Gene mining method combining functional sequence and structure simulation, NADH-preferring glufosinate dehydrogenase mutant and its application

Abstract

The invention discloses a gene mining method combining functional sequence and structure simulation, a NADH-preferring glufosinate ammonium dehydrogenase mutant and application. The gene mining method comprises: (1) analyzing the characteristic sequence that NADH-type glutamate dehydrogenase should have; (2) searching a gene library according to the characteristic sequence; (3) performing cluster analysis and protein structure simulation on the searched genes; (4) selecting genes with high gene polymerization degree and protein structure similar to the known glufosinate ammonium dehydrogenase structure as candidate genes. A wild-type glufosinate ammonium dehydrogenase derived from Lysinibacillus composti with an amino acid sequence as shown in SEQ ID No. 2 is obtained through gene mining, and a NADH-preferring glufosinate ammonium dehydrogenase mutant is obtained through mutation screening, wherein the mutation site is selected from one of the following: (1) A144G-V375F-M91A; (2) A144G-V345A-M91A; (3) A144G, and the mutant enzyme can use the cheap coenzyme NAD for catalytic reaction.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a gene mining method combining functional sequence and structure simulation, an NADH-preferring glufosinate-ammonium dehydrogenase mutant and applications. Background Art

[0002] The chemical name of phosphinothricin (also called glufosinate, abbreviated as PPT) is 2-amino-4-[hydroxy(methyl)phosphonyl]-butyric acid. It is the world's second largest genetically modified crop tolerance herbicide. It was developed and produced by Hoechst (now owned by Bayer after several mergers). It is also known as glufosinate ammonium, Basta, Buster, etc. It belongs to the phosphonic acid herbicide, a non-selective (lethal) contact herbicide and a glutamine synthetase inhibitor.

[0003] There are two optical isomers of glufosinate ammonium, L-glufosinate ammonium and D-glufosinate ammonium. However, only the L-type has physiological activity and is easily decomposed in the soil. It is less toxic to humans and animals, has a wide spectrum of weed control, and is less destructive to the environment.

[0004] At present, the glufosinate sold on the market is generally a racemic mixture. If the glufosinate product can be used in the form of pure optical isomers of L-configuration, the amount of glufosinate used can be significantly reduced, which is of great significance for improving atom economy, reducing use costs, and alleviating environmental pressure.

[0005] There are three main methods for preparing chiral pure L-glufosinate: chiral separation, chemical synthesis and biocatalysis. The biocatalytic method for producing glufosinate has the advantages of strict stereoselectivity, mild reaction conditions and high yield, and is the dominant method for producing L-glufosinate. It mainly includes the following three categories:

[0006] 1) Using L-phosphinothricin derivatives as substrates, it is obtained by direct enzymatic hydrolysis. The main advantages are high conversion rate and high product ee value, but expensive and difficult to obtain chiral raw materials are required as precursors, which increases the cost and is not conducive to industrial production. For example, the simplest method for preparing L-phosphinothricin by biological method is to use protease to directly hydrolyze bialaphos. Bialaphos is a natural tripeptide compound. Under the catalysis of protease, bialaphos removes 2 molecules of L-alanine to generate L-phosphinothricin.

[0007] 2) Using the precursor of racemic glufosinate as a substrate, it is obtained by selective enzyme 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 cause waste of raw materials. For example, Cao et al. (Cao C-H, Cheng F, Xue YP, Zheng YG (2020) Efficient synthesis of L-phosphinothricin using a novel aminoacylase mined from Stenotrophomonas maltophilia. Enzyme and Microbial Technology 135 doi: 10.1016 / j.enzmictec.2019.109493) used a new aminoacylase derived from Stenotrophomonas maltophilia to chirally resolve N-acetyl-PPT to obtain L-glufosinate. Using whole cells for catalysis, the conversion rate was >49% within 4 hours, and optically pure L-PPT (>99.9% ee) was obtained.

[0008] 3) Using α-keto acid-2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid (PPO) as substrate, it is obtained by enzymatic asymmetric synthesis, and the enzymes involved mainly include transaminase and glufosinate dehydrogenase. Bartsch (Bartsch K (2005) Process for the preparation of 1-phosphinothrcine by enzymatic transamination with aspartate. US Patent no. US6936444B1) et al. used PPO as substrate, L-aspartic acid as amino donor, and used transaminase with specific enzyme activity for PPO and L-aspartic acid screened and separated from soil microorganisms for catalysis. When the substrate concentration was 552mM, the reaction was carried out at a very high temperature (80℃) for 4 hours, and the conversion rate still reached 52%, and the space-time yield was 4.5g L-PPT / g·L -1 ·d -1 However, there are two major drawbacks in using transaminase to prepare L-phosphinothricin. First, this is a reversible reaction, and the raw material PPO cannot be completely converted into L-PPT, and the conversion rate cannot reach 100%. Second, in order to make the reversible reaction proceed in the direction of generating L-PPT, at least 2 times more L-aspartic acid needs to be added as an amino donor. Excessive aspartic acid brings great trouble to the separation of L-PPT.

[0009] Among the many enzymatic synthesis routes of glufosinate ammonium, the keto carbonyl group of the keto acid intermediate is a potentially chiral functional group, which can construct a chiral center through enzymatic synthesis. The keto acid route has also become a suitable route for the industrial development and production of L-glufosinate ammonium because of its cheap and readily available raw materials and the avoidance of the use of highly toxic cyanide.

[0010] Amino acid dehydrogenase (EC 1.4.1.X, AADH) is a type of amino acid dehydrogenase that can reversibly deaminate amino acids to produce corresponding keto acids. The reaction requires the participation of nucleoside coenzymes (NAD + ), is widely used in the synthesis of natural and unnatural α-amino acids. According to its substrate specificity, it can be divided into glutamate dehydrogenase, leucine dehydrogenase, alanine dehydrogenase, valine dehydrogenase, etc. If it shows a certain activity towards the glufosinate precursor, it can be called "phosphinothricin dehydrogenase (PPTDH)".

