and the final concentration is shown in Table 1.

[0041] The results showed that the enzyme activity of each mutant was... (AdiA) E95H-E467K-H736E Enzyme activity reaches its peak at pH 7.0, WT, AdiA E95H-E467K AdiA E95H-H736E AdiA E95H-E467K-H736E The enzyme activities were 3 U / g, 15.6 U / g, 17.8 U / g, and 23.4 U / g, respectively.

[0042] Example 3: Production of Butanediamine by Recombinant Bacteria Containing Mutant Arginine Decarboxylase (1) Construction of recombinant bacteria Through primers adiA -F and adiA -R (Table 2) Amplified the mutant gene from the mutant constructed in Example 1, and ligated the mutant gene into the recombinant plasmid pETDuet-speA-speB expressing guanidine amino acid enzyme SpeB and arginine decarboxylase SpeA, to obtain the mutant-containing recombinant plasmid for the preparation of butanediamine: Through primers adiA -F and adiA -R is the mutant pETDuet-adiA constructed from Example 1. E95H-E467K-H736E amplified mutant gene adiA E95H-E467K-H736E ,use Nde I and XhoThe PCR product and the pETDuet-speA-speB plasmid were double-digested. After purification, the mutant gene was ligated into pETDuet-speA-speB using T4 DNA ligase. The ligation product was introduced into E. coli JM109, and screening was performed by colony PCR and Sanger sequencing to obtain the recombinant plasmid pETDuet-speA-adiA. E95H-E467K-H736E -speB. The recombinant plasmid pETDuet-speA-adiA-speB expressing the wild type was prepared using the same method.

[0043] The recombinant plasmid pETDuet-speA-adiA E95H-E467K-H736E -speB conversion to E. coli BL21(DE3) was used to obtain a recombinant strain for butanediamine synthesis. E. coli BL21(DE3) speA-E95H-E467K-H736E-speB .

[0044] Recombinant strains containing wild-type adiA were obtained using the same method. E. coli BL21(DE3) speA-adiA-speB .

[0045] (2) Recombinant bacterial culture The butanediamine recombinant strain preserved in the glycerol tube in step (1) E. coli BL21(DE3) speA-adiA-speB , E. coli BL21(DE3) speA-E95H-E467K-H736E-speB After streaking and isolating on LB solid plates, single colonies are picked and inoculated into 50 mL of LB liquid medium and cultured overnight (12-16 h) at 37°C and 200 r / min on a shaker to obtain primary seed culture.

[0046] The primary seed culture was inoculated into 1 L LB liquid medium at an inoculation rate of 2% (v / v), and ampicillin was added to a final concentration of 100 mg / L. The medium was then incubated in a constant temperature shaker at 37℃ and 250 r / min. When the OD600 of the bacterial culture was ≈15-20, the temperature was lowered to 30℃, and 0.1 mmol / L IPTG was added to induce expression for 16 h to prepare the fermentation broth. 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.

[0047] (2) Whole-cell catalytic synthesis of butanediamine Reaction system: In a 5 L fermenter, the working volume was 3 L, including: arginine total concentration 348.4 g / L (added in batches), 0.1 mmol / L PLP, and finally, the volume was increased to 3 L with PBS buffer (pH 7.0) to achieve cell OD. 600 Approximately 80. Separate groups are set up for CO2 ventilation, argon ventilation, air ventilation, and no ventilation.

[0048] Reaction conditions: Catalytic temperature 42℃, stirring speed 250 r / min. The reaction solution was collected every 12 h, and the yield was determined by HPLC until the arginine in the catalytic system was depleted. Specifically, the CO2-ventilated group continuously introduced CO2 (1 vvm), the argon-ventilated group continuously introduced argon (1 vvm), the air-ventilated group continuously introduced air (1 vvm), and the no-ventilation group did not introduce any gas.

[0049] The results showed that the CO2 ventilation group exhibited the best whole-cell catalytic effect, achieving a butanediamine yield of 131.7 g / L after 12 h. E. coli BL21(DE3) speA-E95H-E467K-H736E-speB ).

