Alcohol dehydrogenase mutants and their application in biological inorganic amination

Abstract

This invention discloses an alcohol dehydrogenase mutant and its application in biological inorganic amination. The alcohol dehydrogenase mutant is obtained by single or multiple mutations at positions 73, 107, 175, 96, or 286 of the amino acid sequence of the alcohol dehydrogenase shown in SEQ ID No. 2. The alcohol dehydrogenase mutant provided by this invention can efficiently and continuously provide NADH to the redox system, ensuring a high-efficiency coenzyme supply in the biological inorganic amination process and reducing the cost of inorganic amination synthesis. The alcohol dehydrogenase mutant of this invention provides NAD... + Its catalytic efficiency is significantly higher than that of the wild type, and its enzyme activity is increased by 2 times. When used in a coenzyme regeneration system and coupled with glufosinate dehydrogenase for glufosinate production, the time required is effectively shortened, showing great application potential.

Description

(I) Technical Field

[0001] This invention relates to biological inorganic amination technology, and particularly to an alcohol dehydrogenase mutant and its application in biological inorganic amination. (II) Background Technology

[0002] Glufosinate, or 4-[Hydroxy(methyl)phosphono]-D,L-homoalanine (PPT), is a phosphorus-containing amino acid herbicide that targets glutamine synthetase. It is characterized by high activity, good absorption, broad-spectrum herbicidal activity, low toxicity, and good environmental compatibility. In recent years, as the world's third-largest non-selective herbicide and the world's second-largest herbicide for genetically modified crops, glufosinate has shown enormous market potential. The main driving force is the widespread adoption of glufosinate-resistant genetically modified crops and the shrinking market for its main competitors (glyphosate and paraquat).

[0003] Of the two configurations of glufosinate, only the L-form has herbicidal activity, and commercially available glufosinate is its racemic form (Herbicidal compositions [P]. Patent application US4265654A, 1981). The use of L-form glufosinate monomers can significantly reduce the amount of glufosinate applied, alleviate environmental pressure, and slow down the development of weed resistance, demonstrating very significant environmental advantages.

[0004] Therefore, the development of L-glufosinate preparation processes is of great significance. There are three main methods for preparing chiral pure L-glufosinate (L-PPT): chiral resolution, chemical synthesis, and biocatalysis. Biocatalysis for glufosinate production offers advantages such as strict stereoselectivity, mild reaction conditions, and high yield, making it the superior method for producing L-glufosinate. It mainly includes the following three categories:

[0005] 1) Using L-glufosinate derivatives as substrates, L-glufosinate can be obtained directly via enzymatic hydrolysis. Its main advantages are high conversion rate and high ee value of the product. However, it requires expensive and difficult-to-obtain chiral raw materials as precursors, resulting in high costs and hindering industrial production. For example, the simplest biological method for preparing L-glufosinate is to directly hydrolyze dialanine using proteases. Dialanine is a natural tripeptide compound; under the catalysis of proteases, dialanine loses two L-alanine molecules to generate L-glufosinate.

[0006] 2) Using racemic glufosinate precursors as substrates, it is obtained through selective enzymatic resolution. The main advantages are that the raw materials are relatively easy to obtain and the catalyst activity is high, but its theoretical yield can only reach 50%, which will lead to waste of raw materials.

[0007] 3) Using α-ketoacid-2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) as a substrate, it is obtained through asymmetric synthesis of enzymes, mainly involving transaminases and glufosinate dehydrogenase. Bartsch et al. (Bartsch K (2005) Process for the preparation of 1-phosphinothrcine by enzymatic transamination with aspartate. US Patent no. US6936444B1) used PPO as a substrate and L-aspartic acid as an amino donor. They used transaminases with specific enzymatic activities for PPO and L-aspartic acid screened and isolated from soil microorganisms for catalysis. When the substrate concentration was 552 mM, the conversion rate still reached 52% after 4 hours of reaction at a very high temperature (80℃), and the space-time yield was only 4.5 g L-PPT / g (catalyst) / h. Furthermore, the preparation of L-glufosinate using transaminase has two major drawbacks. First, it is a reversible reaction, and the raw material PPO cannot be completely converted into L-PPT, so the conversion rate cannot reach 100%. Second, in order to make the reversible reaction proceed in the direction of L-PPT production, at least twice the amount of L-aspartic acid needs to be added as an amino donor. Excessive aspartic acid brings great trouble to the separation of L-PPT.

[0008] Among the many enzymatic synthetic routes for glufosinate, the ketone carbonyl group of the keto acid intermediate is a prochiral functional group, which can be used to construct a chiral center through enzymatic synthesis. The keto acid route is also suitable for the industrial development and production of L-glufosinate because the raw materials are inexpensive and readily available, and it avoids the use of highly toxic cyanide.

[0009] Amino acid dehydrogenases (EC 1.4.1.X, AADH) are a class of amino acid dehydrogenases that reversibly deaminate amino acids to generate corresponding keto acids. The reaction requires the participation of nucleoside coenzymes (NAD(P)+). Utilizing ammonium ions in solution, they deoxygenate and reductively amination the carbonyl group of the keto acid to generate the corresponding amino acid. They are widely used in the synthesis of both natural and non-natural α-amino acids. Based on their substrate specificity, they can be classified into glutamate dehydrogenases, leucine dehydrogenases, alanine dehydrogenases, valine dehydrogenases, etc. If they exhibit high activity towards glufosinate precursors, they can be called "glufosinate dehydrogenase (PPTDH)".

[0010] Alcohol dehydrogenase (EC 1.1.1.1, ADH) is an important coenzyme in biocatalytic redox reactions, used in the regeneration cycle of the coenzyme NAD(P)H in redox catalytic reactions. Isopropanol can be used as a substrate for coenzyme regeneration, and the product acetone has a low boiling point and is easily removed, making it suitable for industrial production. However, the commonly used coenzyme, glucose dehydrogenase, produces byproducts such as gluconic acid, which are often difficult to remove when the main product is an acid.