[0011] Glucose dehydrogenase (EC1.1.1.47, GDH) is an important auxiliary enzyme in biocatalysis and is used for the regeneration cycle of the coenzyme NADH in redox catalytic reactions.

[0012] Although the enzyme activity of NADPH-preferring glufosinate ammonium dehydrogenase is slightly higher than that of NADH-preferring glufosinate ammonium dehydrogenase (more than 50 times), the market price of NADPH (about 20,000 yuan per ton) is 5 times that of NADH. In practical applications, the exogenous addition of NADPH will significantly increase the cost of the product L-glufosinate ammonium. Therefore, the invention of a NADH-preferring high-activity glufosinate ammonium dehydrogenase, combined with the exogenous addition of cheap NADH or NAD, has a good application prospect. Summary of the invention

[0013] The present invention aims to solve the problem that the existing glufosinate ammonium dehydrogenase has low asymmetric amination reduction activity for 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid, and provides an NADH-preferring glufosinate ammonium dehydrogenase mutant and a genetically recombinant bacterium and a crude enzyme solution thereof using the NADH-preferring glufosinate ammonium dehydrogenase mutant as a biocatalyst for the chiral biosynthesis of L-glufosinate ammonium.

[0014] A NADH-preferring glufosinate dehydrogenase mutant is obtained by mutating a wild-type glufosinate dehydrogenase derived from Lysinibacillus composti, wherein the amino acid sequence of the wild-type glufosinate dehydrogenase is shown in SEQ ID No. 2, and the mutation site of the NADH-preferring glufosinate dehydrogenase mutant is selected from the following:

[0015] (1)A144G-V375F-M91A;

[0016] (2)A144G-V345A-M91A;

[0017] (3)A144G.

[0018] The present invention further discloses a gene encoding the NADH-preferring glufosinate-ammonium dehydrogenase mutant.

[0019] The present invention further discloses a genetic engineering bacterium, comprising a host cell and a target gene transferred into the host cell, wherein the target gene comprises the gene.

[0020] Preferably, in the genetic engineering, the target gene also includes a gene encoding glucose dehydrogenase. In this way, the co-expression of the glufosinate ammonium dehydrogenase mutant and the glucose dehydrogenase can be performed. More preferably, the GenBank accession number corresponding to the gene sequence encoding glucose dehydrogenase is KM817194.1.

[0021] The present invention also discloses the use of the NADH-preferring glufosinate-ammonium dehydrogenase mutant, the gene or the genetically engineered bacteria in the preparation of L-glufosinate-ammonium.

[0022] The present invention also discloses a method for preparing L-phosphinothricin ammonium, which uses 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid as a substrate, and uses a catalyst to catalyze a substrate reaction in the presence of an inorganic amino donor, a coenzyme regeneration circulation system and a corresponding auxiliary substrate to obtain L-phosphinothricin ammonium;

[0023] The catalyst is one of the following:

[0024] (1) the NADH-preferring glufosinate dehydrogenase mutant;

[0025] (2) A genetically engineered bacterium capable of expressing the NADH-preferring glufosinate-ammonium dehydrogenase mutant or a crude enzyme solution obtained by lysing the genetically engineered bacterium.

[0026] Preferably, the coenzyme regeneration circulation system uses a coenzyme regeneration circulation system that prefers glucose dehydrogenase, formate dehydrogenase or alcohol dehydrogenase.

[0027] The present invention also provides a gene mining method combining functional sequence and structure simulation, comprising the following steps:

[0028] (1) Analyze the characteristic sequences that NADH-type glutamate dehydrogenase should have, and the characteristic sequences include:

[0029] (1.1) Protein size: The candidate protein is 300-500 amino acids long.

[0030] (1.2) Two necessary characteristic sequences of glufosinate dehydrogenase: the first segment is GGGKGG; the second segment is one of VVTG, FVTG, VLTG, VFTG, FITG, FFTG, VVFG, FVFTG, VLFG, VFFG, FLFG, FFFG,

[0031] (1.3) NADH binding characteristic sequence: GXRVXXG, where the X position represents any amino acid;

[0032] (2) Searching the gene library based on characteristic sequences;

[0033] (3) performing cluster analysis and protein structure simulation on the genes obtained by searching in step (2);

[0034] (4) Genes with high gene aggregation and protein structures similar to the known glufosinate dehydrogenase structures were selected as candidate genes.

[0035] When searching the gene library in step (2), the above characteristic sequences were used to perform iterative PSI-BLAST search and cluster analysis on the NCBI microbial genome resources and then the NCBI NR sequence database (containing about 100 million protein genes), and 15 clusters were obtained. The polymerization degrees of the 15 clusters were 0.82, 0.76, 0.71, 0.66, 0.65, 0.58, 0.43, 0.42, 0.40, 0.39, 0.38, 0.34, 0.33, 0.32, and 0.30, respectively (arranged from high to low).

[0036] Step (3) 36 representative proteins (6 proteins in each cluster) from the 6 clusters with the highest polymerization degree were selected for 3D structural simulation (Tencent tFold protein structure server can be used), and the simulated structure was compared with the known glufosinate ammonium dehydrogenase (PDB database number: 1LEH, 1BW9, 5IJZ). Among them, the structural standard deviation (RMSD) of LcGDH from Lysinibacillus composti and these three known glufosinate ammonium dehydrogenases was less than Therefore, LcGDH gene was selected as the starting gene (amino acid sequence shown in SEQ ID No.2).

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The glufosinate dehydrogenase mutant of the present invention has better catalytic efficiency. When catalyzing the reaction with 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid as substrate, the conversion rate is much higher than that of the wild-type enzyme, and the PPO yield is also greatly improved.

