A L-2,4-diaminobutyrate aminotransferase mutant and its application in the synthesis of ectoine

Abstract

The present invention discloses a L-2,4-diaminobutyrate transaminase mutant and its application in the synthesis of ectoin, which is an amino acid sequence derived from Halomonas sp.7T such as L-2,4-diaminobutyrate transaminase shown in SEQ ID NO:2 as a starting sequence and having an amino acid sequence of corresponding mutation sites (such as E407D or E407K, etc.). The present invention also discloses a coding gene, a vector, a bioengineering bacterium and a method for synthesizing ectoin of the L-2,4-diaminobutyrate transaminase mutant. The advantages of the present invention are good enzyme activity, good thermal stability, and / or high preparation yield.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to a 2,4-diaminobutyrate aminotransferase mutant and application thereof in ectoine synthesis. Background Art

[0002] Ectoin, also known as tetrahydropyrimidine, is an amino acid derivative. As a natural cell protectant, Ectoin can not only balance the cell osmotic pressure, but also has a good protective effect on cells, cell membranes, proteins and nucleic acids under various adverse conditions such as high temperature, high salt and strong ultraviolet rays. It has the functions of skin rejuvenation, anti-aging, moisturizing, whitening, sun protection and anti-inflammatory. These high-value applications have greatly increased its market value and also promoted the development of production methods.

[0003] The traditional "halophilic bacteria milking method" requires the use of high-salt culture medium, which will cause serious corrosion to equipment such as fermentation tanks, increase the difficulty of downstream separation and purification, and also cause environmental pollution. In recent decades, with the elucidation of the biosynthetic pathway of ectoine in the halophilic microorganism Halomonas elongata (e.g. Figure 1 As shown in the figure), as well as the development of heterologous expression and metabolic modification technologies, low-salt fermentation of ectoine has been achieved in non-halophilic microbial cell factories such as Escherichia coli, Corynebacterium glutamicum and yeast. The synthesis of ectoine mainly requires the participation of 2,4-diaminobutyric acid-γ-acetyltransferase (ectA), L-2,4-diaminobutyric acid transaminase (ectB) and ectoine synthase (ectC): L-aspartate-β-semialdehyde (ASA) and glutamate produce L-2,4-diaminobutyric acid (DABA) and α-ketoglutarate (2-OG) under the catalysis of L-2,4-diaminobutyric acid transaminase (ectB); subsequently, DABA and acetyl-CoA produce N-γ-acetyl-L-2,4-diaminobutyric acid (ADABA) and CoA under the catalysis of 2,4-diaminobutyric acid-γ-acetyltransferase (ectA); finally, ADABA is cyclized to produce ectoine under the catalysis of ectoine synthase (ectC). In addition, the precursor of L-aspartate-β-semialdehyde (ASA) can be endogenously derived from the tricarboxylic acid cycle of cells: L-aspartate (Asp) forms phosphorylated L-aspartate under the catalysis of L-aspartate kinase (Ask), and phosphorylated L-aspartate forms ASA, the precursor of ectoine, under the catalysis of L-aspartate-β-semialdehyde dehydrogenase (Asd).