[0011] Although alcohol dehydrogenases have shown great potential in the coenzyme regeneration cycle, wild-type alcohol dehydrogenases have poor cofactor affinity and low enzyme activity, which limits their application and makes them unsuitable as a general tool enzyme for cofactor regeneration. Therefore, molecular modification is needed to improve their substrate affinity and enzyme activity. (III) Summary of the Invention

[0012] The purpose of this invention is to provide an alcohol dehydrogenase mutant and its application in bio-inorganic amination. The bio-inorganic amination method utilizes the alcohol dehydrogenase mutant to improve the efficiency of NADH production, optimize the coenzyme regeneration system, and provide more energy for bio-inorganic amination. This invention provides several alcohol dehydrogenase mutants with significantly increased enzyme activity through site-directed mutagenesis of alcohol dehydrogenase. This not only enables heterologous expression of alcohol dehydrogenase in *E. coli* but also improves the efficiency of NADH production and optimizes the coenzyme regeneration system. These mutants can ensure a higher coenzyme supply in bio-inorganic amination reactions, such as increasing the yield of L-glufosinate, L-glutamic acid, and L-aspartic acid, and have strong industrial application value.

[0013] The technical solution adopted in this invention is:

[0014] The present invention provides an alcohol dehydrogenase mutant, which is obtained by single or multiple mutations at positions 73, 107, 175, 96 or 286 of the amino acid sequence of the alcohol dehydrogenase shown in SEQ ID No. 2.

[0015] Preferably, the alcohol dehydrogenase mutant is formed by mutating the amino acid sequence shown in SEQ ID No. 2 to one of the following: (1) glycine at position 73 is mutated to alanine (G73A); (2) glycine at position 73 is mutated to alanine, and glutamic acid at position 107 is mutated to serine (G73A-E107S); (3) glycine at position 73 is mutated to alanine, glutamic acid at position 107 is mutated to serine, and glycine at position 175 is mutated to aspartic acid (G73A-E107S-G175D); (4) glycine at position 73 is mutated to alanine, glutamic acid at position 107 is mutated to serine, and glycine at position 107 is mutated to aspartic acid (G73A-E107S-G175D). (5) The glutamic acid at position 7 is mutated to serine, the glycine at position 175 is mutated to aspartic acid, and the aspartic acid at position 96 is mutated to glycine (G73A-E107S-G175D-D96G); (6) The glycine at position 73 is mutated to alanine, the glutamic acid at position 107 is mutated to serine, the glycine at position 175 is mutated to aspartic acid, the aspartic acid at position 96 is mutated to glycine, and the valine at position 286 is mutated to alanine (G73A-E107S-G175D-D96G-V286A).

[0016] This invention also relates to the coding gene of an alcohol dehydrogenase mutant, a recombinant vector, and an engineered bacterial strain. The preferred recombinant expression vector is the plasmid pET Duet; the preferred host cell is *Escherichia coli* BL21(DE3). Crude enzyme solution is obtained through protein-induced expression and cell disruption, exhibiting superior catalytic properties compared to the parent alcohol dehydrogenase.

[0017] This invention provides an application of the alcohol dehydrogenase mutant in biological inorganic amination reactions. The application involves using wet bacterial cells obtained through fermentation culture of recombinant bacteria containing both the alcohol dehydrogenase mutant gene and the glufosinate dehydrogenase gene as a catalyst, and using keto acid compounds as a substrate, with the addition of isopropanol and / or NAD. + A reaction system was constructed using a buffer solution with a pH of 7-8 as the reaction medium. The reaction was carried out at 35℃-60℃ and 500-600 rpm until complete. The reaction solution was then separated and purified to obtain the corresponding chiral compound. In the reaction system, the amount of catalyst used was 5-30 g / L (preferably 10 g / L) based on the total weight of wet bacterial cells, the initial concentration of the substrate was 10-500 mM (preferably 200 mM), the amount of NAD added was 0-5 mM (preferably 0.1 mM), and the amount of isopropanol added was 10-500 mM (preferably 300 mM).

[0018] Preferably, the substrate is 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, α-ketoglutaric acid, or oxaloacetic acid, and the corresponding products are L-glufosinate, L-glutamic acid, and L-aspartic acid, respectively.

[0019] This invention also provides an application of the alcohol dehydrogenase mutant in the catalytic preparation of L-glufosinate (L-PPT) from 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid. Using 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid as the reaction substrate and isopropanol as the substrate for the coenzyme regeneration system, the alcohol dehydrogenase mutant catalyzes the regeneration of coenzyme NAD(P)H while the glufosinate dehydrogenase catalyzes the conversion of 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid to L-PPT.

[0020] Preferably, the application is as follows: using wet bacterial cells obtained by fermentation culture of recombinant bacteria containing a co-expression gene of alcohol dehydrogenase mutant and glufosinate dehydrogenase as a catalyst, and using 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid (PPO) as a substrate, with the addition of isopropanol and NAD. + The reaction system is constructed using a buffer solution with a pH of 7-8 as the reaction medium. The reaction is carried out at 35℃-60℃ and 500-600rpm until complete. The reaction solution is then separated and purified to obtain L-glufosinate. In the reaction system, the amount of catalyst is 5-30g / L (preferably 10g / L) based on the total weight of wet cells, the initial concentration of the substrate is 10-500mM (preferably 200mM), the amount of NAD added is 0-5mM (preferably 0.1mM), and the amount of isopropanol added is 10-500mM (preferably 300mM).

[0021] Preferably, the co-expressing recombinant strain containing the alcohol dehydrogenase mutant gene and the glufosinate dehydrogenase gene is constructed by cloning the alcohol dehydrogenase mutant encoding gene into the second multiple cloning site (between the Nde I and Avr II restriction sites) of the pETDuet-PPTDH vector containing the glufosinate dehydrogenase gene using a one-step cloning method, and then transforming it into the host E. coli BL21(DE3) to construct the co-expressing recombinant strain. The nucleotide sequence of the glufosinate dehydrogenase gene is shown in SEQ ID No. 3, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 1.

