WO2022001038A1 - 一种草铵膦脱氢酶突变体、基因工程菌及一锅法多酶同步定向进化方法 - Google Patents

一种草铵膦脱氢酶突变体、基因工程菌及一锅法多酶同步定向进化方法 Download PDF

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WO2022001038A1
WO2022001038A1 PCT/CN2020/139770 CN2020139770W WO2022001038A1 WO 2022001038 A1 WO2022001038 A1 WO 2022001038A1 CN 2020139770 W CN2020139770 W CN 2020139770W WO 2022001038 A1 WO2022001038 A1 WO 2022001038A1
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dehydrogenase
glufosinate
gene
ammonium
genetically engineered
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French (fr)
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薛亚平
程峰
李举谋
李清华
郑裕国
邹树平
徐建妙
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浙江工业大学
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Definitions

  • the invention relates to the technical field of biochemical industry, in particular to a glufosinate-ammonium dehydrogenase mutant, a genetically engineered bacteria and a one-pot multi-enzyme synchronous directional evolution method.
  • glufosinate-ammonium sold on the market is generally a racemic mixture. If the glufosinate-ammonium product can be used in the form of pure optical isomer of L-configuration, the usage amount of glufosinate-ammonium can be significantly reduced, which is of great significance for improving atom economy, reducing use cost and reducing environmental pressure.
  • the precursor of racemic glufosinate-ammonium is used as the substrate, and it is obtained by selective resolution of the enzyme.
  • the main advantages are that the raw materials are relatively easy to obtain and the catalyst activity is high, but the theoretical yield can only reach 50%, which will cause waste of raw materials.
  • Cao et al. Cao CH, Cheng F, Xue YP, Zheng YG (2020) Efficient synthesis of L-phosphinothricin using a novel aminoacylase mined from Stenotrophomonas maltophilia.
  • PPO ⁇ -keto acid-2-carbonyl-4-(hydroxymethylphosphono)butyric acid
  • the main enzymes involved include transaminase and glufosinate-ammonium dehydrogenation enzymes.
  • Bartsch Bartsch K(2005) Process for the preparation of l-phosphinothrcine by enzymatic transamination with aspartate.US Patent no. US6936444B1) etc. used PPO as a substrate and L-aspartic acid as an amino donor, and used the The isolated transaminase with specific enzymatic activity to PPO and L-aspartate was screened and catalyzed.
  • the ketone carbonyl group of the keto-acid intermediate is a latent chiral functional group, and a chiral center can be constructed through the enzymatic synthesis route.
  • the use of highly toxic cyanide has become a suitable route for the industrial development and production of L-Glufosinate-ammonium.
  • Amino acid dehydrogenase (EC 1.4.1.X, AADH) is a class of amino acid dehydrogenases that can reversibly deaminate amino acids to form corresponding keto acids, and its reaction requires the participation of nucleoside coenzymes (NAD(P) + ) , is widely used in the synthesis of natural and unnatural ⁇ -amino acids. According to its substrate specificity, it can be divided into glutamate dehydrogenase, leucine dehydrogenase, alanine dehydrogenase, valine dehydrogenase and so on. If it shows a certain activity to glufosinate precursor, it can be called “glufosinate dehydrogenase (PPTDH)".
  • PPTDH glufosinate dehydrogenase
  • Glucose dehydrogenase (EC1.1.1.47, GDH) is an important auxiliary enzyme in biocatalysis for the regeneration cycle of coenzyme NAD(P)H in redox catalysis reactions.
  • the invention provides a multi-enzyme one-pot synchronous directional evolution method, which can synchronously directional evolution glufosinate-ammonium dehydrogenase, glucose dehydrogenase or formate dehydrogenase and gene elements between them.
  • the present invention provides glufosinate-ammonium dehydrogenase mutants, high-expressing glucose dehydrogenase or formate dehydrogenase mutants and engineering bacteria comprising the genes of these two enzymes and their application in preparing L-glufosinate-ammonium,
  • the two enzymes in the genetically engineered bacteria undergo a one-pot multi-enzyme synchronous evolution, so that the two enzymes have higher synergistic efficiency in the process of synthesizing L-PPT, and the substrate conversion rate when catalyzing the preparation of L-glufosinate is high.
  • the space-time yield is high and the total turnover number is high. And further shorten the reaction process.
  • a glufosinate-ammonium dehydrogenase mutant the amino acid at position 164 of glufosinate dehydrogenase derived from Pseudomonas fluorescens is mutated from alanine to glycine, and the arginine at position 205 is mutated to Lysine, obtained by mutating threonine at position 332 to alanine, the amino acid sequence is shown in SEQ ID No.1.
  • the present invention further provides a gene encoding the glufosinate-ammonium dehydrogenase mutant.
  • the present invention further provides a genetically engineered bacterium, comprising a host cell and a target gene transferred into the host cell, wherein the target gene comprises the gene.
  • the target gene also includes a gene encoding glucose dehydrogenase or a gene encoding formate dehydrogenase.
  • the genetically engineered bacteria use a double-gene expression vector, the gene of the glufosinate-ammonium dehydrogenase mutant is cloned into one of the multiple cloning site regions, the coding gene of glucose dehydrogenase or the coding gene of formate dehydrogenase Cloning into the second multiple cloning site region.
  • GenBank accession number of the coding gene sequence of the glucose dehydrogenase is KM817194.1, and the base length of the connection between the start codon of the coding gene of the glucose dehydrogenase and the corresponding ribosome binding site on the plasmid is 8 ⁇ 10bp. Among them, the best effect is when the length of the ligation base is 8bp.
  • the coding gene sequence of the formate dehydrogenase is shown in SEQ ID No.3.
  • the formate dehydrogenase encoded by the gene sequence shown in SEQ ID No. 3 is obtained by mutating the 224th histidine into glutamine of the formate dehydrogenase derived from Lactobacillus buchneri.
  • the present invention also provides the application of the glufosinate-ammonium dehydrogenase mutant, the gene or the genetically engineered bacteria in preparing L-glufosinate-ammonium.
  • a preparation method of L-glufosinate-ammonium using 2-carbonyl-4-(hydroxymethylphosphono) butyric acid as a substrate, glucose or ammonium formate as an auxiliary substrate, in an inorganic amino donor, and a coenzyme circulatory system Under the condition of existence, utilize the catalyst to catalyze the reaction of the substrate to obtain L-glufosinate-ammonium;
  • the coenzyme recycling system is a glucose dehydrogenase recycling system or a formate dehydrogenase recycling system;
  • the catalyst is the genetically engineered bacteria, the crude enzyme liquid of the genetically engineered bacteria or the immobilized genetically engineered bacteria,
  • the auxiliary substrate is glucose
  • the coenzyme cycle system is the glucose dehydrogenase cycle system
  • the genetically engineered bacteria contain the formate dehydrogenase
  • the auxiliary substrate is ammonium formate
  • the coenzyme cycle system is formate dehydrogenase cycle system.
  • the inorganic amino donor is ammonium sulfate in the glucose dehydrogenase cycle and ammonium formate in the formate dehydrogenase cycle.
  • the present invention also provides a one-pot multi-enzyme synchronous directed evolution method, comprising:
  • the gene of the regenerating enzyme used in the coenzyme recycling system was also cloned into the double gene expression vector and co-expressed with glufosinate-ammonium dehydrogenase; (2) The double enzyme after coupling and co-expression was constructed by error-prone PCR method. (3) Introduce the cloned expression vector with the double gene into the host cell for cultivation, and each obtained single colony is subjected to the L-glufosinate preparation experiment and screened for those with improved preparation efficiency. (4) Determine the mutation site of the glufosinate-ammonium dehydrogenase gene in the screened strain, perform site-directed saturation mutation on the gene, and select the mutant with the highest activity.