[0004] Based on the above research, He et al. introduced the ectABC gene cluster of ectoine synthesis of Halomonas elongata into Escherichia coli BW25113 for induced expression, and used aspartic acid as a substrate for 24 hours to produce 25.1g / L ectoine (Microbial Cell Factories. 2015; 14(1): 55). Patent CN 117866866A disclosed the use of Escherichia coli BL21 (DE3) as a chassis expression cell, by expressing ectA, ectB and ectC enzymes, strengthening the aspartase gene, aspartate kinase gene and aspartate semialdehyde dehydrogenase gene from Escherichia coli, and by continuously feeding glucose and ammonium fumarate, the final fermentation yield reached 30.8g / L in 50 hours. Patent CN 113186143 A constructed a recombinant E. coli ECT-LA that produces ectoine induced by T7 promoter and glucose as substrate, and the yield can reach 60g / L after 56h of batch fed-batch fermentation. Patent CN 116904416A announced that the production of ectoine was increased to 87.36g / L by regulating the expression levels of three enzymes and balancing the cascade pathway reaction by using sodium L-aspartate as substrate and E. coli BL21 as host. Ma et al. heterologously expressed the ectBAC gene from Halomonas elongatus, introduced the de3 gene from Escherichia coli BL21 (DE3), eliminated the lysine transporter lysE, and mutated aspartate kinase to obtain the engineered strain Ect10 (ACSSynth.Biol2024, https: / / doi.org / 10.1021 / acssynbio.4c00019”). Through the optimized fed-batch fermentation process, the final yield reached 115.87 g / L. Although the above method has achieved a certain level of biosynthesis of ectoine in microorganisms such as Escherichia coli and Corynebacterium glutamicum, the low activity and stability of L-2,4-diaminobutyrate aminotransferase (ectB) restrict the efficient synthesis of ectoine, limiting its industrial production and application.

[0005] Therefore, there is a need in the art to develop L-2,4-diaminobutyrate aminotransferase (ectB) with better performance, which can provide more or better options for the biocatalytic synthesis of ectoine. Summary of the invention

[0006] In view of the problems existing in the biocatalytic synthesis of ectoine in the prior art, the present invention provides a class of L-2,4-diaminobutyrate transaminase (ectB) mutants having the characteristics of high catalytic activity, better thermal stability and / or high reaction conversion rate, and also provides a nucleic acid sequence corresponding to the enzyme, a recombinant expression plasmid, a genetically engineered strain and a preparation method thereof, as well as the application of the enzyme in the synthesis of ectoine and a reaction system.

[0007] In a first aspect, the present invention provides a L-2,4-diaminobutyrate aminotransferase mutant, wherein the amino acid sequence of the L-2,4-diaminobutyrate aminotransferase mutant is selected from the following group:

[0008] Mutant 1: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site T15Y;

[0009] Mutant 2: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site Q28R;

[0010] Mutant 3: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site T49A;

[0011] Mutant 5: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site Q61D;

[0012] Mutant 6: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site L95F;

[0013] Mutant 7: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site K96R;

[0014] Mutant 8: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site L274M;

[0015] Mutant 9: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site Y293M;

[0016] Mutant 10: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site N301S;

[0017] Mutant 11: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site K312R;

[0018] Mutant 12: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site K393R;

[0019] Mutant 13: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site E407D;

[0020] Mutant 14: an amino acid sequence corresponding to SEQ ID NO: 2 and having a mutation site E407K;

[0021] Mutant 15: an amino acid sequence corresponding to SEQ ID NO: 2 and having mutation sites N301S / E407K;

[0022] Mutant 16: an amino acid sequence corresponding to SEQ ID NO: 2 and having mutation sites I381V / E407K;

[0023] Mutant 17: an amino acid sequence corresponding to SEQ ID NO: 2 and having mutation sites T15Y / T49A / E407K;

[0024] Mutant 18: an amino acid sequence corresponding to SEQ ID NO: 2 and having mutation sites T15Y / T49A / E407D;

[0025] Mutant 19: an amino acid sequence corresponding to SEQ ID NO: 2 and having mutation sites Q28R / Q61D / I381V / E407K;

[0026] Mutant 20: an amino acid sequence corresponding to SEQ ID NO: 2 and having mutation sites T15Y / T49A / L95F / I381V / E407K;

[0027] Mutant 21: an amino acid sequence corresponding to SEQ ID NO: 2 and having mutation sites Q28R / T49A / L95F / I381V / E407D;

[0028] Mutant 22: an amino acid sequence corresponding to SEQ ID NO: 2 and having mutation sites T15Y / T49A / L95F / K312R / E407D;

[0029] Mutant 23: an amino acid sequence corresponding to SEQ ID NO: 2 and having mutation sites Q28R / T49A / L95F / N301S / I381V / E407D;

[0030] Mutant 24: an amino acid sequence corresponding to SEQ ID NO: 2 and having mutation sites T15Y / T49A / L95F / K312R / K393R / E407D.