[0022] Preferably, the catalyst is prepared as follows: Recombinant bacteria co-expressing the genes of glufosinate dehydrogenase and alcohol dehydrogenase mutants are inoculated into LB liquid medium containing 50 μg / ml ampicillin resistance and cultured at 37°C and 180 rpm for 12 h. Then, at a volume concentration of 2%, the bacteria are inoculated into fresh LB liquid medium containing 50 μg / mL ampicillin resistance and cultured at 37°C and 180 rpm until the bacterial OD600 reaches 0.6-0.8. IPTG is added to a final concentration of 12 μg / mL, and the culture is induced at 28°C for 12 h. The culture is then centrifuged at 4°C and 8000 rpm for 10 min, the supernatant is discarded, and the precipitate is collected and washed twice with pH 7.5, 20 mM sodium phosphate buffer to obtain wet bacterial cells.

[0023] When the substrate of the present invention is 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, the method for separating and purifying the reaction solution is as follows: (1) Add calcium carbonate to the reaction solution to remove sulfate ions in the reaction solution by precipitation. After stirring magnetically at 600 rpm for 2 hours, centrifuge at 8000 rpm for 10 minutes and collect the supernatant. The amount of calcium carbonate added is 0.1 g / mL based on the volume of the reaction solution. (2) The supernatant of step (1) is concentrated by rotary evaporation at 80 rpm and 60℃ for 6 hours to dryness to obtain a concentrate. (3) Add methanol to the concentrate of step (2) and dissolve overnight. Adjust the pH to 2-5, stir at 600 rpm for 4 hours, filter, dry the filter cake, and obtain L-glufosinate-ammonium salt powder. The ratio of methanol to the volume of the reaction solution is 2:1.

[0024] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0025] The alcohol dehydrogenase mutant provided by this invention can efficiently and continuously provide NADH to the redox system, ensuring a high-efficiency coenzyme supply in the inorganic amination process and reducing the cost of inorganic amination synthesis. In the method for preparing L-glufosinate using the alcohol dehydrogenase mutant to construct a coenzyme cycling system, the 200mM substrate conversion rate is as high as 100%, the byproduct acetone has a low boiling point and is easily discharged from the reaction system, and product separation and purification are more convenient. The alcohol dehydrogenase mutant of this invention provides high NAD... + Its catalytic efficiency is significantly higher than that of the wild type, and its enzyme activity is increased by 2 times. When used in a coenzyme regeneration system and coupled with glufosinate dehydrogenase for glufosinate production, the time required is effectively shortened, showing great application potential. (iv) Description of the attached drawings

[0026] Figure 1 SDS-PAGE electrophoresis image of the co-expressed strain of glufosinate dehydrogenase and alcohol dehydrogenase, where lane 1: standard protein molecular weight; lanes 2-8: crude enzyme solution of pETDuet-PPTDH-GstADH.

[0027] Figure 2 A flowchart illustrating the reaction process for preparing L-glufosinate from recombinant genetically engineered bacteria containing glufosinate dehydrogenase and alcohol dehydrogenase.

[0028] Figure 3 This is the NADH standard curve.

[0029] Figure 4 Photo of the finished L-glufosinate powder.

[0030] Figure 5 The images show the HPLC chromatograms of D- and L-glufosinate standards (top) and L-glufosinate ammonium salt powder (bottom). The peak elution time of D-glufosinate is approximately 12.5 minutes, and that of L-glufosinate is approximately 10 minutes.

[0031] Figure 6 X-ray diffraction pattern of L-glufosinate-ammonium salt powder. (V) Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0033] Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0034] Reagents used in upstream genetic engineering operations: The one-step cloning kits used in the embodiments of this invention were all purchased from Vazyme, Nanjing Novizan Biotechnology Co., Ltd.; plasmid extraction kits and DNA recovery and purification kits were purchased from Axygen Hangzhou Co., Ltd.; E. coli BL21(DE3), plasmids, etc., were purchased from Shanghai Sangon Biotech Co., Ltd.; DNA markers, FastPfu DNA polymerase, low molecular weight standard proteins, agarose gel electrophoresis reagents, primer synthesis, and gene sequencing were performed by Hangzhou Qingke Zixi Biotechnology Co., Ltd. Refer to the product instructions for the usage of the above reagents.

[0035] Reagents used in downstream catalytic processes: 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO), D,L-glufosinate, and L-glufosinate (L-PPT) standards were purchased from Sigma-Aldrich; NADH was purchased from Bangtai Biotechnology (Shenzhen) Co., Ltd.; other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0036] Preparation of competent cells: E. coli BL21(DE3) strain, preserved in glycerol tubes, was obtained from a -80℃ freezer. Straw was deposited on antibiotic-free LB agar plates and cultured at 37℃ for 10 h to obtain single colonies. A single colony was picked from the LB agar plate and inoculated into a test tube containing 10 mL of LB medium. The culture was incubated at 37℃ and 180 rpm for 9 h. 2 mL of the bacterial culture was taken from the test tube and inoculated into 100 mL of LB medium. The culture was incubated at 37℃ and 180 rpm until the OD600 reached 0.4-0.6. The bacterial culture was pre-cooled on ice and transferred to sterile centrifuge tubes, then placed on ice for 10 min. Centrifuge at 5000 rpm for 10 min at 4 °C; discard the supernatant, resuspend the precipitated cells in 0.1 mol / L CaCl2 aqueous solution pre-cooled at 4 °C, and place on ice for 30 min; centrifuge at 5000 rpm for 10 min at 4 °C, discard the supernatant, resuspend the precipitated cells in 0.1 mol / L CaCl2 aqueous solution pre-cooled at 4 °C containing 15% glycerol, aliquot 100 μL of the resuspended cells into sterile 1.5 mL centrifuge tubes, and store at -80 °C. Remove as needed.

[0037] The structural formula of D-glufosinate (D-PPT) is shown in formula (1); the structural formula of L-glufosinate (L-PPT) is shown in formula (2); and the structural formula of 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid (PPO) is shown in formula (3).

[0038]

[0039] (3) 2-Carbonyl-4-[hydroxy(methyl)phosphono]butyric acid

[0040] The product concentration was determined by high-performance liquid chromatography (HPLC). The analytical method was as follows:

[0041] (1) Chromatographic conditions: Column type: QS-C18, 5μm, 4.6×250mm. Mobile phase: 50mM ammonium acetate solution: methanol = 10:1. Fluorescence detection wavelength: λex = 340nm, λem = 455nm. Flow rate: 1mL / min. Column temperature: 30℃, L-PPT elution time: 10min, D-PPT elution time: 12.5min.