[0039] (2) The present invention utilizes a mutant of glufosinate ammonium dehydrogenase and a coenzyme circulation system to catalyze the reduction of 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid to L-glufosinate ammonium, thereby achieving asymmetric synthesis of L-glufosinate ammonium.

[0040] (3) The present invention can directly use 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid as a substrate for asymmetric synthesis, without the need for expensive chemical resolution reagents or the synthesis of glufosinate-ammonium derivatives. The inexpensive coenzyme NAD is used for catalytic reaction. Compared with the previous production process using the coenzyme NADP, the cost is significantly reduced and has a high prospect for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the reaction of preparing L-phosphinothricin ammonium by coupling asymmetric amination reduction of the intermediate 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid with a glufosinate dehydrogenase mutant and glucose dehydrogenase.

[0042] Figure 2 This is the SDS-PAGE image of the LcGDH and EsGDH dual enzyme coupling in Example 3. Lane 1: standard protein molecular weight; Lane 2: recombinant E. coli cells containing EsGDH; Lane 3: recombinant E. coli cells without EsGDH expression.

[0043] Figure 3 The reaction process diagram of the asymmetric amination reduction of 2-carbonyl-4-(hydroxymethylphosphonyl)-butyrate by the glufosinate dehydrogenase mutant LcGDH (A144G) coupled with glucose dehydrogenase. 1 mM NAD was added to the reaction system. + Coenzyme.

[0044] Figure 4 The reaction process diagram of the asymmetric amination reduction of 2-carbonyl-4-(hydroxymethylphosphonyl)-butyrate by the glufosinate dehydrogenase mutant LcGDH (A144G-V345A-M91A) coupled with glucose dehydrogenase. 1 mM NAD was added to the reaction system. + Coenzyme.

[0045] Figure 5 The reaction process diagram of the asymmetric amination reduction of 2-carbonyl-4-(hydroxymethylphosphonyl)-butyrate by the glufosinate dehydrogenase mutant LcGDH (A144G-V375F-M91A) coupled with glucose dehydrogenase. 1 mM NAD was added to the reaction system. + Coenzyme. DETAILED DESCRIPTION

[0046] Example 1

[0047] Step 1: Analyze the characteristic sequences that NADH-type glutamate dehydrogenase should have: ① Protein size: the length of the candidate protein (300-500 amino acids), ② Two necessary characteristic sequences of glufosinate dehydrogenase: the first segment is GGGKGG, where the X position represents any amino acid, and the second segment is one of VVTG, FVTG, VLTG, VFTG, FITG, FFTG, VVFG, FVFTG, VLFG, VFFG, FLFG, FFFG, ③ The characteristic sequence of NADH binding: GXRVXXG.

[0048] Step 2: Search the gene library: Use the above characteristic sequences to perform iterative PSI-BLAST search and cluster analysis on the NCBI microbial genome resources and then the NCBINR sequence database (containing about 100 million protein genes), and obtain 15 clusters. The polymerization degrees of the 15 clusters are 0.82, 0.76, 0.71, 0.66, 0.65, 0.58, 0.43, 0.42, 0.40, 0.39, 0.38, 0.34, 0.33, 0.32, and 0.30 (arranged from high to low), respectively.

[0049] Step 3: Select 36 representative proteins (6 proteins in each cluster) from the 6 clusters with the highest polymerization degree for 3D structural simulation (using Tencent tFold protein structure server), and compare the simulated structure with the known glufosinate dehydrogenases (1LEH, 1BW9, 5IJZ). Among them, the structural standard deviation (RMSD) of LcGDH from Lysinibacillus composti and these three known glufosinate dehydrogenases is less than Therefore, LcGDH gene was selected as the starting gene (amino acid sequence shown in SEQ ID No.2).

[0050] Example 2: Construction and screening of a glufosinate dehydrogenase mutant library

[0051] The LcGDH amino acid sequence of Example 1 was codon optimized (the nucleotide sequence after codon optimization is shown in SEQ ID No. 1), and the LcGDH gene obtained by gene synthesis by Hangzhou Qingke Biotechnology Co., Ltd. was cloned into NcoI of MCS1 (multiple cloning site 1) of plasmid pETDuet to construct a recombinant expression vector pETDuet-LcGDH, retaining the His-Tag gene of the plasmid itself, and transformed into Escherichia coli E. coli BL21 (DE3), and sent to Hangzhou Qingke Biotechnology Co., Ltd. to synthesize wild-type glufosinate dehydrogenase engineered bacteria E. coli BL21 (DE3) / pETDuet-LcGDH.

[0052] Then, the glucose dehydrogenase gene EsGDH (nucleotide sequence shown in GenBank accession number KM817194.1) was cloned from Exiguobacterium sibiricum ZJBML01011 and constructed into the NdeI of MCS2 (multiple cloning site 2) of the recombinant expression vector pETDuet-LcGDH using Vazyme's OneStep Cloning Kit to obtain the co-expression vector pETDuet-LcGDH-EsGDH, which was transformed into Escherichia coli BL21 (DE3) to obtain the wild-type glufosinate dehydrogenase and glucose dehydrogenase co-expression strain E. coli BL21 (DE3) / pETDuet-LcGDH-EsGDH. Figure 1 The schematic diagram of the reaction of the strain asymmetric amination reduction of the intermediate product 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid to prepare L-phosphinothricin ammonium.

[0053] The preparation of the glufosinate dehydrogenase mutant library was achieved by 4 rounds of site-directed saturation mutagenesis. The primers were designed as shown in Table 1 (wherein, in the degenerate bases involved in the primer sequence, N represents A, C, G or T; K represents G or T; M represents A or C). The vector pETDuet-LcGDH-EsGDH was used as a template, and the sequence (A144) in Table 1 was used as a primer. After saturation mutation PCR, it was digested with DpnI and transformed into E. coli BL21 (DE3). The LB plate containing 50 μg / mL ampicillin was coated, and the strains were picked for screening of dominant strains using a high-throughput screening method. The second, third, and fourth rounds of site-directed saturation mutagenesis were repeated according to the above steps to screen dominant strains with higher activity.