[0031] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is selected from mutants 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.

[0032] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is selected from mutants 3, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24; preferably, the L-2,4-diaminobutyrate aminotransferase mutant is selected from mutants 5, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24; more preferably, the L-2,4-diaminobutyrate aminotransferase mutant is selected from mutants 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.

[0033] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is selected from mutants 2, 4, 6, 7, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24; preferably, the L-2,4-diaminobutyrate aminotransferase mutant is selected from mutants 2, 7, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.

[0034] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is selected from mutants 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.

[0035] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 13.

[0036] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 14.

[0037] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 15.

[0038] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 16.

[0039] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 17.

[0040] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 18.

[0041] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 19.

[0042] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 20.

[0043] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 21.

[0044] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 22.

[0045] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 23.

[0046] In some specific embodiments, the L-2,4-diaminobutyrate aminotransferase mutant is mutant 24.

[0047] The L-2,4-diaminobutyrate transaminase mutant provided by the present invention is obtained by molecularly modifying L-2,4-diaminobutyrate transaminase ectB (whose amino acid sequence is shown in SEQ ID NO: 2) derived from Halomonas p.7T, thereby obtaining a mutant of L-2,4-diaminobutyrate transaminase with improved enzyme activity and / or thermal stability.

[0048] The present invention uses wild-type L-2,4-diaminobutyrate transaminase ectB from Halomonas sp.7T as a template and uses a random point mutagenesis kit ( II Site-Directed Mutagenesis Kit) for error-prone PCR, or by semi-rational design to mutate the wild-type ectB, to obtain a plasmid library containing the evolved ectB enzyme gene. High-throughput screening was used to obtain mutation sites with improved enzyme activity, and a mutation library was established for these mutation sites to screen out mutants with better enzyme activity and / or thermal stability.

[0049] Compared with the wild-type ectB enzyme, the L-2,4-diaminobutyrate aminotransferase mutant provided by the present invention has higher enzyme activity, better thermal stability, and higher yield of ectoine.

[0050] In a second aspect, the present invention also provides a gene encoding the L-2,4-diaminobutyrate aminotransferase mutant described in the first aspect. The nucleotide sequence of the encoding gene is based on the sequence shown in SEQ ID NO: 1 as the starting sequence, and the codons are mutated accordingly (e.g., base substitution) according to the difference between the amino acid sequence of the L-2,4-diaminobutyrate aminotransferase mutant and SEQ ID NO: 2.

[0051] The present invention also provides an isolated nucleic acid, which is a nucleic acid encoding the L-2,4-diaminobutyrate aminotransferase mutant described in the first aspect.

[0052] Among them, the preparation method of the nucleic acid can be a conventional preparation method in the art, and the preparation method preferably includes: obtaining a nucleic acid molecule encoding the L-2,4-diaminobutyrate aminotransferase by gene cloning technology, or obtaining a nucleic acid molecule encoding the L-2,4-diaminobutyrate aminotransferase by an artificial full sequence synthesis method.

[0053] In a third aspect, the present invention further provides an expression vector, wherein the expression vector carries a gene encoding the L-2,4-diaminobutyrate aminotransferase mutant described in the second aspect.

[0054] In a preferred embodiment, the expression vector is a recombinant plasmid.

[0055] In a preferred embodiment, the expression vector is a pETDuet-1 vector.

[0056] In a preferred embodiment, the expression vector is simultaneously loaded with the coding gene of the L-2,4-diaminobutyrate aminotransferase (ectB) mutant described in the second aspect, the coding gene of 2,4-diaminobutyrate-γ-acetyltransferase (ectA) (the exemplary nucleotide sequence is shown in SEQ ID NO: 3) and the coding gene of ectoine synthase (ectC) (the exemplary nucleotide sequence is shown in SEQ ID NO: 5).

[0057] The recombinant expression vector can be obtained by conventional methods in the art, and is generally constructed by connecting the nucleic acid to various expression vectors.