[0042] (2) Derivatization reagent: Weigh 0.1g of o-phthalaldehyde and 0.12g of N-acetyl-L-cysteine, respectively, dissolve them with 10mL of ethanol, then add 40mL of 0.1mol / L borate buffer (pH 9.8), shake to dissolve completely, and store at 4℃ for later use (not exceeding 4 days).

[0043] (3) Derivatization reaction and HPLC determination: The reaction solution was made up to 1 mL with ultrapure water, that is, the reaction solution was diluted 10 times. The diluted sample was first derivatized, specifically: 200 μL of the diluted reaction solution was added to 400 μL of derivatization reagent and derivatized at 30℃ for 5 min. Then, 400 μL of ultrapure water was added to make up to 1 mL. The sample was centrifuged at 12000 rpm for 1 min. The supernatant was taken and filtered through a 0.22 μm microfiltration membrane as the liquid phase sample. PPO, L-PPT, D-PPT and ee value were detected by HPLC.

[0044] High-performance liquid chromatography (HPLC) was used to determine the substrate concentration. The analytical method was as follows:

[0045] Column type: QS-C18, 5μm, 4.6×250mm. Mobile phase: 50mM ammonium dihydrogen phosphate and 10mM tetrabutylammonium bromide were dissolved in 800mL of ultrapure water, the pH was adjusted to 3.8 with phosphoric acid, and the volume was brought to 1000mL. This solution was then mixed with acetonitrile at a volume ratio of 88:12. Detection wavelength: 232nm, flow rate: 0.8mL / min, column temperature: 40℃, elution time: 10.0min.

[0046] Example 1: Construction of glufosinate dehydrogenase genetically engineered bacteria

[0047] Glufosinate dehydrogenase (GenBank No.: RQW74141.1) derived from Lysinibacillus composti was synthesized in its entirety by Hangzhou Qingke Biotechnology Co., Ltd. (nucleotide sequence as shown in SEQ ID No. 3, amino acid sequence as shown in SEQ ID No. 1). The obtained glufosinate dehydrogenase gene was cloned into the pETDuet plasmid between the EcoRI and AflII restriction sites to construct the expression vector pETDuet-PPTDH. This vector was then transformed into E. coli BL21(DE3) to obtain the starting strain E. coli BL21(DE3) / pETDuet-PPTDH.

[0048] SEQ ID No.1

[0049] MAENLNLFTSTQEVVKEALNKLGYDEAMYELLKEPLRLLKVRIPVKMDDGTTQVFTGYRAQHSDAVGPTKGGVRFHPMVSEDEVKALSMWMTLKCGIVDLPYG GGKGGIICDPRQMSMGELERLSRGYVRAISQIVGPTKDIPGPDVFTNAQIMAWMMDEYSRMDEFNSPGFITGKPLVLGGSKGRDRATAEGVTIVIQEAAKKRNI DIKGARVVIQGFGNAGSFLAKFMSDLGAKVIGISDAYGALHDPNGLDIDYLLDRRDSFGTVTTLFENTITNQELLELDCDILVPAAIENQITAENAHNIKATI VVEAANGPTTSEATKILTERGILLVPDVLASAGGATVSYFEWVQNNMGYYWEEEEVQEKLYKKMYDSFEAVYTTATTRNIDMRLAAYMVGVRRTAEASRFRGWV

[0050] SEQ ID NO.3

[0051]

[0052] Example 2: Construction of engineered bacteria for alcohol dehydrogenase

[0053] The nucleic acid sequence of the alcohol dehydrogenase GstADH (NCBI accession number WP_001058802.1) from *Geobacillus stearothermophilus* was synthesized in its entirety by Hangzhou Qingke Biotechnology Co., Ltd. The obtained GstADH gene (nucleotide sequence as shown in SEQ ID No. 4, amino acid sequence as shown in SEQ ID No. 2) was cloned between the Nco I and Xho I restriction sites of plasmid pET-28a to construct the recombinant plasmid pET-28a-GstADH. The recombinant plasmid was transformed into *E. coli* to obtain the starting strain *E. coli* BL21(DE3) / pET28a-GstADH.

[0054] SEQ ID No.2

[0055] MKAAVVEQFKEPLKIKEVEKPTISYGEVLVRIKACGVCHTDLHAAHGDWPVKPKLPLIPGHEGVGIVEEVGPGVTHLKVGDRVGIPWLYSACGHCDYCLSGQETLCEHQKNAGYSVDGGYAEYCRAAAADYVVKIPDNLSFEEAAPIFCAGVTTYKALKVTGAKPGEWVA IYGIGGLGHVAVQYAKAMGLNVVAVDIGDEKLELAKELGADLVVNPLKEDAAKFMKEKVGGVHAAVVTAVSKPAFQSAYNSIRRGGACVLVGLPPEEMPIPIFDTVLNGIKIIGSIVGTRKDLQEALQFAAEGKVKTIIEVQPLEKINEVFDRMLKGQINGRVVLTLEDK

[0056] SEQ ID NO.4

[0057]

[0058] Example 3: Construction and screening of alcohol dehydrogenase mutant libraries

[0059] In the first round, using the codon-optimized alcohol dehydrogenase gene (nucleotide sequence as shown in SEQ ID No. 4) obtained from the whole-gene synthesis in Example 2 as a template, E. coli BL21(DE3) was transformed by site-directed mutagenesis PCR using the primers in Table 1 used for mutating G73M and G73A, respectively, and plated on LB plates to obtain pET28a-GstADH-G73A and pET28a-GstADH-G73M mutants. The enzyme activity of the pET28a-GstADH-G73A mutant was determined to be 8.53 U / mg using the methods of Examples 4 and 5, and the enzyme activity of the pET28a-GstADH-G73M mutant was 7.25 U / mg. The dominant strain was obtained by screening and the mutant with the G73A mutation was named the alcohol dehydrogenase mutant GstADH-G73A. The engineered strain E. coli BL21(DE3) / pET28a-GstADH-G73A was constructed using the method of Example 2 and was denoted as strain E1.