[0054] Table 1 Primer design for site-directed saturation mutagenesis of glufosinate dehydrogenase

[0055]

[0056] The mutation PCR system (100 μL) was: 25 μL of 2x Phanta Max buffer, 1 μL of dNTPs, 1 μL of each mutation upper and lower primers, 1 μL of template, 0.5 μL of Phanta Super-Fidelity DNA polymerase, and ddH2O to 50 μL. The PCR conditions were: 95°C pre-denaturation for 5 minutes, 30 cycles of 90°C for 30 seconds, 62°C for 30 seconds, 72°C for 7 minutes, and a final extension at 72°C for 5 minutes. The PCR results were verified by DNA agarose gel electrophoresis, and the PCR products were digested with DpnI enzyme templates, inactivated at 37°C for 1 hour, 220 rpm, and 65°C for 1 minute. The PCR products were heat-shocked and transformed, and E. coli BL21 (DE3) was activated, placed at 37°C and 220 rpm for 1 hour, and spread on LB plates containing 50 μg / mL ampicillin resistance, and incubated at 37°C overnight. The obtained mutants were screened for dominant mutants according to the method of Example 3, and the obtained dominant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing confirmation and preservation. The following co-expression strains with mutant glufosinate dehydrogenase genes were screened:

[0057] (1) E. coli BL21(DE3) / pETDuet-1-LcGDH(A144G)-EsGDH: The expressed glufosinate dehydrogenase (LcGDH) has the mutation A144G.

[0058] (2) E. coli BL21(DE3) / pETDuet-1-LcGDH(A144G-V345A-M91A)-EsGDH: The expressed glufosinate dehydrogenase (LcGDH) has three mutations in amino acid residues, namely A144G, V345A and M91A.

[0059] (3) E. coli BL21(DE3) / pETDuet-1-LcGDH(A144G-V375F-M91A)-EsGDH: The expressed glufosinate dehydrogenase (LcGDH) had three mutations in amino acid residues, namely A144G, V375F and M91A.

[0060] Example 3: Induced expression of engineered bacteria of phosphinothricin dehydrogenase mutant

[0061] The wild-type glufosinate ammonium dehydrogenase and glucose dehydrogenase co-expression strain E. coli BL21 (DE3) / pETDuet-1-LcGDH-EsGDH and the glufosinate ammonium dehydrogenase mutant and glucose dehydrogenase co-expression strain in Example 2 were used:

[0062] E.coli BL21(DE3) / pETDuet-1-LcGDH(A144G)-EsGDH、

[0063] E.coli BL21(DE3) / pETDuet-1-LcGDH(A144G-V345A-M91A)-EsGDH,

[0064] E.coli BL21(DE3) / pETDuet-1-LcGDH(A144G-V375F-M91A)-EsGDH,

[0065] The cells were inoculated into LB liquid culture medium containing a final concentration of 50 μg / mL ampicillin, and cultured at 37°C for 8 hours. The cells were inoculated into fresh LB liquid culture medium containing a final concentration of 50 μg / mL ampicillin at an inoculum concentration of 2% by volume, and cultured at 37°C and 180 rpm for 2 hours. IPTG with a final concentration of 0.1 mM was then added to the culture medium. After culturing at 18°C ​​for 14 hours, the cells were centrifuged at 4°C and 8000 rpm for 10 minutes to obtain the corresponding wet bacteria.

[0066] The cells obtained above produce corresponding proteins, which can be used for the preparation of pure protein enzyme solution, and can also be used for the asymmetric amination of 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid with crude enzyme solution to prepare L-phosphinothricin ammonium. Figure 2 This is a SDS-PAGE image of LcGDH and EsGDH dual enzyme coupling. Lane 1: standard protein molecular weight; Lane 2: recombinant E. coli cells containing EsGDH; Lane 3: recombinant E. coli cells without EsGDH expression.

[0067] Example 4: Screening of mutation library

[0068] The wild-type glufosinate dehydrogenase prepared by the method of Example 3 was co-expressed with glucose dehydrogenase wet bacteria or the glufosinate dehydrogenase mutant was co-expressed with glucose dehydrogenase wet bacteria as a catalyst, the intermediate product 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid was used as a substrate, glucose was used as a coenzyme regeneration substrate, ammonium sulfate was added, a trace amount of NADH was added exogenously, and pH 7.4, 100mM phosphate buffer was used as a reaction medium to form a 1mL reaction system, the catalyst dosage was 20g / L based on the final concentration of the wet bacteria, the final concentration of the substrate was 100mM, the final concentration of glucose was 125mM, the final concentration of ammonium sulfate was 150mM, 35°C, 600 rpm for 5min, 50μL of the reaction solution was taken, 5μL of hydrochloric acid was added to terminate the reaction, the reaction solution was diluted 100 times, 200μl of the diluted reaction solution + 400μL of a derivatization reagent (containing 15mM o-phthalaldehyde, 15mM The mixture was derivatized with N-acetyl-L-cysteine ​​(pH=9.8 boric acid buffer) at 30°C for 5 min, and then 400 μL ultrapure water was added to make up to 1 mL, and the mixture was centrifuged at 12000 rpm for 1 min. The supernatant was taken and passed through a 0.22 μM microfiltration membrane. The filtrate was collected as a liquid sample, and HPLC was used to detect 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid, L-phosphinothricin, D-phosphinothricin and ee values. The concentration of the product L-phosphinothricin and the enantiomeric excess ee were used as indicators to screen the dominant mutants. The experimental results are shown in Table 2.