  • glufosinate dehydrogenase gene derived from Pseudomonas fluorescens, NCBI accession number WP_150701510.1, the glucose dehydrogenase gene derived from Exiguobacterium sibiricum, GenBank number ACB59697.1, derived from Lactobacillus buchneri, NCBI Formate dehydrogenase with accession number WP_013726924.1, glufosinate-ammonium dehydrogenase gene was introduced into MCS1 (multiple cloning site 1) on pETDuet-1 plasmid, glucose dehydrogenase or formate dehydrogenase were respectively introduced with glufosinate-ammonium dehydrogenase Phosphine dehydrogenase vector pETDuet-1 plasmid on MCS2 (multiple cloning site 2), then the pETDuet-1 plasmid was transformed into Escherichia
  • Glucose dehydrogenase can catalyze glucose to gluconic acid or formate dehydrogenase can catalyze ammonium formate to generate ammonium ion, carbon dioxide and water, and at the same time convert NADP + into NADPH, thereby forming a coenzyme cycle in the cell ( Figure 4).
  • glufosinate-ammonium dehydrogenase has significantly improved the activity of PPO after the mutant A164G_R205K_T332A, when it is coupled with glucose dehydrogenase or formate dehydrogenase to prepare L-PPT by one-pot method, the overall enzyme of the two enzymes The activity still needs to be improved, so it is necessary to catalyze the generation of L-PPT by simultaneous directed evolution of two enzymes, glufosinate-ammonium dehydrogenase and glucose dehydrogenase or formate dehydrogenase. This method is very novel and will have strong industrial application value.
  • the present invention has the following beneficial effects:
  • the obtained PPTDH-A164G_R205K_T332A and EsGDH are coupled and co-expressed, and at the same time, the ribosome binding site and the start codon of the EsGDH encoding gene are optimized.
  • the base length can further overexpress EsGDH, or mutate LbFDH to obtain mutant LbFDH-H224Q, thereby further improving the conversion rate and space-time yield of one-pot biosynthesis of L-PPT.
  • the L-PPT preparation method provided by the present invention has high conversion rate of raw materials, high yield, and easy separation and purification of products.
  • the L-PPT preparation method provided by the present invention has relatively simple process, high conversion rate of raw materials, conversion rate up to 100%, and high optical purity of products (ee >99%).
  • Figure 1 is a high performance liquid chromatography (HPLC) spectral analysis diagram of a PPO sample.
  • Figure 2 shows the high performance liquid chromatography (HPLC) spectral analysis of D, L-PPT samples.
  • Example 3 is the plasmid map of the PPTDH and EsGDH coupled expression vector obtained in Example 1.
  • FIG. 4 is a schematic diagram of the reaction of preparing L-PPT by using PPTDH and EsGDH double-enzyme coupling to catalyze the asymmetric amination reduction of PPO in Example 1.
  • FIG. 4 is a schematic diagram of the reaction of preparing L-PPT by using PPTDH and EsGDH double-enzyme coupling to catalyze the asymmetric amination reduction of PPO in Example 1.
  • FIG. 5 is the double-enzyme-coupled SDS-PAGE chart of PPTDH and EsGDH in Example 4.
  • Lane 1 standard protein molecular weight
  • lane 2 recombinant E. coli cells before EsGDH overexpression
  • lane 3 recombinant E. coli cells after EsGDH overexpression.
  • Figure 6 is an SDS-PAGE graph of the wild type and mutants of glufosinate-ammonium dehydrogenase.
  • Lane M standard protein molecular weight
  • Lane 1 wild-type crude enzyme liquid supernatant of glufosinate-ammonium dehydrogenase
  • Lane 2 wild-type purified enzyme liquid of glufosinate-ammonium dehydrogenase
  • Lane 3 containing wild-type grass Escherichia coli with ammonium phosphine dehydrogenase
  • lane 4 supernatant of the crude enzyme liquid of the mutant of glufosinate dehydrogenase A164G_R205K_T332A
  • lane 5 purified enzyme liquid of the mutant A164G_R205K_T332A of glufosinate dehydrogenase
  • lane 6 E. coli containing the glufosinate-ammonium dehydrogenase mutant A164G_R205K_T3
  • Figure 7 is a diagram showing the reaction progress of asymmetric reductive amination synthesis of L-PPT with recombinant E. coli BL21(DE3)/pETDuet-PPTDH-EsGDH.
  • the experimental methods in the present invention are conventional methods unless otherwise specified, and the specific gene cloning operation can be found in the "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.
  • the concentration of substrate PPO was detected by high performance liquid chromatography (HPLC), and the analysis method was as follows: Column type: QS-C18, 5 ⁇ m, 4.6 ⁇ 250 mm. Mobile phase: dissolve 50mM dihydrogen phosphate in 800mL ultrapure water, add 10mL tetrabutylammonium hydroxide (10%) to dilute with water and dilute to 1000mL, adjust pH to 3.8 with phosphoric acid, and acetonitrile in a volume ratio of 88:12 mix. The detection wavelength was 232 nm, and the flow rate was 1.0 mL/min. Column temperature: 40°C, 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid (PPO) peak time: 10.3 minutes (Fig. 1).
  • HPLC high performance liquid chromatography
  • the chirality analysis and concentration analysis of the product are carried out by pre-column derivatization high performance liquid chromatography, and the specific analysis method is as follows:
  • pETDuet-PPTDH-LbFDH with glufosinate dehydrogenase was obtained between Bg1II and PacI of the second multiple cloning site of the pETDuet-1 vector.
  • the operation of the PCR program was as follows: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15s, annealing at 53-58°C for 15s, extension at 72°C for 1.5 minutes, a total of 25 cycles; then extension at 72°C for 10 minutes.
  • the preparation method of competent cells is as follows: obtain the E.coli BL21 (DE3) strain stored in a glycerol tube from a -80°C refrigerator, streak it on an anti-anti-LB plate, and cultivate it at 37°C for 10 hours to obtain a single colony; pick the LB plate A single colony was inoculated into a test tube containing 5 mL of LB medium, and cultured at 37°C and 180 rpm for 9 h; 200 ⁇ L of bacterial liquid was taken from the test tube, inoculated into 50 mL of LB medium, and incubated at 37°C and 180 rpm for an OD 600 to 0.4- 0.6; Pre-cool the bacterial solution on ice, take the bacterial solution into a sterilized centrifuge tube, place on ice for 10 min, and centrifuge at 4°C and 5000 rpm for 10 min; pour out the supernatant, taking care to prevent bacterial contamination, use pre-cooled Resuspend the pelleted cells
  • PPTDH and EsGDH were respectively subjected to directed evolution using the multi-enzyme stepwise directed evolution method. After screening 20,000 clones, PPTDH (PfGluDH) and EsGDH with significantly improved activity were not obtained.
  • PPTDH PPTDH
  • EsGDH E. coli cells
  • the conversion of 100 mM PPO to L-PPT was 37.3% after 12 hours (without the addition of exogenous NADP + ).
  • the conversion rate decreased to 11.5% and 7.7%, respectively (without the addition of exogenous NADP + ), and the ee value reached more than 99%. Therefore, there is a need to improve the efficiency of multi-enzyme-catalyzed reactions for L-PPT synthesis.
  • the strategy adopted in the present invention is to use the expression recombinant plasmid pETDuet-1-PPTDH-EsGDH obtained in Example 1 as the starting plasmid, and perform simultaneous analysis of glufosinate dehydrogenase and glucose dehydrogenase and the gene expression regulatory elements between them. Wrong PCR, screening for strains with improved activity. There are two rounds of PCR in error-prone PCR.
  • the first round of PCR error-prone PCR is performed on the target gene, and the concentration of Mn 2+ added is 0.15mM;
  • the second round of PCR the first round of error-prone PCR products were cloned into the expression vector pETDuet-1 using the megaprimer method to obtain a recombinant plasmid with an error-prone target gene (Miyazaki K, Takenouchi M. 2002. Creating Random Mutagenesis Libraries Using Megaprimer PCR of Whole Plasmid. BioTechniques 33:1033-1038.).
  • the second round PCR product digested with DpnI was transformed into E. coli BL21(DE3) for expression and screening.
  • reaction solution 500 ⁇ L was added to each well of the 96-well plate, and at the same time, the bacteria collected in the 96-well plate were resuspended by repeated pipetting with a pipette. After 1 h of reaction on a shaker, the supernatant was collected by centrifugation for high-throughput screening.
  • mutants About 8,000 mutants were initially screened by high-throughput screening and liquid-phase re-screening, and finally four mutants with improved activity were screened, which increased the yield of L-PPT by at least three times.