[0058] The present invention also provides a recombinant expression transformant comprising the above expression vector. The recombinant expression transformant comprises a host cell and a target gene transferred into the host cell, wherein the target gene comprises the coding gene of the L-2,4-diaminobutyrate transaminase mutant described in the second aspect. Preferably, the target gene comprises the coding gene of the L-2,4-diaminobutyrate transaminase mutant described in the second aspect, the coding gene of 2,4-diaminobutyrate-γ-acetyltransferase (ectA) (the exemplary nucleotide sequence is shown in SEQ ID NO:3) and the coding gene of ectoine synthase (ectC) (the exemplary nucleotide sequence is shown in SEQ ID NO:5).

[0059] The preparation method of the recombinant expression transformant is generally to transform the expression vector into a host microorganism. The host microorganism is preferably Escherichia coli (E. coli), more preferably Escherichia coli BL21 (DE3). The recombinant expression vector (such as a plasmid) is transformed into Escherichia coli BL21 (DE3) to obtain the preferred genetically engineered strain of the present invention. The transformation method can be selected from conventional methods in the art, such as electroporation, heat shock, etc.

[0060] In a fourth aspect, the present invention provides the use of the L-2,4-diaminobutyrate aminotransferase mutant described in the first aspect as a catalyst in the preparation of L-2,4-diaminobutyric acid via a biocatalytic reaction using L-aspartate-β-semialdehyde (ASA) as a substrate.

[0061] Specifically, the present invention also provides the use of the L-2,4-diaminobutyrate transaminase mutant described in the first aspect above in combination with 2,4-diaminobutyrate-γ-acetyltransferase (ectA) and ectoine synthase (ectC) as catalysts in the preparation of ectoine by biocatalytic reaction using L-aspartate-β-semialdehyde (ASA) as a substrate.

[0062] The specific method is a biocatalytic reaction method, which comprises the following steps:

[0063] (a) in a liquid reaction system, L-aspartate-β-semialdehyde (ASA) is converted under the catalysis of L-2,4-diaminobutyrate transaminase to obtain L-2,4-diaminobutyric acid;

[0064] Wherein, the L-2,4-diaminobutyrate aminotransferase is the L-2,4-diaminobutyrate aminotransferase mutant described in the first aspect above.

[0065] Preferably, the biocatalytic reaction method further comprises the following steps:

[0066] (b) converting the L-2,4-diaminobutyric acid obtained in step (a) under the catalysis of 2,4-diaminobutyric acid-γ-acetyltransferase (ectA) to obtain N-acetyl-L-2,4-diaminobutyric acid. In some specific embodiments, the amino acid sequence of the 2,4-diaminobutyric acid-γ-acetyltransferase (ectA) is shown in SEQ ID NO:4.

[0067] Preferably, the biocatalytic reaction method further comprises the following steps:

[0068] (c) Under the catalysis of ectoine synthase (ectC), the N-acetyl-L-2,4-diaminobutyric acid obtained in step (b) is converted to obtain ectoine. In some specific embodiments, the amino acid sequence of the ectoine synthase (ectC) is shown in SEQ ID NO:6.

[0069] Preferably, the biocatalytic reaction method further comprises the following steps:

[0070] (d) optionally separating ectoine from the reaction system after the reaction of step (c).

[0071] The main technical effects of the present invention include one or more of the following:

[0072] 1) The L-2,4-diaminobutyrate transaminase mutant screened in this application has better thermal stability.

[0073] 2) The L-2,4-diaminobutyrate transaminase mutant screened in the present application exhibits better reaction activity in the reaction of catalyzing L-aspartate-β-semialdehyde (ASA) to prepare L-2,4-diaminobutyric acid.

[0074] 3) The L-2,4-diaminobutyrate transaminase mutant screened in this application and the preparation method provided by the present invention are used to prepare ectoine. Compared with the existing technology level, the yield of ectoine is increased to 141.5 g / L, with high yield and less impurities, and has good prospects for industrial application.