[0050] The argon-ventilated group also showed good whole-cell catalytic performance, achieving a butanediamine yield of 108.9 g / L after 12 h. E. coli BL21(DE3) speA-E95H-E467K-H736E-speB ).

[0051] Under hypoxic conditions (non-ventilated group), the yield of butanediamine increased with time, reaching 105.3 g / L after 24 h. E. coli BL21(DE3) speA-E95H-E467K-H736E-speB It is evident that under hypoxic conditions, the yield of butanediamine is lower than under anaerobic conditions (CO2 ventilation group). Under oxygen-enriched conditions (ventilated group), the yield of butanediamine was the lowest, with a maximum yield of only 89.4 g / L at 36 h. E. coli BL21(DE3) speA-E95H-E467K-H736E-speB ).

[0052] 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 mutant was obtained by simultaneously mutating glutamic acid at position 95 to histidine, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid, based on the amino acid sequence shown in SEQ ID NO.

1. Alternatively, based on the amino acid sequence shown in SEQ ID NO.1, the glutamic acid at position 95 is mutated to histidine, and the glutamic acid at position 467 is mutated to lysine. Alternatively, it can be obtained by simultaneously mutating glutamic acid at position 95 to histidine and histidine at position 736 to glutamic acid, based on the amino acid sequence shown in SEQ ID NO.

1.

2. A gene encoding the arginine decarboxylase mutant of claim 1, or a vector of the gene of claim 2.

3. Recombinant cells expressing the arginine decarboxylase mutant of claim 1, or the vector of claim 2; Preferably, the recombinant cells use bacteria or fungi as host cells.

4. A recombinant bacterial strain, characterized in that, The recombinant strain expresses the arginine decarboxylase mutant, arginine decarboxylase SpeA, and guanidine aminoase SpeB as described in claim 1. Preferably, the amino acid sequence of the guanidine amino acid enzyme SpeB is shown in SEQ ID NO.3, and the amino acid sequence of the arginine decarboxylase SpeA is shown in SEQ ID NO.

4.

5. A whole-cell catalyst, characterized in that, The whole-cell catalyst contains the recombinant strain of claim 4.

6. A method for producing butanediamine, characterized in that, The method involves using arginine as a substrate and catalyzing a reaction with the mutant described in claim 1, the recombinant strain described in claim 4, or the whole-cell catalyst described in claim 5 to prepare butanediamine, with the reaction maintained in a CO2 atmosphere or an inert atmosphere throughout. Preferably, the inert atmosphere includes at least one of argon and nitrogen.

7. The method according to claim 6, characterized in that, The CO2 atmosphere is continuously introduced at a flow rate of 0.5~2 vvm.

8. A method for increasing the activity of arginine decarboxylase, characterized in that, The method involves mutating glutamic acid at position 95 of the arginine decarboxylase shown in SEQ ID NO.1 to histidine, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid. Alternatively, based on the amino acid sequence shown in SEQ ID NO.1, the glutamic acid at position 95 is mutated to histidine, and the glutamic acid at position 467 is mutated to lysine. Alternatively, it can be obtained by simultaneously mutating glutamic acid at position 95 to histidine and histidine at position 736 to glutamic acid, based on the amino acid sequence shown in SEQ ID NO.

1.

9. A method for increasing the yield of butanediamine, characterized in that, The method involves introducing CO2 during the preparation of butanediamine using the recombinant strain of claim 4 or the whole-cell catalyst of claim 5. Preferably, the CO2 is continuously introduced at a ventilation rate of 0.5~2 vvm.

10. The arginine decarboxylase mutant of claim 1, or the gene or the vector of claim 2, or the recombinant cell of claim 3, or the recombinant strain of claim 4, or the whole-cell catalyst of claim 5, used in the preparation of butanediamine or products containing butanediamine or in the preparation of nylon products; Preferably, the nylon product includes, but is not limited to, PA46, PA410, and PA4T.

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