  • Three of the mutants are A164G, R205K and T332A mutants from glufosinate dehydrogenase, and the fourth mutant is derived from the deletion of the base between RBS and glucose dehydrogenase from the second cloning site resulting in increased activity , the analysis reason is that the expression of glucose dehydrogenase is increased due to the deletion of bases, which improves the efficiency of coenzyme cycle.
  • the two beneficial mutation sites A164 and R205 of glufosinate-ammonium dehydrogenase obtained in Example 2 were subjected to site-directed saturation mutation for further screening.
  • the PCR primers were designed as shown in Table 1.
  • PCR system 50 ⁇ L: 25 ⁇ L of 2* Phanta Max buffer, 1 ⁇ L of dNTPs, 1 ⁇ L of upper and lower primers for mutation, 1 ⁇ L of template (starting strain), 0.5 ⁇ L of Pfu DNA polymerase, and supplemented with ddH2O to 50 ⁇ L.
  • PCR conditions were: pre-denaturation at 95 °C for 3 min: denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 7 min for 20 s, 30 cycles; final extension at 72 °C for 10 min.
  • the PCR product was verified by DNA agarose gel electrophoresis, and after digesting the template with DpnI, the PCR product was transformed into E.coli BL21(DE3) competent cells, and the transformed product was spread on 50 ⁇ g/mL ampicillin containing The obtained mutants were screened for dominant mutants, and the dominant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing confirmation and stored.
  • primer name Primer sequence (5'-3') PfGluDH-205-F GGCAGTTTGATTNNKCCAGAAGCTACC PfGluDH-205-R GGTAGCTTCTGGMNNAATCAAACTGCC PfGluDH-164-F GTCGATGTGCCANNKGGAGATATTGGCG PfGluDH-164-R ACGCCAATATCTCCMNNTGGCACATCGA
  • the high-throughput screening method and reaction system for measuring enzyme activity are the same as those in Example 2.
  • the glufosinate dehydrogenase mutant A164G can increase the conversion rate of 300 mM PPO from 11.5% to 63.6%.
  • other strains with improved viability were not screened by A164 point saturation mutagenesis and screening.
  • a mutant of glufosinate dehydrogenase R205K increased the conversion rate from 11.5% to 21.3% in 300 mM PPO without the addition of exogenous NADP +, however, by R205 point saturation mutagenesis and screening, there was also no screening to other strains with increased vigor. Then the two mutation points A164G and R205K were combined and mutated, and it was found that the activity was further improved, and the conversion rate of 300mM PPO reached 68.1%.
  • homology modeling and molecular docking of glufosinate-ammonium dehydrogenase were also carried out through semi-rational design, and some other potential activity-improving mutation sites were selected for site-directed saturation mutation.
  • Homology modeling By finding the protein crystal structure with the highest homology to glufosinate dehydrogenase in the PDB database as a template, using Modeller 9.22 for homology modeling, and using autock vina for molecular docking to select appropriate mutations site, design mutation primers, perform site-directed saturation mutagenesis, and screen by high-throughput methods.
  • the mutant PPTDH-T332A with improved activity was finally screened.
  • the activity of the three-enzyme mutant A164G_R205K_T332A was further improved compared with the wild type, and the conversion rate of 300mM PPO reached 71.2%. Therefore, the glufosinate-ammonium mutant was finally selected as A164G_R205K_T332A.
  • primer name Primer sequence 10bp-F TATAATTCATATACATCCATGGGTTATAATTC 10bp-R GAATTATAACCCATGGATGTATATGAATTATA 8bp-F TATAATTCATATACATATGGGTTATAATTC 8bp-R GAATTATAACCCATATGTATATGAATTATA 6bp-F TATAATTCATATACATGGGTTATAATTC
  • TTN total turnover numbers
  • TTN ⁇ mol L-PPT/ ⁇ mol catalyst
  • TTN (L-PPT(mol/L))/(PPTDH(g/L) ⁇ 49060(mol/g))
  • NADP+ -dependent formate dehydrogenase from Lactobacillus buchneri was obtained through gene mining, but the original enzyme activity was low. Therefore, homology modeling and molecular docking semi-rational design were used to select suitable mutation sites. Mutation was used to screen mutants with improved activity, and the identified mutation sites were S148, Q222, R223, H224, M334, T338, K380 and T383.
  • a high-throughput screening method for NADPH was established. Since the molar extinction coefficient ( ⁇ ) of NADPH at 340nm is large, which is always 6220L mol -1 cm -1 , the OD 340 and NADPH concentration are proportional to each other, so the determination reaction can be used. The OD 340 value in the solution was used to reflect the activity of the enzyme.
  • the crude enzyme solution was centrifuged at 4°C and 12,000 rpm for 20 min, and the supernatant was taken.
  • the mutant protein was purified using Ni affinity column (1.6 ⁇ 10cm, Bio-Rad Company, USA), the specific operation was as follows: 1 Use 5 times the column volume of binding buffer (pH 8.0, 50mM sodium phosphate buffer containing 0.3M NaCl) ) Equilibrate the Ni column until the baseline is stable; 2 Load the sample at a flow rate of 1 mL/min, and the loading amount is 25-40 mg/mL protein, so that the target protein is adsorbed on the Ni column; 3 Use 6 times the column volume of buffer A ( Wash the impurity protein with 0.3M NaCl, 30mM imidazole pH 8.0, 50mM sodium phosphate buffer) at a flow rate of 1mL/min until the baseline is stable; 4Use buffer B (pH 8.0, 50mM sodium phosphate containing 0.3M NaCl, 500mM imi
  • LB medium tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, the solvent is distilled water, pH 7.4.
  • LB plate tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, 18g/L agar, the solvent is distilled water, pH 7.4.
  • Reaction system substrate PPO concentration 2-30 mM, 50 mM ammonium sulfate, 1-15 mM NADPH and a certain amount of purified enzyme, the reaction medium is 100 mM phosphate buffer (pH 7.0), and the reaction temperature is 40 ° C. Fit the obtained initial velocity data to the following equation:
  • [A] and [B] are the concentrations of NADPH and PPO, and Km A and Km B are the apparent substrate affinities for NADPH and PPO.
  • Vmax is the maximum reaction rate of the enzyme when the substrate reaches saturation, and Ks A represents the dissociation constant between glufosinate-ammonium dehydrogenase and NADPH.
  • L-PPT was synthesized by asymmetric reductive amination with recombinant E. coli BL21(DE3)/pETDuet-1-PPTDH_A164G_R205K_T332A-EsGDH/LbFDH_H224Q.
  • Reaction system 100mM-1M PPO, 120mM-1.2M glucose, 150mM-1M ammonium sulfate, 0.5mM NADP + , recombinant E.coli BL21(DE3)/pETDuet-1-PPTDH_A164G_R205K_T332A-EsGDH (high expression level) (0.5g /L stem cells).
  • Reaction conditions constant temperature water bath at 40°C, rotating speed at 500rpm. During the whole reaction process, the pH value of the reaction solution was kept at 7.5 by adding NH 3 ⁇ H 2 O by flow.
  • Reaction system 400mM PPO, 800mM ammonium formate, recombinant Escherichia coli E.coli BL21(DE3)/pETDuet-1-PPTDH_A164G_R205K_T332A-LbFDH_H224Q 25g ⁇ L -1 catalyst, reaction conditions: constant temperature water bath 45°C, rotating speed 600rpm reaction. The changes of the concentration and ee value of the product L-PPT during the reaction were detected by high performance liquid phase method.