[0075] 4) The present invention provides a new class of L-2,4-diaminobutyrate transaminase mutants, which have the advantages of high catalytic activity and / or good thermal stability, providing more and better options for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Diagram of the biosynthetic pathway of ectoine.

[0077] Figure 2 A schematic diagram of an exemplary recombinant plasmid. DETAILED DESCRIPTION

[0078] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0079] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials involved in the present invention can be obtained from commercial channels unless otherwise specified.

[0080] The nucleotide sequence or amino acid sequence involved in the present invention is shown in the following table.

[0081] Table 1: Sequence information

[0082]

[0083]

[0084] Example 1: Construction, fermentation and transformation of Ekdoin engineered bacteria

[0085] The genes encoding 2,4-diaminobutyrate-γ-acetyltransferase (ectA), L-2,4-diaminobutyrate aminotransferase (ectB), and ectoine synthase (ectC) from Halomonas sp. 7T (see Table 2) were commissioned to a commercial company for full gene synthesis and cloned into the pETDuet-1 vector to obtain the plasmid pETDuet-1-EctABC (see Figure 2 As shown). The plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, spread on ampicillin resistance plates (50 μg / mL), and cultured at 37°C overnight. Pick the positive transformation single colony, culture it in LB, extract the plasmid and sequence it to obtain the recombinant genetic engineering bacteria E1 containing the ectoine synthesis pathway. According to the same process as above, the ectABC gene sequence from Chromohalobacter sp.NJS-2 and the ectABC gene sequence of Halomonas salifodinae were connected to the vector pACYCDuet-1 to obtain the recombinant genetic engineering bacteria E2 and E3 containing the ectoine synthesis pathway, respectively, as shown in Table 2.

[0086] Cultivation of recombinant genetically engineered bacteria E1, E2, and E3: The glycerol bacteria of E1, E2, and E3 prepared above were inoculated into 5 mL of LB medium (containing 50 μg / mL ampicillin), respectively, and cultured at 37° C., 220 rpm for 14 h as seed liquid for shake flask fermentation; the prepared seed liquid was inoculated into 50 mL of fermentation medium (containing 50 μg / mL ampicillin) at an inoculum amount of 1.5% (v / v), cultured at 37° C. for 3 h, isopropylthiogalactoside (IPTG) was added at a final concentration of 1 mM, induced at 28° C. for 20 h, the bacteria were collected by centrifugation, and stored at -20° C. Among them, the LB medium (g / L) formula: tryptone 10.0, yeast extract 5.0, NaCl 10.0, deionized water 1L, pH 7.0; the fermentation medium formula (g / L) formula: yeast extract 24.0, soy peptone 12.0, sodium chloride 3.0, glycerol 5.0, dipotassium hydrogen phosphate 2.0, magnesium sulfate heptahydrate 0.5, ferrous sulfate 0.06, deionized water 1L, pH 7.0.

[0087] Whole cell transformation of ectoine: The whole cells of E1, E2 and E3 collected above were suspended as whole cell catalysts in phosphate buffer (50mM, pH 7.0) containing 50g / L sodium L-aspartate and 25g / L glucose, and the transformation reaction was carried out at 30°C for 20h; the yield of ectoine was detected by high performance liquid chromatography, and the results are shown in Table 2. The optimal recombinant strain E1 can produce 11.2g / L ectoine.

[0088] Table 2: Gene information and yield of E1, E2 and E3 strains

[0089]

[0090] Example 2: Construction and screening of L-2,4-diaminobutyrate aminotransferase (ectB) mutant library