[0069] 2-Carbonyl-4-(hydroxymethylphosphonyl)-butyric acid Liquid phase detection conditions: Chromatographic column C18 (4.6×250 mm, Acchrom, China) column, mobile phase acetonitrile: 50 mM diammonium phosphate solution (pH 3.8, containing 10% tetrabutylammonium hydroxide) volume ratio of 12:88. Flow rate 1 mL / min, detection wavelength 232 nm, injection volume 10 μL, column temperature 30 ° C, 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid retention time: 9.7 minutes.

[0070] Glufosinate liquid phase detection conditions: chromatographic column C18 (4.6×250 mm, Acchrom, China) column, mobile phase methanol: 0.05 M ammonium acetate (pH 5.7) volume ratio of 10:90, flow rate 1.0 mL / min, detection wavelength E x =340nm, E m =450nm, injection volume 10μL, column temperature 35°C. The retention times of L-phosphinothricin ammonium and D-phosphinothricin ammonium were 10.6 minutes and 12.6 minutes respectively.

[0071] Table 2 Catalytic performance and stereoselectivity of wild-type LcGDH and its mutant whole cells

[0072]

[0073] Note: A144G-V375F-M91A means that the amino acid residue A at position 144 of LcGDH mutated to G, the amino acid residue V at position 375 mutated to F, and the amino acid residue M at position 91 mutated to A.

[0074] Example 4: Purification of wild-type phosphinothricin dehydrogenase and its mutants

[0075] The glufosinate dehydrogenase engineered bacteria and the dominant mutant constructed in Example 1 were prepared into corresponding wet cells according to the method of Example 2. 0.2 g of the wet cells of the wild-type glufosinate dehydrogenase engineered bacteria and the glufosinate dehydrogenase mutant engineered bacteria were suspended in 10 ml of binding buffer (pH 7.4, 100 mM sodium phosphate buffer containing 0.3 M NaCl), ultrasonically disrupted for 15 minutes (ice bath, power 400 W, disruption for 1 second, pause for 5 seconds), centrifuged at 4°C and 12,000 rpm for 20 minutes, and the supernatant was taken as a sample. The protein was purified using a Ni affinity column (1.6×10 cm, Bio-Rad, USA) with the following specific steps: ① Equilibrate the Ni column with 5 column volumes of binding buffer (pH 7.4, 50 mM sodium phosphate buffer containing 0.3 M NaCl) until the baseline is stable; ② Load the sample at a flow rate of 1 mL / min with a loading amount of 25-40 mg / mL protein to allow the target protein to adsorb on the Ni column; ③ Wash the impurities with 6 column volumes of buffer A (pH 7.4, 50 mM sodium phosphate buffer containing 0.3 M NaCl and 30 mM imidazole) at a flow rate of 1 mL / min until the baseline is stable; ④ Elute with buffer B (pH 7.4, 50 mM sodium phosphate buffer containing 0.3 M NaCl and 500 mM imidazole) at a flow rate of 1 mL / min to collect the target protein. The target protein was dialyzed overnight in pH 7.4, 20 mM phosphate buffer, and the retentate was collected to obtain 10 ml of pure wild-type glufosinate dehydrogenase enzyme and 10 ml of pure glufosinate dehydrogenase mutant enzyme, respectively; ⑤ The Ni column was rinsed with 5 column volumes of binding buffer (pH 8.0, 50 mM sodium phosphate buffer containing 0.3 M NaCl) until the baseline was stable, and the Ni column was preserved with 5 column volumes of ultrapure water containing 20% ​​ethanol.

[0076] Example 5: Determination of the specific enzyme activity of wild-type glufosinate dehydrogenase and its mutants

[0077] The enzyme activity unit (U) is defined as: the amount of enzyme required to generate 1 μmol of L-phosphinothion per minute at 35°C and pH 7.4, U. The specific enzyme activity is defined as the number of activity units per milligram of enzyme protein, U / mg.

[0078] Standard conditions for enzyme activity detection: 100 mM 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid, 10 mM NADH, 0.02 μg / μL enzyme solution (prepared by the method in Example 4), 30° C., pH 7.4, 600 rpm for 10 minutes, and HPLC detection and analysis using the method in Example 3.

[0079] The protein concentration was determined using a BCA protein assay kit (Nanjing KeyGen Biotechnology Development Co., Ltd., Nanjing), as shown in Table 3.

[0080] Table 3 Specific enzyme activity of wild-type glufosinate dehydrogenase and its mutants

[0081]

[0082]

[0083] a : The initial enzyme activity of each wild-type glufosinate dehydrogenase under standard conditions was assigned as 100%.

[0084] Example 6: Determination of kinetic parameters of wild-type glufosinate dehydrogenase and its mutants

[0085] The kinetic parameters of wild-type glufosinate dehydrogenase and its mutants were investigated, with 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid as the substrate, the concentration was set to 2-10 mM (2, 4, 6, 8, 10 mM), sufficient coenzyme (10 mM) was added, and 100 μL of pure enzyme solution (collected by the method in Example 4) was added.

[0086] The reaction system was selected as 500 μL. The pure enzyme solution collected in Example 4 was diluted 10 times with pH 7.4, 100 mM phosphate buffer, and 100 μL was taken. Substrate and exogenous coenzyme NADPH were added. pH 7.4, 100 mM phosphate buffer was used as the reaction medium. The reaction was carried out at 35° C. and 600 rpm for 10 min. The reaction solution was sampled and the L-phosphinothricin concentration was detected by HPLC (same as in Example 3).