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Abstract

一种草铵膦脱氢酶突变体、基因工程菌及一锅法多酶同步定向进化方法。所述草铵膦脱氢酶突变体由来源于荧光假单胞菌的草铵膦脱氢酶第164位氨基酸由丙氨酸突变为甘氨酸所得，第205位精氨酸突变为赖氨酸，第332位苏氨酸突变为丙氨酸获得的所得，氨基酸序列如SEQ ID No.1所示。所述基因工程菌为将所述草铵膦脱氢酶突变体的基因导入宿主细胞。宿主细胞中还可以将葡萄糖脱氢酶的编码基因或甲酸脱氢酶的编码基因也导入，进行同步定向进化，使双基因过表达。所述一锅法多酶同步定向进化方法可以筛选到活性大大提高的基因工程菌。L-PPT制备方法与转氨酶等催化工艺相比，工艺相对简单，原料转化率高，转化率可达100％，且立体选择性高。

Description

一种草铵膦脱氢酶突变体、基因工程菌及一锅法多酶同步定向进化方法 技术领域
本发明涉及生物化工技术领域，特别是涉及一种草铵膦脱氢酶突变体、基因工程菌及一锅法多酶同步定向进化方法。
背景技术
草铵膦(phosphinothricin，也叫glufosinate，简称PPT)的化学名称为2-氨基-4-[羟基(甲基)膦酰基]-丁酸，是世界第二大转基因作物耐受除草剂，由赫斯特公司(几经合并后现归属拜耳公司)开发生产，又称草铵膦铵盐、Basta、Buster等，属膦酸类除草剂，非选择性(灭生性)触杀型除草剂是谷氨酰胺合成酶抑制剂。
草铵膦有两种光学异构体，分别为L-草铵膦和D-草铵膦。但只有L-型具有生理活性，且在土壤中易分解，对人类和动物的毒性较小，除草谱广，对环境的破坏力小。
目前，市场上销售的草铵膦一般都是外消旋混合物。若草铵膦产品能以L-构型的纯光学异构体形式使用，可显著降低草铵膦的使用量，这对于提高原子经济性、降低使用成本、减轻环境压力具有重要意义。
手性纯L-草铵膦的制备方法主要有三种：手性拆分法，化学合成法和生物催化法。生物催化法生产草铵膦则具有立体选择性严格、反应条件温和、收率高等优点，是生产L-草铵膦的优势方法。主要包括以下三类：
1)以L-草铵膦的衍生物为底物，通过酶法直接水解获得，主要的优点转化率高，产物e.e.值较高，但需要昂贵且不易获得的手性原料作为前体，成本加高，不利于工业化生产。例如生物法制备L-草铵膦最简单的方法就是利用蛋白酶直接水解双丙氨膦。双丙氨膦是一种天然的三肽化合物，在蛋白酶的催化下，双丙氨膦脱去2分子L-丙氨酸，生成L-草铵膦。
2)以外消旋草铵膦的前体为底物，通过酶的选择性拆分获得。主要优点为原料相对易得，催化剂活力高，但其理论收率只能达到50％，会造成原料的浪费。例如Cao等人(Cao C-H,Cheng F,Xue Y-P,Zheng Y-G(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)采用一种来源于Stenotrophomonas maltophilia的新型的氨基酰化酶手性拆分N-乙酰-PPT，得到L-草铵膦。用全细胞进行催化，在4小时内转化率>49％，获得了光学纯的L-PPT(>99.9％e.e.)。
3)以α-酮酸-2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)为底物，通过酶的不对称合成获得，主要涉及的酶包括转氨酶与草铵膦脱氢酶。Bartsch(Bartsch K(2005)Process for the preparation of l-phosphinothrcine by enzymatic transamination with aspartate.US Patent no.US6936444B1)等利用PPO为底物，L-天冬氨酸为氨基供体，利用从土壤微生物中筛选分离出来的对PPO和L-天冬氨酸有特异性酶活的转氨酶进行催化，当底物浓度为552mM时，在非常高的温度(80℃)下反应4小时，转化率仍达到52％，时空产率为4.5g L-PPT/g of biocatalyst/h。但利 用转氨酶制备L-草铵膦有两大缺陷，其一是这是一个可逆反应，原料PPO不能完全转化为L-PPT，转化率无法达到100％；其二是要使可逆反应向生成L-PPT的方向进行，需要加入至少2倍以上的L-天冬氨酸作为氨基供体，过量的天冬氨酸给L-PPT的分离带来了很大的麻烦。
在诸多草铵膦的酶法合成路线中，酮酸中间体的酮羰基是潜手性官能团，能够通过酶法合成途径构建手性中心，酮酸路线也因原料价廉易得，且可以避免使用剧毒氰化物，而成为适宜L-草铵膦工业化开发生产的路线。
氨基酸脱氢酶(EC 1.4.1.X，AADH)是一类能将氨基酸可逆的脱氨生成对应酮酸的氨基酸脱氢酶，其反应需要核苷类辅酶的参与(NAD(P) +)，被广泛的应用于天然与非天然α-氨基酸的合成。根据其底物特异性，可分为谷氨酸脱氢酶、亮氨酸脱氢酶、丙氨酸脱氢酶、缬氨酸脱氢酶等。如果其表现出对草铵膦前体具有一定的活力，就可称之为“草铵膦脱氢酶(PPTDH)”。
葡萄糖脱氢酶(EC1.1.1.47，GDH)是生物催化的重要辅助酶，用于氧化还原催化反应中辅酶NAD(P)H的再生循环。
发明内容
本发明提供了一种多酶一锅同步定向进化法，可以同步定向进化草铵膦脱氢酶和葡萄糖脱氢酶或者甲酸脱氢酶以及它们之间的基因元件。本发明提供了草铵膦脱氢酶突变体，高表达葡萄糖脱氢酶或者甲酸脱氢酶突变体以及包含这两种酶的基因的工程菌及其在制备L-草铵膦中的应用，该基因工程菌中的两种酶经过了一锅法多酶同步进化，使两种酶在合成L-PPT过程中协同效率更高，催化制备的L-草铵膦时底物转化率高，时空产率高、总转换数高。且进一步缩短了反应进程。
一种草铵膦脱氢酶突变体，由来源于荧光假单胞菌(Pseudomonas fluorescens)的草铵膦脱氢酶第164位氨基酸由丙氨酸突变为甘氨酸，第205位精氨酸突变为赖氨酸，第332位苏氨酸突变为丙氨酸获得的所得，氨基酸序列如SEQ ID No.1所示。
本发明又提供了编码所述草铵膦脱氢酶突变体的基因。
本发明又提供了一种基因工程菌，包括宿主细胞和转入宿主细胞的目的基因，所述目的基因包含所述的基因。
所述的基因工程菌，目的基因还包括葡萄糖脱氢酶的编码基因或甲酸脱氢酶的编码基因。
所述的基因工程菌，使用双基因表达载体，所述草铵膦脱氢酶突变体的基因克隆到其中一个多克隆位点区域，葡萄糖脱氢酶的编码基因或甲酸脱氢酶的编码基因克隆到第二个多克隆位点区域。
所述葡萄糖脱氢酶的编码基因序列GenBank登录号为KM817194.1，所述葡萄糖脱氢酶的编码基因起始密码子与质粒上对应核糖体结合位点之间的连接碱基长度为8～10bp。其中，效果最好的情况是该连接碱基长度为8bp时。
所述甲酸脱氢酶的编码基因序列如SEQ ID No.3所示。如SEQ ID No.3所示基因序列编码的甲酸脱氢酶由来源于布赫内氏乳杆菌(Lactobacillus buchneri)的甲酸脱氢酶第224位组氨酸突变成谷氨酰胺所得。
本发明还提供了所述草铵膦脱氢酶突变体、所述基因或所述基因工程菌在制备L-草铵膦中的应用。
一种L-草铵膦的制备方法，以2-羰基-4-(羟基甲基膦酰基)丁酸为底物，葡萄糖或甲酸铵为辅助底物，在无机氨基供体、和辅酶循环系统存在的条件下，利用催化剂催化底物反应获得L-草铵膦；
所述辅酶循环系统为葡萄糖脱氢酶循环系统或甲酸脱氢酶循环系统；所述催化剂为所述的基因工程菌、该基因工程菌的粗酶液或固定化的所述基因工程菌，
当所述基因工程菌中含有所述葡萄糖脱氢酶的编码基因时，辅助底物为葡萄糖，辅酶循环系统为葡萄糖脱氢酶循环系统；当所述基因工程菌中含有所述甲酸脱氢酶的编码基因时，辅助底物为甲酸铵，辅酶循环系统为甲酸脱氢酶循环系统。无机氨基供体在葡萄糖脱氢酶循环系统中是硫酸铵，在甲酸脱氢酶循环系统中是甲酸铵。