[0091] Using wild-type L-2,4-diaminobutyrate aminotransferase (ectB) from Halomonas sp.7T as template, random point mutagenesis kit ( II Site-Directed Mutagenesis Kit) for error-prone PCR or by semi-rational design to mutate the wild-type EctB to obtain a plasmid library containing the evolved EctB enzyme gene. The constructed plasmid library was transferred into Escherichia coli BL21 (DE3), then spread on LB solid medium containing 50 μg / mL ampicillin, and cultured overnight in a 37°C incubator. Pick a single colony to a 96-well plate containing 400 μL LB medium (containing 50 μg / mL ampicillin), and culture at 37°C, 200 rpm overnight to obtain the ectB enzyme mutant seed solution. Pipette 10 μL of EctB enzyme mutant seed solution and transfer it to a 96-well plate containing 400 μL fermentation medium (containing 50 μg / mL ampicillin), and culture at 37°C, 800 rpm until OD 600 Value>0.8. The mutant was induced to express with isopropylthiogalactoside (IPTG) at a final concentration of 0.5 mM at 28°C, and then cultured for 20 h. After the fermentation culture was completed, the 96-well plate was placed in a centrifuge and centrifuged at 3000 g for 30 min to collect the bacteria, and then the bacteria were resuspended with 200 μL lysis buffer (0.1 M phosphate buffer containing 1000 U lysozyme, pH 7.0), and then lysed at 30°C for 1 h, and then centrifuged at 4°C, 4000 g for 30 min in a centrifuge, and the clarified supernatant was aspirated to determine the activity of the mutant.

[0092] Detection method of L-diaminobutyrate aminotransferase (ectB) enzyme activity:

[0093] The activity of L-diaminobutyric acid transaminase was calculated by measuring the production of L-2,4-diaminobutyric acid from L-aspartate-β-semialdehyde catalyzed by ectB in 15 minutes. The reaction system contained 50mM substrate, 100mM glutamate and an appropriate amount of ectB enzyme solution. After the reaction was completed for 15 minutes, ectA and ectC crude enzyme solutions were added to continue the reaction. After the reaction, the amount of ectoine generated was determined by HPLC, and the productivity of L-2,4-diaminobutyric acid was calculated. The activity of ectB enzyme was defined as the amount of enzyme required to generate 1μmol L-2,4-diaminobutyric acid in 1 minute.

[0094] HPLC detection method of ectoine: the sample is diluted with acetonitrile / water (70:30 v / v), filtered through a 0.22 μm microporous filter membrane, and then the ectoine content in the sample is determined using an Agilent 1260 liquid chromatography system. The chromatographic column is an Agilent ZOBAX NH2 column (2.6×250 mm, 5 um), the flowability is acetonitrile: water = 70:30 (v / v), the column temperature is 30°C, the flow rate is 1 mL / min, the injection volume is 20 μL, and the UV detection wavelength is 210 nm.

[0095] Thermal stability test method: The supernatant of the wild-type enzyme and mutant enzyme was incubated at 45°C for 1 hour and then the activity was measured. The enzyme solution without heat treatment was used as a reference to calculate the relative activity of the wild-type enzyme and the mutant residual enzyme activity. The activity measurement system was the same as above.

[0096] The relative activities and relative thermal stability of each mutant are shown in Table 2.

[0097] Table 2 Exemplary EctB enzyme mutants and their relative activities and relative thermal stability

[0098]

[0099]

[0100] *The activity and thermostability of the wild-type ectB enzyme (SEQ ID NO: 2) were set to 100%.

[0101] The data in Table 2 show that the activity-enhancing sensitive mutation residue positions of L-2,4-diaminobutyrate transaminase include L95F, Q169K, Y293M, N301S, K312R, E407D, E407K, etc., and their relative activity is >200%, especially the mutants of E407D and E407K mutation sites have better activity. The thermal stability-enhancing sensitive mutation residue positions of L-2,4-diaminobutyrate transaminase include Q28R, Q61D, K96R, L274M, K312R, E407D, E407K, etc., and their relative thermal stability is >150%. Moreover, it was unexpectedly found that E407D and E407K are better in both relative activity and relative thermal stability. Compared with the wild type, the representative L-2,4-diaminobutyrate transaminase mutants provided in this embodiment have better activity and thermal stability.