[0060] In the second round, using the mutant GstADH-G73A as a template, and with the primers listed in Table 1 for mutating E107D, E107N, E107S, and E107K, respectively, site-directed mutagenesis PCR was performed, followed by transformation into E. coli BL21(DE3), and plated on LB plates to obtain the mutants pET28a-GstADH-G73A-E107D, pET28a-GstADH-G73A-E107N, pET28a-GstADH-G73A-E107S, and pET28a-GstADH-G73A-E107K. The enzyme activities were measured using the methods in Examples 4 and 5 to be 8.56 U / mg, 8.55 U / mg, 8.79 U / mg, and 8.62 U / mg, respectively. The dominant strain was a mutant with double mutations of G73A and E107S. The plasmid of this dominant mutant was named alcohol dehydrogenase mutant GstADH-G73A-E107S.

[0061] In the third round, using the mutant GstADH-G73A-E107S as a template, and with the primers listed in Table 1 for mutating G175A, G175E, and G175D, site-directed mutagenesis PCR was performed, followed by transformation into E. coli BL21(DE3). The resulting mutants were plated on LB agar plates, yielding pET28a-GstADH-G73A-E107D-G175A, pET28a-GstADH-G73A-E107D-G175E, and pET28a-GstADH-G73A-E107D-G175D mutants. The enzyme activities were measured using the methods in Examples 4 and 5, and were 8.81 U / mg, 8.86 U / mg, and 8.86 U / mg, respectively. 9.24 U / mg, the dominant strain was obtained by screening and found to be a mutant with triple mutations of G73A, E107S and G175D. The plasmid of this dominant mutant was named alcohol dehydrogenase mutant GstADH-G73A-E107S-G175D. The engineered strain E. coli BL21(DE3) / pET28a-GstADH-G73A-E107S-G175D was constructed using the method in Example 2 and denoted as strain E2.

[0062] In the fourth round, using the mutant GstADH-G73A-E107S-G175D as a template, and with the primers listed in Table 1 for mutating D96A, D96G, and D96E, respectively, site-directed mutagenesis PCR was performed, followed by transformation into E. coli BL21(DE3), and plated on LB plates to obtain pET28a-GstADH-G73A-E107D-G175D-D96A, pET28a-GstADH-G73A-E107D-G175D-D96G, and pET28a-GstADH-G73A-E107D-G175D-D96E. The enzyme activities were determined using the methods in Examples 4 and 5 to be 9.64 U / mg, 11.36 U / mg, and 9.83 U / mg, respectively. The dominant strain obtained by screening was a strain with four mutations: G73A, E107S, G175D, and D96G. The plasmid of this dominant mutant was named GstADH-G73A-E107S-G175D-D96G, which is an alcohol dehydrogenase mutant. The engineered strain E. coli BL21(DE3) / pET28a-GstADH-G73A-E107S-G175D-D96G was constructed using the method in Example 2 and was designated as strain E3.

[0063] In the fifth round, using the mutant GstADH-G73A-E107S-G175D-D96G as a template, and with the primers listed in Table 1 for mutating V286A, V286E, and V286T, site-directed mutagenesis PCR was performed, followed by transformation into E. coli BL21(DE3), and plated on LB plates to obtain pET28a-GstADH-G73A-E107D-G175D-D96A-V286A, pET28a-GstADH-G73A-E107D-G175D-D96A-V286E, and pET28a-GstADH-G73A-E107D-G175D-D96A-V286T. The enzyme activities were determined using the methods in Examples 4 and 5, and were 13.76 U / mg, 11.63 U / mg, and 11.54 U / mg, respectively. The dominant strain obtained by screening was a strain with five mutations: G73A, E107S, G175D, D96G, and V286A. The plasmid of this dominant mutant was named alcohol dehydrogenase mutant GstADH-G73A-E107S-G175D-D96G-V286A. The engineered strain E. coli BL21(DE3) / pET28a-GstADH-G73A-E107S-G175D-D96G-V286A was constructed using the method in Example 2 and designated as strain E4. The dominant single mutants in subsequent experiments were all constructed using the same method.

[0064] The PCR reaction system was as follows: 2×Phanta Max buffer: 25 μL; dNTPs: 1 μL; upstream primer: 2 μL; downstream primer: 2 μL; template: 1 μL; Phanta Super-Fidelity DNA polymerase: 0.5 μL; ddH2O: 18.5 μL.

[0065] PCR reaction conditions: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 3 min, for a total of 30 cycles; final extension at 72℃ for 10 min; store at 4℃.

[0066] The PCR results were verified positive by DNA agarose gel electrophoresis, showing that the amplified product was a single band, approximately 2200 bp in size. The PCR product was then digested with Dpn I enzyme, and the amplified product was purified using a DNA purification kit. Specific steps were followed according to the kit's instructions.

[0067] Table 1. Mutation sites and primer sequences

[0068]

[0069]

[0070] Example 4: Induction of expression of alcohol dehydrogenase parent and mutant

[0071] The starting strain E. coli BL21(DE3) / pET28a-GstADH from Example 2 and the mutant strains (E1, E2, E3, E4) constructed in Example 3 were inoculated into LB liquid medium containing a final concentration of 50 μg / mL ampicillin and cultured at 37°C for 8 hours. Then, they were inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL ampicillin at a volume fraction of 2% (v / v) and cultured at 37°C and 180 rpm for 2 hours. IPTG was then added to the culture medium to a final concentration of 0.1 mM and cultured at 28°C for 14 hours. After that, the culture was centrifuged at 4°C and 8000 rpm for 10 minutes to obtain the corresponding wet bacterial cells.

[0072] After culturing, the collected wet bacterial cells were washed twice with phosphate-buffered saline (50 mM) at pH 8. Then, the cells were resuspended in PBS (50 mM) at pH 8 and sonicated 30 times under the following conditions: 400 W power, 2 s disruption, 5 s interval. The cell lysate was centrifuged at 8000 rpm for 10 min at 4°C to remove the precipitate. The supernatant obtained was the crude enzyme solution. Crude enzyme solutions were obtained for the following mutant strains: E1 (2.72 mg / mL), E2 (2.83 mg / mL), E3 (2.78 mg / mL), and E4 (2.68 mg / mL).