本发明还提供了一种一锅法多酶同步定向进化方法，包括：
(1)将辅酶循环系统所用再生酶的基因也克隆到双基因表达载体中与草铵膦脱氢酶进行共表达；(2)将偶联共表达后的双酶利用易错PCR方法构建易错PCR库并克隆到双基因表达载体中；(3)将克隆有双基因的表达载体导入宿主细胞中进行培养，得到的各单菌落分别进行L-草铵膦制备实验并筛选制备效率提高的菌株；(4)确定筛选到的菌株中草铵膦脱氢酶基因的突变位点，对该基因进行定点饱和突变，筛选获得活性最高的突变体。
本申请中，克隆了来源于Pseudomonas fluorescens、NCBI登录号为WP_150701510.1的草铵膦脱氢酶基因，来源于Exiguobacterium sibiricum、GenBank编号为ACB59697.1的葡萄糖脱氢酶，来源于Lactobacillus buchneri、NCBI登录号为WP_013726924.1的甲酸脱氢酶，草铵膦脱氢酶基因导入pETDuet-1质粒上的MCS1(多克隆位点1)，葡萄糖脱氢酶或甲酸脱氢酶分别导入带有草铵膦脱氢酶载体pETDuet-1质粒上的MCS2(多克隆位点2)上，然后将pETDuet-1质粒转化进在大肠杆菌(Escherichia coli)中实现该基因的异源表达，草铵膦脱氢酶能够催化PPO不对称胺化还原为L-PPT，同时辅酶NADPH变成NADP +。葡萄糖脱氢酶能够催化葡萄糖生成葡萄糖酸或甲酸脱氢酶能催化甲酸铵生成铵根离子，二氧化碳和水，同时使NADP +变成NADPH，从而在胞内形成辅酶循环(图4)。虽然该草铵膦脱氢酶经过突变体A164G_R205K_T332A之后对PPO的活力有了明显提高，但是和葡萄糖脱氢酶或者甲酸脱氢酶一锅法偶联制备L-PPT时，两种酶整体的酶活还有待提高，所以需要将草铵膦脱氢酶和葡萄糖脱氢酶或者甲酸脱氢酶两种酶同时一锅法同步定向进化催化生成L-PPT。这种方法很新颖，而且将具有较强的工业应用价值。
与现有技术相比，本发明具有以下有益效果：
(1)本发明通过发展一锅法多酶同步定向进化技术，通过将获得的PPTDH-A164G_R205K_T332A和EsGDH偶联共表达，同时优化了核糖体结合位点和EsGDH编码基因起始密码子之 间的碱基长度，使EsGDH进一步过表达，或者突变LbFDH，获得突变体LbFDH-H224Q，从而使一锅法生物合成L-PPT的转化率和时空产率进一步提高。通过一锅法多酶同步定向进化后，总转换数较之前单酶突变体PPTDH-A164G_R205K_T332A提高了5.8倍，最终L-PPT时空产率高达7110g·L –1·d –1，且产物L-PPT的e.e.值大于99％。
(2)本发明提供的L-PPT制备方法原料转化率高、收率高、产物易于分离提纯。
(3)本发明方提供的L-PPT制备方法与化学法及其他生物法(转氨酶等)工艺相比，工艺相对简单，原料转化率高，转化率可达100％，产物光学纯度高(e.e.>99％)。
附图说明
图1为PPO样品的高效液相色谱(HPLC)光谱分析图。
图2为D,L-PPT样品的高效液相色谱(HPLC)光谱分析图。
图3为实施例1获得的PPTDH和EsGDH偶联表达载体的质粒图谱。
图4为实施例1利用PPTDH与EsGDH双酶偶联催化PPO不对称胺化还原制备L-PPT的反应示意图。
图5为实施例4中PPTDH和EsGDH双酶偶联SDS-PAGE图。其中泳道1：标准蛋白分子量；泳道2：EsGDH过表达之前的重组大肠杆菌细胞；泳道3：EsGDH过表达之后的重组大肠杆菌细胞。
图6为草铵膦脱氢酶的野生型和突变体的SDS-PAGE图。泳道M：标准蛋白分子量；泳道1：草铵膦脱氢酶的野生型粗酶液上清液；泳道2：草铵膦脱氢酶的野生型纯化后酶液；泳道3：含有野生型草铵膦脱氢酶的大肠杆菌；泳道4：草铵膦脱氢酶的突变体A164G_R205K_T332A粗酶液上清液；泳道5：草铵膦脱氢酶的突变体A164G_R205K_T332A纯化后酶液；泳道6：含有草铵膦脱氢酶突变体A164G_R205K_T332A的大肠杆菌。
图7为用重组大肠杆菌E.coli BL21(DE3)/pETDuet-PPTDH-EsGDH不对称还原胺化合成L-PPT反应进程图。反应体系中外加了0.5mM NADP +辅酶，反应条件：40℃，pH＝7.5。LE：低表达；HE：过表达。
具体实施方式
本发明中的实验方法如无特别说明均为常规方法，基因克隆操作具体可参见J.萨姆布鲁克等编的《分子克隆实验指南》。
上游基因工程操作所用试剂：本发明实施例中使用的一步克隆试剂盒均购自Vazyme，南京诺唯赞生物科技有限公司；质粒提取试剂盒、DNA回收纯化试剂盒购自Axygen杭州有限公司；质粒等购自上海生工；DNA marker、Fast Pfu DNA聚合酶、低分子量标准蛋白、琼脂糖电泳试剂、引物合成与基因测序以及基因合成工作由杭州擎科梓熙生物技术有限公司完成。以上试剂使用方法参考商品说明书。
下游催化工艺所用试剂：2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)，D,L-PPT、L-PPT标准品购自Sigma-Aldrich公司；NADPH购于邦泰生物工程(深圳)有限公司；其他常用试剂购 自国药集团化学试剂有限公司。
Figure PCTCN2020139770-appb-000001
下列实施例中高效液相色谱的检测方法如下：
高效液相色谱(HPLC)检测底物PPO浓度，分析方法为：色谱柱型号：QS-C18，5μm，4.6×250mm。流动相：将50mM磷酸二氢胺溶于800mL超纯水中，加入10mL四丁基氢氧化铵(10％)用水稀释并定容至1000mL，用磷酸调pH到3.8，与乙腈以体积比88∶12混合。检测波长为232nm，流速：1.0mL/min。柱温：40℃，2-羰基-4-(羟基甲基氧膦基)-丁酸(PPO)出峰时间为：10.3分钟(图1)。
产物的手性分析及浓度分析通过柱前衍生化高效液相色谱进行，具体的分析方法为：
(1)色谱条件：色谱柱型号：QS-C18，5μm，4.6×250mm。流动相：50mM乙酸铵溶液∶甲醇＝10∶1。荧光检测波长：λ ex＝340nm，λ em＝455nm。流速：1mL/min。柱温：30℃，L-PPT出峰时间为11min，D-PPT出峰时间为13.4min(图2)。
(2)衍生化试剂：分别称取0.1g邻苯二甲醛与0.12g N-乙酰-L-半胱氨酸，用10mL乙醇助溶，再加入40mL 0.1moL/L硼酸缓冲液(pH 9.8)，振荡使其充分溶解，4℃冰箱保存备用(不超过4天)。
(3)衍生化反应与HPLC测定：以超纯水补足至1mL，即反应液稀释10倍，稀释后的样品先经过衍生化处理，取200μL稀释后的反应液加400μL衍生化试剂30℃衍生化5min，再加400μL超纯水补足至1mL，12000转/分钟离心1分钟，取上清，过0.22μM微滤膜，作为液相样品，HPLC检测PPO、L-PPT、D-PPT及e.e.值。
实施例1
草铵膦脱氢酶和葡萄糖脱氢酶或者甲酸脱氢酶偶联表达载体和工程菌的构建。
构建表达载体：以下所述的草铵膦脱氢酶，葡萄糖脱氢酶和甲酸脱氢酶基因合成工作均由杭州擎科梓熙生物技术有限公司完成。将来源于荧光假单胞菌(Pseudomonas fluorescens)的草铵膦脱氢酶基因(NCBI登录号为WP_150701510.1)通过PCR无缝克隆到pETDuet-1载体的第一个多克隆位点的SacI和NotI之间，将来源于Exiguobacterium sibiricum的葡萄糖脱氢酶基因(NCBI登录号：KM817194.1)或者来源于Lactobacillus buchneri的(NCBI登录号为WP_013726924.1)的甲酸脱氢酶，通过PCR无缝克隆到pETDuet-1载体的第二个多克隆位点的Bg1II和PacI之间，获得带有草铵膦脱氢酶的表达载体pETDuet-PPTDH-EsGDH(图3)或者pETDuet-PPTDH-LbFDH。PCR程序的操作如下：95℃预变性3分钟；95℃变性15s，53-58℃退火15s，72℃延伸1.5分钟，共25个循环；然后以72℃延伸10分钟。