[0102] Example 3: Construction of genetically engineered bacteria E4 producing ectoin

[0103] According to the method of Example 1, the genes encoding 2,4-diaminobutyric acid-γ-acetyltransferase (ectA), L-2,4-diaminobutyric acid aminotransferase (ectB) mutant 24 and ectoine synthase (ectC) from Halomonas sp.7T were cloned into the pETDuet-1 vector to obtain the plasmid pETDuet-1-EctAB 6M C. The plasmid was transferred into the Escherichia coli BL21 (DE3) host, and the recombinant genetic engineering bacteria E4 containing ectB mutant 24 was obtained by sequencing verification. The E4 wet bacteria were obtained by fermentation induction according to the method of Example 1. The E4 wet bacteria were used as whole cell catalysts for the preparation of ectoin.

[0104] Example 4: Fermentation evaluation of recombinant genetically engineered bacteria E4

[0105] The recombinant engineered bacteria E4 was fermented at a high density using a 5 L fermentor and the yield of ectoin was detected: the engineered bacteria E4 obtained in Example 4 was activated on an LB solid plate; a shovel of bacteria was scraped into a 25 mL seed culture medium containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 25 μg / mL chloramphenicol and 50 μg / mL ampicillin, and cultured at 37 degrees and 220 rpm for 12 h to prepare the engineered strain seed solution; then, the 25 mL seed solution was transferred to a 2 L fermentation medium (20 g / L glucose, 10 g / L ammonium sulfate, 5 g / L yeast powder, 5 g / L peptone, 2 g / L K2HPO4, 0.5 g / LMgSO4·7H2O, 0.1 g / L FeSO4·7H2O, 0.1 g / L mnSO4·H2O, 1 mg / L vitamin B6), cultured at 37°C, 200rpm, 2 L / min aeration, serial rotation speed and dissolved oxygen (lower limit of dissolved oxygen is 30%), pH maintained at 7.0 with ammonia water during the culture process. 70% glucose was continuously added, and samples were taken regularly to measure biomass and glucose content, and the glucose content was controlled at about 0.5%. When OD 600 When the value of reached about 15, the temperature was lowered to 25°C and the inducer IPTG was added. After 5 hours of induction culture, the glucose feeding rate was increased to 10g / L / h. After 24 hours of fermentation, the glucose feeding rate was increased to 15g / L / h. Finally, after 72 hours of fermentation, the yield of ectoine reached 120g / L.

[0106] Example 5: Fermentation evaluation of recombinant genetically engineered bacteria E4

[0107] The recombinant engineered bacteria E4 was fermented at a high density using a 5L fermentor and the yield of ectoin was detected: the engineered bacteria E4 obtained in Example 4 was activated on an LB solid plate; a shovel of bacteria was scraped into a 25mL seed culture medium containing 10g / L tryptone, 5g / L yeast extract, 10g / L NaCl, 25μg / mL chloramphenicol and 50μg / mL ampicillin, and cultured at 37 degrees and 220 rpm for 12h to prepare the engineered strain seed solution; then, the 25mL seed solution was transferred to a 2L fermentation medium (20g / L glucose, 10g / L ammonium sulfate, 10g / L yeast powder, 5g / L peptone, 2g / L K2HPO4, 0.5g / L MgSO4·7H2O, 0.1g / L FeSO4·7H2O, 0.1g / L MnSO4·H2O, 0.1g / L sodium citrate, 1mg / L vitamin B6), cultured at 37°C, 200rpm, 2L / min aeration, serial rotation speed and dissolved oxygen (lower limit of dissolved oxygen is 30%), pH maintained at 7.0 with ammonia water during the culture process. 70% glucose and ammonium sulfate were continuously added, and samples were taken regularly to measure biomass and glucose content, and the glucose content was controlled at about 0.5%. When OD 600 When the value of reached about 15, the temperature was lowered to 25°C and the inducer IPTG was added. After 36 hours of fermentation, the glucose feeding rate was increased to 15g / L / h. Finally, after 84 hours of fermentation, the yield of ectoine reached 141.5g / L.