[0073] Example 5: Determination of alcohol dehydrogenase activity

[0074] Enzyme activity definition: The 1961 International Enzyme Conference defined one unit of enzyme activity as the amount of enzyme that converts 1 micromolar of substrate in 1 minute under specific conditions (30°C), or the amount of enzyme that converts 1 micromolar of the relevant groups in the substrate.

[0075] Alcohol dehydrogenase activity assay: Take 950 μL of isopropanol and add 10 mM NAD. + 25 μL of aqueous solution was placed in a metal bath shaker and kept at 30 °C for 10 min; 25 μL of the corresponding crude enzyme solution was added, the mixture was quickly removed and shaken by hand, poured into a cuvette, and quickly placed in a spectrophotometer to detect the absorbance at 340 nm. The enzyme activity was calculated according to the NADH standard curve.

[0076] NADH standard curve: 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, and 0.8 mM NADH solutions were prepared using ultrapure water. Appropriate amounts were added to cuvettes, which were then quickly placed in a spectrophotometer. The absorbance at 340 nm was measured. The absorbance was plotted with time on the x-axis (in minutes) and absorbance on the y-axis. Figure 3 The NADH standard curve is shown in Table 2. The enzyme activity measurement results are shown in Table 2. The mutant strain E4 was selected for subsequent experiments.

[0077] Table 2. Enzyme activity assay results (mg refers to the protein content in the crude enzyme solution).

[0078] serial number Mutation type Enzyme activity (U / mg) Starting strain No mutation 6.80 E1 G73A; 8.53 E2 G73A-E107S-G175D 9.24 E3 G73A-E107S-G175D-D96G 11.36 E4 G73A-E107S-G175D-D96G-V286A 13.76

[0079] Example 6: Construction of recombinant engineered bacteria co-expressing glufosinate dehydrogenase and alcohol dehydrogenase

[0080] The GstADH-G73A-E107S-G175D-D96G-V286A gene was cloned into the second multiple cloning site (between the Nde I and Avr II restriction sites) of the pETDuet-PPTDH vector containing the glufosinate dehydrogenase gene constructed in Example 1 using a one-step cloning method. This clone was then transformed into the host E. coli BL21(DE3) to construct the co-expressing recombinant engineered strain E. coli BL21(DE3) / pETDuet-PPTDH-GstADH-G73A-E107S-G175D-D96G-V286A. The specific procedures are as follows:

[0081] 1. Primer design

[0082] Primers 1, 2, 3, and 4 were designed based on the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4. Approximately 20 bp of Nde I and Avr II restriction sites contained in the vector pETDuet-PPTDH were added as homologous arms to the 5' ends of the alcohol dehydrogenase gene-specific forward / reverse amplification primer sequences (primer 1 and primer 2).

[0083] Primer 1: 5'-taacctaggctgctgccaccgctgagcaataa-3';

[0084] Primer 2: 5'-CATatgtatatctccttcttatacttaactaatatact-3';

[0085] Primer 3: 5'-gtataagaaggagatatacatATGAAGGCGGCTGTCGTAGAACAGT-3';

[0086] Primer 4: 5'-tggcagcagcctaggttaTTTGTCTTCCAGCGT-3';

[0087] 2. Fragment amplification

[0088] (1) pETDuet-PPTDH vector

[0089] Using pETDuet-PPTDH constructed in Example 1 as a template, primers 1 and 2 were used to amplify the DNA using high-fidelity Pfu DNA polymerase. DPN I was added to digest the DNA to obtain the pETDuet-PPTDH vector.

[0090] (2) GstADH fragment

[0091] Using pET-28a-GstADH-G73A-E107S-G175D-D96G-V286A constructed in Example 5 as a template, primers 3 and 4 were used to amplify the DNA using high-fidelity Pfu DNA polymerase. DPN I was added to digest the DNA to obtain the alcohol dehydrogenase GstADH fragment with homologous arms.

[0092] (3) Single-fragment homologous recombination

[0093] The nucleic acid concentration of each fragment in steps (1) and (2) was determined using a NanoDropone micro spectrophotometer (TermoFisherScientific, USA), and the single-fragment homologous recombination reaction system was prepared according to the concentration in Table 3.

[0094] Optimal cloning vector usage = {0.02 * number of base pairs in the cloning vector} ng (0.03 pmol)

[0095] Optimal insert size = {0.04 * number of insert base pairs} ng (0.06 pmol)

[0096] Table 3 Reaction System

[0097]

[0098] Note: X represents the amount of linearized vector added, Y represents the amount of inserted fragments, and n is the number of inserted fragments.

[0099] The prepared reaction system was gently mixed using a pipette, and after a brief centrifugation, the reaction solution was collected at the bottom of the tube. The reaction system was placed in a 50°C water bath and allowed to stand for 5 minutes, then immediately cooled on ice. The three different systems were transformed into Escherichia coli BL21(DE3) (42°C, 90 s), plated on LB agar plates containing 100 μg / mL ampicillin resistance, and incubated at 37°C for 12–16 h. Clones were randomly selected, plasmids were extracted, and sequenced for identification. Recombinant E. coli E. coli BL21(DE3) / pETDuet-PPTDH-GstADH-G73A-E107S-G175D-D96G-V286A containing glufosinate dehydrogenase and alcohol dehydrogenase genes were screened.

[0100] Using the same method, recombinant E. coli BL21(DE3) / pETDuet-PPTDH-GstADH was constructed.