感受态细胞的制备方法为：从-80℃冰箱中获得甘油管保藏的E.coli BL21(DE3)菌株，在无抗LB平板上划线，37℃培养10h，获取单菌落；挑取LB平板的单菌落，接种至含5mL的LB培养基的试管中，37℃、180rpm培养9h；从试管中取200μL菌液，接种到50mL的LB培养基中，37℃、180rpm培养OD 600至0.4-0.6；将菌液在冰上预冷，取菌液至灭菌的离 心管中，冰上放置10min，4℃、5000rpm离心10min；将上清液倒出，注意防止染菌，用预冷的0.1mol/L的CaCl2水溶液重悬沉淀细胞，并在冰上放置30min；4℃、5000rpm离心10min，弃上清，用预冷的含15％甘油的0.1mol/L的CaCl 2水溶液重悬沉淀细胞，取100μL重悬细胞分装至灭菌的1.5mL离心管中，保藏于-80℃冰箱，需要时取出。
构建工程菌文库：将储藏于-80℃的大肠杆菌BL21(DE3)(Invitrogen)感受态细胞在0℃冰浴10min，然后在超净台内分别加入5μL的带有草铵膦脱氢酶和葡萄糖脱氢酶的表达载体pETDuet-1-PPTDH-EsGDH，0℃冰浴30min，42℃水浴中热击90s，0℃冰浴2min，加入600μL的LB培养基，在37℃、200rpm摇床培养1h；涂布于含有50μg/ml氨苄霉素抗性的LB平板，37℃下培养8-12h，获得含有表达重组质粒的重组大肠杆菌E.coli BL21(DE3)/pETDuet-1-PPTDH-EsGDH或者pETDuet-PPTDH-LbFDH工程菌。
实施例2
草铵膦脱氢酶和葡萄糖脱氢酶/甲酸脱氢酶双酶偶联基因文库的构建和筛选。
一)高通量筛选方法的建立
配制50mL工作液(衍生化试剂)：邻苯二甲醛0.013g，N-乙酰-L-半胱氨酸0.032g，用pH＝9.8硼酸缓冲液溶解定容到50mL，振荡使其充分溶解，4℃冰箱保存备用(不超过4天)，作为高通量工作液，也叫做衍生化试剂。吸取50μL样品反应液加入50μL工作液震荡反应30s，再加入100μL ddH2O，在λ ex＝340nm，λ em＝455nm条件下测定荧光值。
二)一锅法多酶同步定向进化
对比例：，使用多酶分步定向进化方法，对PPTDH和EsGDH分别进行定向进化，在筛选2万个克隆后，未获得活力明显提高的PPTDH(PfGluDH)和EsGDH。利用PPTDH(PfGluDH)和EsGDH的野生型冻干大肠杆菌细胞作为生物催化剂，在12小时后(未添加外源NADP +)，100mM PPO到L-PPT的转化率为37.3％。当PPO浓度增加到300和500mM时，转化率分别降低到11.5％和7.7％(不添加外源NADP +)，e.e.值达到99％以上。因此，需要提高用于L-PPT合成的多酶催化反应的效率。
本发明采用的策略是将实例1获取的获得含有表达重组质粒pETDuet-1-PPTDH-EsGDH为出发质粒，对草铵膦脱氢酶和葡萄糖脱氢酶以及之间的基因表达调控元件同时进行易错PCR，筛选活力提高菌株。易错PCR存在两轮PCR过程，第一轮PCR过程中，对目的基因进行易错PCR，选择添加的Mn 2+的浓度为0.15mM；。第二轮PCR过程中，采用的大引物方法将第一轮易错PCR产物克隆到表达载体pETDuet-1上，获得带有易错目的基因的重组质粒(Miyazaki K,Takenouchi M.2002.Creating Random Mutagenesis Libraries Using Megaprimer PCR of Whole Plasmid.BioTechniques 33:1033-1038.)。将经过DpnI消化过的第二轮PCR产物转化到大肠杆菌BL21(DE3)中进行表达和筛选。
配置反应液：终浓度300mM底物PPO(2-羰基-4-(羟基甲基膦酰基)丁酸)，750mM的硫酸铵，360mM的葡萄糖，pH＝7.5的磷酸缓冲液为反应介质构成反应液。
待平板中长出菌落后，从平板中挑出单个菌落，并接种到96孔板中，每个孔中含有1.0mL LB培养基(含135μM氨苄青霉素)。在37℃下培养8小时后，取出200μL菌液转移到另一 个含有800μL LB培养基(含135μM氨苄青霉素和0.1mM IPTG)的96孔板中，并在22℃，200rpm条件下培养16小时。将培养好的菌液以4000rpm离心10分钟，倒掉上清液，收集菌体于孔底。接下来进行催化反应验证，96深孔板每孔加入500μL的反应液，同时用移液枪反复吹打，将96孔板收集的菌体重悬，然后将96深孔板放入40℃、200rpm的摇床上反应1h后，离心取上清，进行高通量筛选。
初筛：吸取50μL样品反应上清液加入50μL工作液震荡反应30s，再加入100μL ddH 2O，在λ ex＝340nm，λ em＝455nm条件下测定荧光值。筛选荧光值高于pETDuet-1-PPTDH-EsGDH野生型反应液的菌株，找到相对应的保藏菌种，划线培养，然后吸取高浓度菌液和30％甘油以1∶1的比例加入甘油管中，最后放到-80℃冰箱中保藏。
通过高通量筛选方法初筛以及液相复筛了大约8000多突变体，最后筛选到四个活力提高突变株，使L-PPT的产率至少提高了三倍以上。其中三个突变体是来自草铵膦脱氢酶的A164G，R205K和T332A突变体，第四个突变体是来自第二个克隆位点RBS到葡萄糖脱氢酶之间碱基的缺失造成活力提升，分析原因是因为碱基的缺失造成葡萄糖脱氢酶的表达量提升，提高了辅酶循环效率。
实施例3
多酶偶联反应体系中草铵膦脱氢酶定点饱和突变。
为了进一步筛选潜在活力提升菌株，先将实施例2中得到的草铵膦脱氢酶的A164和R205两个有益突变位点进行定点饱和突变，做进一步的筛选，PCR引物设计如表1所示，PCR体系(50μL)为：2*Phanta Max缓冲液25μL，dNTPs 1μL，突变上下引物各1μL，模板(出发菌株)1μL，Pfu DNA聚合酶0.5μL，补ddH2O至50μL。PCR条件为：95℃预变性3min：95℃变性15s，60℃退火15s，72℃延伸7min20s，30个循环；72℃终延伸10min。PCR产物经过DNA琼脂糖凝胶电泳验证，并通过DpnI消化模板后，将PCR产物转化到大肠杆菌E.coli BL21(DE3)感受态细胞中，转化后的产物涂布于含50μg/mL氨苄霉素抗性的LB平板上，37℃倒置培养过夜，对获得的突变体进行优势突变体的筛选，将获得优势菌株送杭州擎科生物技术有限公司进行测序确认，并保存。
表1草铵膦脱氢酶定点饱和突变引物设计
引物名称 引物序列(5’-3’)
PfGluDH-205-F GGCAGTTTGATTNNKCCAGAAGCTACC
PfGluDH-205-R GGTAGCTTCTGGMNNAATCAAACTGCC
PfGluDH-164-F GTCGATGTGCCANNKGGAGATATTGGCG
PfGluDH-164-R ACGCCAATATCTCCMNNTGGCACATCGA
高通量筛选方法和测酶活反应体系同实施例2。
表2草铵膦脱氢酶定点饱和突变后多酶催化反应的转化率
草铵膦脱氢酶164位突变 转化率(％) 草铵膦脱氢酶205位突变 转化率(％)
WT 11.5 WT 11.5
A164R N.D. R205A N.D.