[0087] K can be calculated by double reciprocal plotting cat 、v max , K m , the results are shown in Table 4. By comparing k cat and K m It can be found that the Km value of LcGDH for 2-carbonyl-4-(hydroxymethylphosphonyl)-butyrate is 8.56mM, and the other mutants have an increasing affinity for 2-carbonyl-4-(hydroxymethylphosphonyl)-butyrate. The catalytic efficiency k of the mutant LcGDH-(A144G-V375F-M91A)-EsGDH for 2-carbonyl-4-(hydroxymethylphosphonyl)-butyratecat / K m Reach 169.25mM -1 , maternal parent (k cat / K m =1.15 mM -1 ) increased by 147.17 times.

[0088] Table 4 Comparison of kinetic parameters of parent LcGDH and its mutants

[0089] Enzymes <![CDATA[k cat (s -1 ) a ]]> <![CDATA[K m (mM)]]> <![CDATA[k cat / K m (s -1 *mM)]]> LqCy 9.88 8.56 1.15 LcGDH(A144G) 428.12 4.54 94.30 LcGDH(A144G+V345A+M91A) 533.25 4.22 126.36 LcGDH(A144G+V375F+M91A) 736.22 4.35 169.25

[0090] Example 7: Asymmetric amination reduction of 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid by coupling the glufosinate dehydrogenase mutant LcGDH-A144G with glucose dehydrogenase

[0091] 1 g of wet E. coli BL21 (DE3) / LcGDH (A144G) -EsGDH prepared by the method of Example 2 was resuspended in 40 mL of phosphate buffer (100 mM) at pH 7.4, and 50 mL of reaction system was formed by adding 2-carbonyl-4- (hydroxymethylphosphonyl) butyric acid at a final concentration of 100 mM, glucose at a final concentration of 125 mM, and ammonium sulfate at a final concentration of 125 mM. The reaction was carried out at 35°C and a magnetic stirring speed of 600 rpm, and ammonia water was added to maintain the pH of the reaction solution at 7.4. The formation of the product L-phosphinothricin ammonium and the change of the ee value during the reaction were detected by the liquid phase method shown in Example 3. The reaction progress curve is shown in FIG. Figure 3 As shown in the figure, the product concentration gradually increases with time, the reaction is completed within 6 hours, the substrate conversion rate is greater than 99%, and the product ee value is always maintained above 99.5%.

[0092] Example 8: Asymmetric amination reduction of 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid by glufosinate dehydrogenase mutant LcGDH (A144G-V345A-M91A)-EsGDH coupled with glucose dehydrogenase

[0093] 1 g of wet E. coli BL21 (DE3) / LcGDH-(A144G-V345A-M91A) -EsGDH prepared by the method of Example 2 was resuspended in 40 mL of phosphate buffer (100 mM) at pH 7.4, and 50 mL of reaction system was formed by adding 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid at a final concentration of 100 mM, glucose at a final concentration of 125 mM, and ammonium sulfate at a final concentration of 125 mM. The reaction was carried out at 35°C and a magnetic stirring speed of 600 rpm, and ammonia water was added to maintain the pH of the reaction solution at 7.4. The formation of the product L-phosphinothricin ammonium and the change of the ee value during the reaction were detected by the liquid phase method shown in Example 3. The reaction progress curve is shown in FIG. Figure 4As shown in the figure, the product concentration gradually increases with time, the reaction is completed within 5.5 hours, the substrate conversion rate is greater than 99%, and the product ee value is always maintained above 99.5%.

[0094] Example 9: Asymmetric amination reduction of 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid by glufosinate dehydrogenase mutant LcGDH-(A144G-V375F-M91A)-EsGDH coupled with glucose dehydrogenase