[0108] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A L-2,4-diaminobutyrate aminotransferase mutant, characterized in that: The amino acid sequence of the L-2,4-diaminobutyrate transaminase mutant is selected from the following group: Mutant 13: amino acid sequence having the sequence of SEQ ID NO: 2 and the mutation site being E407D; Mutant 18: amino acid sequence having the sequence of SEQ ID NO: 2 and mutation sites of T15Y / T49A / E407D; Mutant 21: amino acid sequence having the sequence of SEQ ID NO: 2 and mutation sites of Q28R / T49A / L95F / I381V / E407D; Mutant 22: amino acid sequence having the sequence of SEQ ID NO: 2 and mutation sites of T15Y / T49A / L95F / K312R / E407D; Mutant 23: amino acid sequence having the sequence of SEQ ID NO: 2 and mutation sites of Q28R / T49A / L95F / N301S / I381V / E407D; Mutant 24: The amino acid sequence of SEQ ID NO: 2 and the mutation sites are T15Y / T49A / L95F / K312R / K393R / E407D.

2. A gene encoding the L-2,4-diaminobutyrate aminotransferase mutant according to claim 1.

3. An expression vector, characterized in that: The expression vector is loaded with the gene encoding the L-2,4-diaminobutyrate aminotransferase mutant according to claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector is a recombinant plasmid.

5. The expression vector according to claim 3, characterized in that The expression vector is pETDuet-1 vector.

6. The expression vector according to claim 3, characterized in that The expression vector is also loaded with a gene encoding 2,4-diaminobutyric acid-γ-acetyltransferase and a gene encoding ectoine synthase.

7. The expression vector according to claim 6, wherein The nucleotide sequence of the gene encoding the 2,4-diaminobutyrate-γ-acetyltransferase is shown in SEQ ID NO:3, and the nucleotide sequence of the gene encoding the ectoine synthase is shown in SEQ ID NO:

5.

8. A genetically engineered bacterium, characterized in that: The invention comprises a host cell and a target gene transferred into the host cell, wherein the target gene comprises a gene encoding the L-2,4-diaminobutyrate aminotransferase mutant according to claim 2.

9. The genetically engineered bacterium according to claim 8, characterized in that The host cell is Escherichia coli.

10. The genetically engineered bacterium according to claim 8, characterized in that: The target genes also include the coding gene of 2,4-diaminobutyric acid-γ-acetyltransferase and the coding gene of ectoine synthase.

11. The genetically engineered bacterium according to claim 10, characterized in that: The nucleotide sequence of the gene encoding the 2,4-diaminobutyrate-γ-acetyltransferase is shown in SEQ ID NO:3, and the nucleotide sequence of the gene encoding the ectoine synthase is shown in SEQ ID NO:

5.

12. A biocatalytic reaction method, characterized in that: The following steps are involved: (a) in a liquid reaction system, L-aspartate-β-semialdehyde is converted under the catalysis of L-2,4-diaminobutyric acid transaminase to obtain L-2,4-diaminobutyric acid; Wherein, the L-2,4-diaminobutyrate aminotransferase is the L-2,4-diaminobutyrate aminotransferase mutant according to claim 1.

13. The method according to claim 12, characterized in that The biocatalytic reaction method further comprises the following steps: (b) converting the L-2,4-diaminobutyric acid obtained in step (a) under the catalysis of 2,4-diaminobutyric acid-γ-acetyltransferase to obtain N-acetyl-L-2,4-diaminobutyric acid.

14. The method according to claim 13, wherein: The amino acid sequence of the 2,4-diaminobutyrate-γ-acetyltransferase is shown in SEQ ID NO:

4.

15. The method according to claim 13 or 14, characterized in that The biocatalytic reaction method further comprises the following steps: (c) Under the catalysis of ectoine synthase, the N-acetyl-L-2,4-diaminobutyric acid obtained in the reaction of step (b) is converted to obtain ectoine.

16. The method according to claim 15, wherein: The amino acid sequence of the ectoine synthase is shown in SEQ ID NO:

6.

17. The method according to claim 15, characterized in that The biocatalytic reaction method further comprises the following steps: (d) isolating ectoine from the reaction system after the reaction of step (c).

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