A164N 1.2 R205N 1.6
A164D N.D. R205D N.D.
A164C 4.3 R205C N.D.
A164Q 5.7 R205Q 3.4
A164E N.D. R205E N.D.
A164G 63.6 R205G N.D.
A164H N.D. R205H 10.8
A164I 10.4 R205I N.D.
A164L 9.9 R205L N.D.
A164K N.D. R205K 21.3
A164M 7.3 R205M 2.6
A164F N.D. R205F N.D.
A164P N.D. R205P N.D.
A164S N.D. R205S N.D.
A164T N.D. R205T 2.9
A164W 3.0 R205W N.D.
A164Y N.D. R205Y N.D.
A164V 10.9 R205V N.D.
N.D.：检测不到
通过表2可以看出，在未添加外源NADP +条件下，草铵膦脱氢酶突变体A164G，可以把300mM PPO的转化率从11.5％提高到了63.6％。但是，通过A164点饱和诱变和筛选，并没有筛选到其他活力提高的菌株。在未添加外源NADP +条件下，草铵膦脱氢酶R205K的突变体，使300mM PPO的转化率从从11.5％提高到21.3％，但是，通过R205点饱和诱变和筛选，也没有筛选到其他活力提高的菌株。然后将A164G和R205K两个突变点进行组合突变，发现活力进一步提高，300mM PPO的转化率达到68.1％。
除了利用易错PCR技术，还通过半理性设计，对草铵膦脱氢酶进行同源建模和分子对接，选择一些其他潜在活力提高的突变位点进行定点饱和突变。同源建模：通过在PDB数据库中找到与草铵膦脱氢酶同源性最高的蛋白晶体结构作为模板，利用Modeller 9.22进行同源建模，并利用autock vina进行分子对接，选择合适的突变位点，设计突变引物，进行定点饱和突变，并通过高通量方法进行筛选。
经过多次定点饱和突变和筛选后，最终筛选到活力提高突变体PPTDH-T332A，三酶突变体A164G_R205K_T332A活力较野生型进一步的提高，300mM PPO的转化率达到71.2％。因此，最终选择草铵膦突变体为A164G_R205K_T332A。
实施例4
多酶偶联反应体系中葡萄糖脱氢酶的过表达。
在双酶偶联表达载体pETDuet-1上，EsGDH蛋白表达量不高，造成辅酶循环效率较差，实施例2中筛选到活力提高菌株是因为第二个克隆位点RBS到葡萄糖脱氢酶之间碱基的缺失，因此推断GDH表达量与葡萄糖脱氢酶到RBS序列之间的碱基长度有关。因此对葡萄糖脱氢酶到RBS序列之间的碱基长度做了进一步的优化。引物设计如表3所示，实验结果如表4所示。
表3多酶催化反应中碱基数量优化引物设计
引物名称 引物序列
10bp-F TATAATTCATATACATCCATGGGTTATAATTC
10bp-R GAATTATAACCCATGGATGTATATGAATTATA
8bp-F TATAATTCATATACATATGGGTTATAATTC
8bp-R GAATTATAACCCATATGTATATGAATTATA
6bp-F TATAATTCATATACATGGGTTATAATTC
6bp-R GAATTATAACCCATGTATATGAATTATA
4bp-F TATAATTCATATATGGGTTATAATTC
4bp-R GAATTATAACCCATATATGAATTATA
表4多酶催化反应中碱基数量优化后转化率汇总表
编号 连接序列(5’-3’) 连接长度(bp) 转化率(％)
1 ATATACATCCAGAT 14 11.5
2 ATATACATCCAGA 13 35.1
3 ATATACATCC 10 78.8
4 ATATACAT 8 99.1
5 ATATAC 6 26.0
6 ATAT 4 4.3
从表4可知，当葡萄糖脱氢酶到RBS序列之间的碱基长度为8bp时，EsGDH基因表达有了较大提高，SDS-PAGE分析见附图5。双酶偶联催化效率有了较大提高，300mM PPO转化率达到了99.1％。
实施例5
PPO胺化总转换数[total turnover numbers(TTN)]测量和比较。
通过测定PPO胺化总转换数来具体反应草铵膦脱氢酶及突变体和PPTDH-EsGDH及突变体双酶偶联催化效率。当只有草铵膦脱氢酶及突变体时，反应体系为：100mM PPO，0.5mM NADPH，400mM NH 4 +，0.5g/L干细胞。当有草铵膦脱氢酶和葡萄糖脱氢酶时，反应体系中还需要额外添加120mM的葡萄糖。反应5分钟后，取反应液样品进行处理，然后用HPLC测定L-PPT的浓度。
单酶或者多酶时TTN(μmol L-PPT/μmol催化剂)的计算公式为：
TTN＝(L-PPT(mol/L))/(PPTDH(g/L)·49060(mol/g))
表5三种不同的酶的野生型和突变体A164G_R205K_T332A对应的TTN值
Figure PCTCN2020139770-appb-000002
从表5中数据可知，当只有PPTDH时，没有NAPDH再生系统(EsGDH)，TTN保持在较低水平，PPTDH_WT的TTN和变体PPTDH_A164G_R205K_T332A的TTN值均低于200。PPTDH_A164G_R205K_T332A和低表达量的EsGDH偶联催化反应时，TTN增加到5846，当PPTDH_A164G_R205K_T332和高表达的EsGDH偶联催化反应时TTN达到最大值33950，这表明PPTDH_A164G_R205K_T332A和高表达的EsGDH偶联催化反应制备L-PPT达到了最佳偶联效率。
实施例6
多酶偶联反应体系中甲酸脱氢酶的定点饱和突变。
首先，通过基因挖掘获得来源于Lactobacillus buchneri的NADP +依赖型甲酸脱氢酶，但原始酶活较低，因此利用同源建模与分子对接半理性设计来选择合适的突变位点，通过定点饱和突变来筛选活力提高突变株，确定的突变位点为S148、Q222、R223、H224、M334、T338、 K380和T383。
建立了NADPH高通量筛选方法，由于在340nm处NADPH的摩尔消光系数(ε)较大，始终恒为6220L mol -1cm -1，故OD 340和NADPH浓度成正比关系，因此可以利用测定反应液中OD 340的值来反应酶的活力大小。测定酶活反应体系为1ml，包含100mM甲酸铵，1mM NADP +，100mM磷酸缓冲液(pH＝7)，以及适量的菌体，反应温度为30℃。
表6多酶偶联反应体系中甲酸脱氢酶的定点饱和突变引物设计表
引物名称 引物序列
FDH-S148-F GAAGTGACCTATAGCAATNNKGTTAGTGTTGC
FDH-S148-R CTGCTTCTGCAACACTAACMNNATTGCTATAG
FDH-Q222-F CGTTAAACTGGTGTATAATNNKCGCCATCAGC
FDH-Q222-R CCGGCAGCTGATGGCGMNNATTATACACC
FDH-R223-F CTGGTGTATAATCAGNNKCATCAGCTGCCG
FDH-R223-R CTTCATCCGGCAGCTGATGMNNCTGATTATAC
FDH-H224-F GGTGTATAATCAGCGCNNKCAGCTGCCGG
FDH-H224-R CAACTTCATCCGGCAGCTGMNNGCGCTGATTA
FDH-M334-F GAAGCAATGACCCCGCATNNKAGTGGCACC
FDH-M334-R CTCAGGGTGGTGCCACTMNNATGCGGGGTC
FDH-T338-F CCCGCATATGAGTGGCACCNNKCTGAGTGCC
FDH-T338-R GCGTGCCTGGGCACTCAGMNNGGTGCCACTC