[0095] 1 g of wet E. coli BL21 (DE3) / LcGDH-(A144G-V375F-M91A) -EsGDH prepared by the method of Example 2 was resuspended in 40 mL of phosphate buffer (100 mM) at pH 7.4, and 50 mL of reaction system was formed by adding 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid at a final concentration of 100 mM, glucose at a final concentration of 125 mM, and ammonium sulfate at a final concentration of 125 mM. The reaction was carried out at 35°C and a magnetic stirring speed of 600 rpm, and ammonia water was added to maintain the pH of the reaction solution at 7.4. The formation of the product L-phosphinothricin ammonium and the change of the ee value during the reaction were detected by the liquid phase method shown in Example 3. The reaction progress curve is shown in FIG. Figure 5 As shown in the figure, the product concentration gradually increases with time, the reaction is completed within 5 hours, the substrate conversion rate is greater than 99%, and the product ee value is always maintained above 99.5%. Sequence Listing <110> Zhejiang University of Technology <120> Gene mining method combining functional sequence and structure simulation, NADH-preferring glufosinate dehydrogenase mutant and its application <160> twenty four <170> SIPOSequenceListing 1.0 <210> 1 <211> 1242 <212> DNA <213> Artificial Sequence <400> 1 atggcagaaa acctgaactt atttacgagc acccaggagg ttgtgaaaga agcgctgaac 60 aaactgggtt atgatgaggc aatgtacgaa ctgctgaaag aaccgctgcg cctgctgaaa 120 gtgcgtattc ctgtgaagat ggacgatggc accacacagg tgtttacggg ttatcgcgca 180 caacattccg atgcagtagg tcccaccaaa ggtggcgtgc gttttcatcc tatggtttct 240 gaaggaag ttaaagcact gagcatgtgg atgaccctga agtgcgggat tgtagatctg 300 ccttatggtg gtggtaaagg tggcattatt tgtgatccgc gtcagatgag catgggggaa 360 ttagaacgtc tgagccgtgg atatgttcgg gcaattagtc agattgttgg gccgaccaaa 420 gatataccgg caccggatgt ttttaccaat gcacaaatta tggcatggat gatggatgag 480 tatagccgta tggatgaatt taatagtccg ggttttataa ccggtaaacc tctggtgctg 540 ggcggtagta aagggcgtga tcgggcgacg gcagaaggtg ttacgattgt tattcaggag 600 gcagcaaaaa agaaatat cgatatcaaa ggtgcacgcg ttgttatca agggttcggt 660 aatgccggca gttttttagc aaagtttatg agtgatctgg gcgcgaaggt tataggaata 720 agtgatgcat acggggccct gcacgatccg aatggtttag atattgatta tctgctggac 780 agacgtgata gttttggtac cgttaccacg ctgtttgaaa ataattac gaatcaggag 840 ctgctggaac tggattgtga tattctggtg ccggccgcaa ttgagaatca gattacggca 900 gaaaatgcac ataatattaa ggcaaccata gttgtggaag cagcgaacgg cccaaccacc 960 tctgaagcaa ccaaaattct gaccgaacgt ggtattctgt tagtgccaga cgttttagca 1020 agcgcaggtg gggttacagt tagctacttt gagtgggttc aaaataatat gggctattac 1080 tgggaagaag aagaggttca agaaaaactg tacaaaaaaa tggtggatag ctttgaagca 1140 gtatatacaa ccgcaaccac gcgcaatata gatatgcgtc tggcagcgta tatggtggga 1200 gtgagaagaa cagcagaagc gagccgtttc cggggctggg tg 1242 <210> 2 <211> 414 <212> PRT <213> Lysinibacillus composti <400> 2 Met Ala Glu Asn Leu Asn Leu Phe Thr Ser Thr Gln Glu Val Val Lys 1 5 10 15 Glu Ala Leu Asn Lys Leu Gly Tyr Asp Glu Ala Met Tyr Glu Leu Leu 20 25 30 Lys Glu Pro Leu Arg Leu Leu Lys Val Arg Ile Pro Val Lys Met Asp 35 40 45 Asp Gly Thr Thr Gln Val Phe Thr Gly Tyr Arg Ala Gln His Ser Asp 50 55 60 Ala Val Gly Pro Thr Lys Gly Gly Val Arg Phe His Pro Met Val Ser 65 70 75 80 Glu Asp Glu Val Lys Ala Leu Ser Met Trp Met Thr Leu Lys Cys Gly 85 90 95 Ile Val Asp Leu Pro Tyr Gly Gly Gly Lys Gly Gly Ile Ile Cys Asp 100 105 110 Pro Arg Gln Met Ser Met Gly Glu Leu Glu Arg Leu Ser Arg Gly Tyr 115 120 125 Val Arg Ala Ile Ser Gln Ile Val Gly Pro Thr Lys Asp Ile Pro Ala 130 135 140 Pro Asp Val Phe Thr Asn Ala Gln Ile Met Ala Trp Met Met Asp Glu 145 150 155 160 Tyr Ser Arg Met Asp Glu Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys 165 170 175 Pro Leu Val Leu Gly Gly Ser Lys Gly Arg Asp Arg Ala Thr Ala Glu 180 185 190 Gly Val Thr Ile Val Ile Gln Glu Ala Ala Lys Lys Arg Asn Ile Asp 195 200 205 Ile Lys Gly Ala Arg Val Val Ile Gln Gly Phe Gly Asn Ala Gly Ser 210 215 220 Phe Leu Ala Lys Phe Met Ser Asp Leu Gly Ala Lys Val Ile Gly Ile 225 230 235 240 Ser Asp Ala Tyr Gly Ala Leu His Asp Pro Asn Gly Leu Asp Ile Asp 245 250 255 Tyr Leu Leu Asp Arg Arg Asp Ser Phe Gly Thr Val Thr Thr Leu Phe 260 265 270 Glu Asn Thr Ile Thr Asn Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile 275 280 285 Leu Val Pro Ala Ala Ile Glu Asn Gln Ile Thr Ala Glu Asn Ala His 290 295 300 Asn Ile Lys Ala Thr Ile Val Val Glu Ala Ala Asn Gly Pro Thr Thr 305 310 315 320 Ser Glu Ala Thr Lys Ile Leu Thr Glu Arg Gly Ile Leu Leu Val Pro 325 330 335 Asp Val Leu Ala Ser Ala Gly Gly Val Thr Val Ser Tyr Phe Glu Trp 340 345 350 Val Gln Asn Asn Met Gly Tyr Tyr Trp Glu Glu Glu Glu Val Gln Glu 355 360 365 Lys Leu Tyr Lys Lys Met Val Asp Ser Phe Glu Ala Val Tyr Thr Thr 370 375 380 Ala Thr Thr Arg Asn Ile Asp Met Arg Leu Ala Ala Tyr Met Val Gly 385 390 395 400 Val Arg Arg Thr Ala Glu Ala Ser Arg Phe Arg Gly Trp Val 405 410 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <400> 3 Gly Gly Gly Lys Gly Gly 1 5 <210> 4 <211> 4 <212> PRT <213> Artificial Sequence <400> 4 Val Val Thr Gly 1 <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <400> 5 Phe Val Thr Gly 1 <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <400> 6 Val Leu Thr Gly 1 <210> 7 <211> 4 <212> PRT <213> Artificial Sequence <400> 7 Val Phe Thr Gly 1 <210> 8 <211> 4 <212> PRT <213> Artificial Sequence <400> 8 Phe Ile Thr Gly 1 <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <400> 9 Phe Phe Thr Gly 1 <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <400> 10 Val Val Phe Gly 1 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <400> 11 Phe Val Phe Thr Gly 1 5 <210> 12 <211> 4 <212> PRT <213> Artificial Sequence <400> 12 Val Leu Phe Gly 1 <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <400> 13 Val Phe Phe Gly 1 <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <400> 14 Phe Leu Phe Gly 1 <210> 15 <211> 4 <212> PRT <213> Artificial Sequence <400> 15 Phe Phe Phe Gly 1 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (2)..(2) <223> The 'Xaa' at location 2 stands for any kind of Amino acid residue. <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 2 stands for any kind of Amino acid residue. <220> <221> UNSURE <222> (6)..(6) <223> The 'Xaa' at location 2 stands for any kind of Amino acid residue. <220> <221> UNSURE <222> (2)..(2) <223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (6)..(6) <223> The 'Xaa' at location 6 stands for Gln, Arg, Pro, or Leu. <400> 16 Gly Xaa Arg Val Xaa Xaa Gly 1 5 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (5)..(5) <223> n stands for a, c, g or t. <220> <221> misc_feature <222> (6)..(6) <223> n stands for a, c, g or t. <220> <221> misc_feature <222> (7)..(7) <223> k stands for g or t. <400> 17 accgnnkccg gatgttttta ccaatgc 27 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (6)..(6) <223> m stands for a or c. <220> <221> misc_feature <222> (7)..(7) <223> n stands for a, c, g or t. <220> <221> misc_feature <222> (8)..(8) <223> n stands for a, c, g or t. <400> 18 tccggmnncg gtatatcttt ggtcggc 27 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (5)..(5) <223> n stands for a, c, g or t. <220> <221> misc_feature <222> (6)..(6) <223> n represents a, c, g or t. <220> <221> misc_feature <222> (7)..(7) <223> k represents g or t. <400> 19 tgggnnkaca gttagctact ttgagtgg 28 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (7)..(7) <223> m represents a or c. <220> <221> misc_feature <222> (8)..(8) <223> n represents a, c, g or t. <220> <221> misc_feature <222> (9)..(9) <223> n represents a, c, g or t. <400> 20 aactgtmnnc ccacctgcgc ttgctaa 27 <210> 21 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (7)..(7) <223> n represents a, c, g or t. <220> <221> misc_feature <222> (8)..(8) <223> n stands for a, c, g or t. <220> <221> misc_feature <222> (9)..(9) <223> k stands for g or t. <400> 21 aaaatgnnkg atagctttga agcagtata 29 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (7)..(7) <223> m stands for a or c. <220> <221> misc_feature <222> (8)..(8) <223> n stands for a, c, g or t. <220> <221> misc_feature <222> (9)..(9) <223> n stands for a, c, g or t. <400> 22 gctatcmnnc atttttttgt acagtttttc 30 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (8)..(8) <223> n represents a, c, g or t. <220> <221> misc_feature <222> (9)..(9) <223> n represents a, c, g or t. <220> <221> misc_feature <222> (10)..(10) <223> k represents g or t. <400> 23 catgtggnnk accctgaagt gcgggatt 28 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (7)..(7) <223> m represents a or c. <220> <221> misc_feature <222> (8)..(8) <223> n represents a, c, g or t. <220> <221> misc_feature <222> (9)..(9) <223> n represents a, c, g or t. <400> 24 cagggtmnnc cacatgctca gtgctttaa 29