[0101] Example 7: Induction of expression of recombinant engineered bacteria co-expressing glufosinate dehydrogenase and alcohol dehydrogenase

[0102] The *E. coli* BL21(DE3) / pETDuet-PPTDH-GstADH and *E. coli* BL21(DE3) / pETDuet-PPTDH-GstADH-G73A-E107S-G175D-D96G-V286A strains constructed in Example 6 were inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance and cultured at 37°C and 200 rpm for 12 h. Then, they were inoculated into fresh LB liquid medium containing 50 μg / mL ampicillin resistance at a volume concentration of 2% and cultured at 37°C and 180 rpm until the bacterial OD600 reached 0.6-0.8. IPTG was added to a final concentration of 12 μg / mL, and the culture was induced at 28°C for 12 h. After incubation, the cells were centrifuged at 4°C and 8000 rpm for 15 min, the supernatant was discarded, the precipitate was collected, and washed twice with pH 7.5, 20 mM sodium phosphate buffer to obtain wet bacterial cells.

[0103] The bacterial cells were broken up using the method described in Example 4. Figure 1 For the corresponding gel electrophoresis results, lane 1 is Maker; lanes 2, 3, 4, and 5 are pETDuet-PPTDH-GstADH; and lanes 6, 7, and 8 are pETDuet-PPTDH-GstADH-G73A-E107S-G175D-D96G-V286A.

[0104] Example 8: Preparation of L-glufosinate by co-expression of glufosinate dehydrogenase and alcohol dehydrogenase in recombinant engineered bacteria

[0105] Using PPO as a substrate, and wet bacterial cells obtained by fermentation culture of recombinant engineered bacteria co-expressing glufosinate dehydrogenase and alcohol dehydrogenase as a biocatalyst, L-glufosinate was generated. The specific operation is as follows:

[0106] First, 10 g DCW / L of the co-expressed recombinant engineered strain *E. coli* BL21(DE3) / pETDuet-PPTDH-GstADH-G73A-E107S-G175D-D96G-V286A, prepared according to the method in Example 7, was dissolved sequentially in 1 L of 100 mM, pH 7.5 sodium phosphate buffer, along with 300 mM isopropanol and 0.1 mM NAD. Then, 1 L of substrate PPO (36.2 g / L) was added to a final concentration of 200 mM to form a coenzyme regeneration system. The reaction was carried out at 55°C and 600 rpm for 8 h. The reaction solution was sampled and the formation of L-glufosinate was detected by high-performance liquid chromatography. Under the same conditions, the co-expressed recombinant engineered strain *E. coli* BL21(DE3) / pETDuet-PPTDH-GstADH was used as a control. The results are as follows Figure 2 As shown.

[0107] Figure 2 The results showed that the product concentration of strain E. coli BL21(DE3) / pETDuet-PPTDH-GstADH-G73A-E107S-G175D-D96G-V286A gradually increased over time during the reaction, and the reaction was completed within 8 hours, with a substrate conversion rate greater than 99%. In the comparative experiment, the conversion rate of strain E. coli BL21(DE3) / pETDuet-PPTDH-GstADH was only 65% ​​after 6 hours of reaction.

[0108] Example 9: Preparation of L-glutamic acid by co-expression of glufosinate dehydrogenase and alcohol dehydrogenase in recombinant engineered bacteria.

[0109] Using α-ketoglutarate as a substrate, and wet cells obtained from fermentation culture of recombinant engineered bacteria co-expressing glufosinate dehydrogenase and alcohol dehydrogenase as a biocatalyst, L-glutamic acid was generated. The specific operation is as follows:

[0110] First, 10 g DCW / L of glufosinate dehydrogenase and alcohol dehydrogenase co-expressing recombinant engineered bacteria E. coli BL21(DE3) / pETDuet-PPTDH-GstADH-G73A-E107S-G175D-D96G-V286A prepared by the method in Example 7, 300 mM isopropanol, and 0.1 mM NAD were sequentially dissolved in 1 L of 100 mM, pH 7.5 sodium phosphate buffer. Then, 1 L of substrate α-ketoglutarate with a final concentration of 200 mM was added to form a coenzyme regeneration system. The reaction was carried out at 55 °C and 600 rpm for 8 h. The reaction solution was sampled and the formation of L-glutamic acid was detected by high performance liquid chromatography.

[0111] Strain E.coli BL21(DE3) / pETDuet-PPTDH-GstADH-G73A-E107S-G175D-

[0112] During the D96G-V286A reaction, the product concentration gradually increased over time, and the reaction was completed within 6 hours, with a substrate conversion rate greater than 99%. In the comparative experiment, the conversion rate of E. coli BL21(DE3) / pETDuet-PPTDH-GstADH strain was only 76% after 6 hours of reaction.

[0113] Example 10: Preparation of L-Aspartic Acid by Co-expression of Glufosinate Dehydrogenase and Alcohol Dehydrogenase in Recombinant Engineered Bacteria

[0114] Using oxaloacetic acid as a substrate, and wet bacterial cells obtained by fermentation culture of recombinant engineered bacteria co-expressing glufosinate dehydrogenase and alcohol dehydrogenase as a biocatalyst, L-aspartic acid was generated. The specific operation is as follows:

[0115] First, 10 g DCW / L of the recombinant engineered strain *E. coli* BL21(DE3) / pETDuet-PPTDH-GstADH-G73A-E107S-G175D-D96G-V286A, prepared by the method in Example 7, was dissolved sequentially in 1 L of 100 mM, pH 7.5 sodium phosphate buffer, along with 300 mM isopropanol and 0.1 mM NAD. Then, 1 L of substrate oxaloacetic acid (200 mM) was added to form a coenzyme regeneration system. The reaction was carried out at 55°C and 600 rpm for 8 h. The reaction solution was sampled and the formation of L-aspartic acid was detected by high-performance liquid chromatography (HPLC). Under the same conditions, the recombinant engineered strain *E. coli* BL21(DE3) / pETDuet-PPTDH-GstADH, co-expressing glufosinate dehydrogenase and alcohol dehydrogenase, served as a control. In the reaction of strain E. coli BL21(DE3) / pETDuet-PPTDH-GstADH-G73A-E107S-G175D-D96G-V286A, the product concentration gradually increased over time, and the reaction was completed within 6 hours, with a substrate conversion rate greater than 99%. In the comparative experiment, the conversion rate of strain E. coli BL21(DE3) / pETDuet-PPTDH-GstADH was only 61% after 6 hours of reaction.

[0116] Example 11: Purification and crystallization of high-concentration L-glufosinate from reaction solution using methanol crystallization method

[0117] Add 50g of calcium carbonate to 50mL of the reaction solution obtained in Example 7 to precipitate sulfate ions. After magnetic stirring at 600rpm for 2h, centrifuge at 8000rpm for 10min. Concentrate the supernatant by rotary evaporation at 80rpm and 60℃ for 6h until dry. Dissolve in 600mL of methanol overnight. Filter under vacuum to obtain a methanol solution of L-glufosinate. Adjust the pH to 4.2 with concentrated sulfuric acid, stir magnetically at 600rpm for 4h, filter, and dry the filter cake at 65℃ for 12h to obtain off-white crystals of L-glufosinate. Figure 4 As shown.

[0118] The purity of the prepared L-glufosinate was then verified. 0.01 g of the off-white crystals of L-glufosinate was dissolved in 10 mL of ultrapure water to prepare a 1 g / L L-glufosinate aqueous solution, which was then analyzed by high performance liquid chromatography. The results showed that the mass fraction of L-glufosinate was >95%.

[0119] Example 12: Characterization of L-Glufosinate-ammonium salt form

[0120] 1. HPLC characterization

[0121] The off-white crystals of L-glufosinate prepared in Example 11 were determined by liquid chromatography, and the results are as follows: Figure 5 As shown, L-glufosinate-ammonium salt was successfully obtained by the method described in Example 11.

[0122] 2. XRD characterization

[0123] The L-glufosinate-ammonium salt crystals obtained in Example 11 were pretreated by grinding (drum milling) and sieving. Liquid nitrogen or dry ice was used to make them brittle, and then they were ground using a mortar and pestle (ball mill) until the powder was <360 mesh and felt smooth to the touch, indicating that the crystal size met the requirements. During the grinding process, the powder was continuously sieved to separate the refined particles.

[0124] Sprinkle the sample powder onto a microscope slide approximately 25×35×1mm in size (the powder should be sprinkled at the position corresponding to the window opening of the sample frame). Then, add a sufficient amount of acetone to make the powder a thin slurry. Spread it evenly to form a single-particle layer. After the acetone evaporates, the powder will adhere to the glass slide.

[0125] The prepared L-glufosinate-ammonium salt crystals were subjected to Cu-Ka radiation at 25°C. The X-ray powder diffraction pattern, expressed in 2θ angles, is shown below. Figure 6 As shown. Figure 6 This demonstrates that L-glufosinate was successfully obtained using the method described in Example 11.

[0126] Example 13: Hygroscopicity test of L-glufosinate crystals

[0127] 50g of L-glufosinate prepared in Example 11 was taken and kept under different humidity conditions (Table 4) for 15 days. The mass was tested for any significant changes and the mass change rate was calculated. The results are shown in Table 4.

[0128] Table 4 Hygroscopicity Data

[0129]

[0130] As can be seen from Table 4 above, the L-glufosinate-ammonium salt crystals prepared in Example 11 showed a mass change of less than 0.1% after being stored at a relative humidity of 60% and a room temperature (25°C) for 15 days, and had no obvious hygroscopicity.

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The alcohol dehydrogenase mutant is the amino acid sequence shown in SEQ ID No. 2, which is mutated into one of the following: (1) the 73rd glycine is mutated into alanine; (2) the 73rd glycine is mutated into alanine, the 107th glutamic acid is mutated into serine, and the 175th glycine is mutated into aspartic acid; (3) the 73rd glycine is mutated into alanine, the 107th glutamic acid is mutated into serine, the 175th glycine is mutated into aspartic acid, and the 96th aspartic acid is mutated into glycine; (4) the 73rd glycine is mutated into alanine, the 107th glutamic acid is mutated into serine, the 175th glycine is mutated into aspartic acid, the 96th aspartic acid is mutated into glycine, and the 286th valine is mutated into alanine.

2. A recombinant genetically engineered bacterium containing the coding gene of the alcohol dehydrogenase mutant of claim 1.

3. Use of the alcohol dehydrogenase mutant of claim 1 in a bio-inorganic amination reaction, characterized in that, The application is: using wet bacteria obtained by fermenting and culturing recombinant bacteria containing co-expression of alcohol dehydrogenase mutant gene and glufosinate dehydrogenase gene as catalyst, using keto acid compound as substrate, adding isopropyl alcohol and NAD + buffer with pH value of 7-8 as reaction medium to form a reaction system, reacting under the condition of 35-60 DEG C and 500-600 rpm, separating and purifying the reaction liquid to obtain corresponding chiral compounds; the substrate is 2-carbonyl-4-(hydroxymethyl phosphine) butyric acid, alpha-ketoglutaric acid, oxaloacetic acid; the glufosinate dehydrogenase gene nucleotide sequence is shown in SEQ ID No.

3.

4. The use according to claim 3, wherein the compound is ###0002### The co-expression recombinant bacteria of the alcohol dehydrogenase mutant gene and the glufosinate dehydrogenase gene is that the alcohol dehydrogenase mutant gene is cloned into the second multi-cloning site of the pETDuet-PPTDH vector containing the glufosinate dehydrogenase gene by using one-step cloning method, and then the host E. coli BL21(DE3), and the co-expression recombinant bacteria is constructed.

5. The use according to claim 3, wherein the compound is ###0002### The catalyst is prepared as follows: the co-expression recombinant bacterium containing the phosphoramidion dehydrogenase gene and the alcohol dehydrogenase mutant gene is inoculated into LB liquid medium containing 50 μg / ml ampicillin resistance, and cultured at 37 ℃, 200 rpm for 12 h, then inoculated into fresh LB liquid medium containing 50 μg / mL ampicillin resistance at a volume concentration of 2% inoculation amount, and cultured at 37 ℃, 180 rpm until the OD600 of the bacterium reaches 0.6-0.8, then 12 μg / mL IPTG is added, and the culture is induced at 28 ℃ for 12 h, then centrifuged at 4 ℃, 8000 rpm for 10 min, the supernatant is discarded, the precipitate is washed twice with pH 7.5, 20 mM sodium phosphate buffer, and wet bacterium is obtained.

Images