Claims

1. A NADH-preferring glufosinate dehydrogenase mutant, characterized in that: Obtained by mutation of wild-type glufosinate ammonium dehydrogenase derived from Lysinibacillus composti, the amino acid sequence of the wild-type glufosinate ammonium dehydrogenase is shown in SEQ ID No. 2, and the mutation site of the NADH-preferring glufosinate ammonium dehydrogenase mutant is selected from the following: (1)A144G-V375F-M91A; (2)A144G-V345A-M91A; (3)A144G.

2. A gene encoding the NADH-preferring glufosinate dehydrogenase mutant as described in claim 1.

3. A genetically engineered bacterium comprising a host cell and a target gene transferred into the host cell, characterized in that: The target gene comprises the gene according to claim 2.

4. The genetic engineering according to claim 3, characterized in that The target gene also includes a gene encoding glucose dehydrogenase.

5. The genetic engineering according to claim 4, characterized in that The GenBank accession number corresponding to the gene sequence encoding glucose dehydrogenase is KM817194.

1.

6. Use of the NADH-preferring glufosinate dehydrogenase mutant of claim 1, the gene of claim 2 or the genetically engineered bacteria of any one of claims 3 to 5 in the preparation of L-glufosinate.

7. A method for preparing L-phosphinothricin ammonium, characterized in that: Using 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid as a substrate, in the presence of an inorganic amino donor, a coenzyme regeneration circulation system and a corresponding auxiliary substrate, a catalyst is used to catalyze the substrate reaction to obtain L-phosphinothricin ammonium; The catalyst is one of the following: (1) The NADH-preferring glufosinate dehydrogenase mutant according to claim 1; (2) A genetically engineered bacterium capable of expressing the NADH-preferring glufosinate-ammonium dehydrogenase mutant of claim 1, or a crude enzyme solution obtained by lysing the genetically engineered bacterium.

8. The preparation method according to claim 7, characterized in that: The coenzyme regeneration circulation system uses a coenzyme regeneration circulation system that prefers glucose dehydrogenase, formate dehydrogenase or alcohol dehydrogenase.

9. A gene mining method combining functional sequence and structure simulation, characterized in that: The following steps are involved: (1) Analyze the characteristic sequences that NADH-type glutamate dehydrogenase should have, and the characteristic sequences include: (1.1) Protein size: The candidate protein is 300-500 amino acids long. (1.2) Two essential characteristic sequences of glufosinate dehydrogenase: the first segment is GGGKGG, wherein the X position represents any amino acid, and the second segment is one of VVTG, FVTG, VLTG, VFTG, FITG, FFTG, VVFG, FVFTG, VLFG, VFFG, FLFG, and FFFG, (1.3) NADH binding characteristic sequence: GXRVXXG; (2) Searching the gene library based on characteristic sequences; (3) performing cluster analysis and protein structure simulation on the genes obtained in step (2); (4) Genes with high gene aggregation and protein structures similar to the known glufosinate dehydrogenase structures were selected as candidate genes.

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