FDH-K380-F GGCCGGTACCGGTGCCNNKAGTTATACCG
FDH-K380-R CCTTTTTTCACGGTATAACTMNNGGCACCGG
FDH-T383-F CGGTGCCAAAAGTTATNNKGTGAAAAAAGG
FDH-T383-R GGTTTCTTCGCCTTTTTTCACMNNATAACTTT
通过高通量筛选方法进行筛选，结果显示大部分突变体是无义突变，，仅有H224Q为有益突变位点，甲酸脱氢酶的相对酶活提高了23％。
实施例7
草铵膦脱氢酶的表达和纯化。
1、含草铵膦脱氢酶基因-葡萄糖脱氢酶基因的湿菌体：分别将实施例1获得的重组大肠杆菌E.coli BL21(DE3)/pETDuet-PPTGDH-EsGDH、接种至含有50μg/mL氨苄霉素抗性的LB液体培养基，37℃，200rpm下培养12h，再以1％(v/v)接种量接种至新鲜的含有50μg/mL氨苄霉素抗性的LB液体培养基中，于37℃，150rpm下培养至菌体OD 600达0.6-0.8，加入终浓度为0.1mM的IPTG，22℃下诱导培养16h后，4℃、8000rpm离心20min，弃去上清液，收集沉淀，用pH 7.5、20mM磷酸盐缓冲液(PBS)洗涤两次，即获得含草铵膦脱氢酶-葡萄糖脱氢酶重组菌株E.coli BL21(DE3)/pETDuet-PPTGDH-EsGDH的湿菌体；将湿菌体加入pH7.5、100mM的PBS中重悬，在冰水混合物上超声破碎10min，超声破碎条件：功率为400W，破碎1s，暂停5s，获得粗酶液。
粗酶液通过4℃、12000转/分钟离心20min，取上清。使用Ni亲和柱(1.6×10cm，Bio-Rad公司，美国)纯化突变体蛋白，具体操作如下：①用5倍柱体积的结合缓冲液(含0.3M NaCl的pH 8.0、50mM磷酸钠缓冲液)平衡Ni柱，至基线稳定；②样品上样，流速1mL/min，上样量在25-40mg/mL蛋白，使目标蛋白吸附于Ni柱上；③用6倍柱体积的缓冲液A(含0.3M NaCl、30mM咪唑的pH 8.0、50mM磷酸钠缓冲液)冲洗杂蛋白，流速1mL/min，至基线稳 定；④用缓冲液B(含0.3M NaCl、500mM咪唑的pH 8.0、50mM磷酸钠缓冲液)洗脱，流速1mL/min，收集目的蛋白。将目的蛋白置于pH 7.5、20mM磷酸盐缓冲液中透析过夜，获得纯化酶，电泳图见图6所示。
LB培养基：胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L，溶剂为蒸馏水，pH 7.4。
LB平板：胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L，18g/L琼脂，溶剂为蒸馏水，pH 7.4。
实施例8
草铵膦脱氢酶野生型及其突变体动力学参数测定
反应体系:底物PPO浓度2-30mM，50mM的硫酸铵，1～15mM的NADPH和一定量的纯化酶，反应介质为100mM磷酸盐缓冲液(pH 7.0)，反应温度为40℃。将获得的初始速度数据拟合到下面的方程式：
Figure PCTCN2020139770-appb-000003
其中[A]和[B]是NADPH和PPO的浓度，Km A和Km B是对NADPH和PPO的表观底物亲和力。Vmax是底物达到饱和时酶的最大反应速率，Ks A表示草铵膦脱氢酶和NADPH之间的解离常数。
表7草铵膦脱氢酶野生型及其突变体(A164G_R205K_T332A)动力学参数
Figure PCTCN2020139770-appb-000004
从表7数据可知，草铵膦脱氢酶突变体A164G_R205K_T332A的催化常数(k cat)有了很大的提高，从7.47s –1(WT)增加到951.85s –1(A164G_R205K_T332A)，草铵膦脱氢酶突变体A164G_R205K_T332A的底物亲和力比WT略有提高(K m值：5.35mM对6.12mM)。高k cat和适度的底物亲和力导致对PPO有非常高的催化效率(k cat/K m)，从1.22s -1mM -1(WT)显着提高到177.92s -1mM -1(A164G_R205K_T332A)，草铵膦脱氢酶突变体A164G_R205K_T332A催化效率相对于野生型提高了145.83倍。
实施例9
用重组大肠杆菌E.coli BL21(DE3)/pETDuet-1-PPTDH_A164G_R205K_T332A-EsGDH/LbFDH_H224Q不对称还原胺化合成L-PPT。
反应体系100mM-1M PPO，120mM-1.2M葡萄糖，150mM-1M硫酸铵，0.5mM NADP +，重组大肠杆菌E.coli BL21(DE3)/pETDuet-1-PPTDH_A164G_R205K_T332A-EsGDH(高表达量)(0.5g/L干细胞)。反应条件：恒温水浴40℃，转速500rpm。在整个反应过程中，通过流加 NH 3·H 2O控制反应液pH值保持在7.5。在固定的时间间隔取样品(100μl)，加入5μL 6M浓盐酸终止反应，然后处理样品，通过使用HPLC测量L-PPT浓度来确定转化率，反应进程曲线如图7所示。
当PPO底物浓度为100mM时，使用0.5g·L –1催化剂可在5分钟内将底物完全转化为L-PPT(转化率>99％和e.e.>99％)。在相同的反应条件下，使用初始菌体(草铵膦脱氢酶WT和表达低的EsGDH)，转化率只能达到≈26％(3h后为25.68mM)。当PPO底物浓度增加到500mM(89.04g·L –1)和1M(178.08g·L –1)时，使用0.5g·L –1催化剂可在在20分钟和40分钟后将底物完全转化，e.e.>99％。后者的时空产率(STY)达到7110g·L -1·d -1，比初始菌体(37g·L -1·d -1)高173倍。与酰胺酶和转氨酶催化的反应相比，草铵膦脱氢酶偶联葡萄糖脱氢酶仍然显示出最高的转化率和最短的反应时间，如表8所示。
表8和其它文献报道过通过改造酶制备L-PPT的对比
Figure PCTCN2020139770-appb-000005
注：[a]纯化后的酰胺酶作催化剂；[b]固定化转氨酶做催化剂；[c]干细胞做催化剂；[d]外消旋-4-[羟基(甲基)磷酰基]-2-(2-苯基乙酰胺基)丁酸。
反应体系：400mM的PPO，800mM的甲酸铵，重组大肠杆菌E.coli BL21(DE3)/pETDuet-1-PPTDH_A164G_R205K_T332A-LbFDH_H224Q 25g·L –1的催化剂，反应条件：恒温水浴45℃，转速600rpm下进行反应。通过高效液相方法检测反应过程中产物L-PPT的浓度和e.e.值的变化情况。
结果显示产物浓度随时间的推移而逐渐升高，3.5h内反应完成，底物转化率大于99％，产物e.e.值始终保持在99.5％以上。

Claims (10)

  1. 一种草铵膦脱氢酶突变体，其特征在于，由来源于荧光假单胞菌(Pseudomonas fluorescens)的草铵膦脱氢酶第164位氨基酸由丙氨酸突变为甘氨酸，第205位精氨酸突变为赖氨酸，第332位苏氨酸突变为丙氨酸获得的所得，氨基酸序列如SEQ ID No.1所示。
  2. 编码如权利要求1所述草铵膦脱氢酶突变体的基因。
  3. 一种基因工程菌，包括宿主细胞和转入宿主细胞的目的基因，其特征在于，所述目的基因包含如权利要求2所述的基因。
  4. 如权利要求3所述的基因工程菌，其特征在于，目的基因还包括葡萄糖脱氢酶的编码基因或甲酸脱氢酶突变体的编码基因。
  5. 如权利要求4所述的基因工程菌，其特征在于，使用双基因表达载体，所述草铵膦脱氢酶突变体的基因克隆到其中一个多克隆位点区域，葡萄糖脱氢酶的编码基因或甲酸脱氢酶突变体的编码基因克隆到第二个多克隆位点区域。
  6. 如权利要求5所述的基因工程菌，其特征在于，所述葡萄糖脱氢酶的编码基因序列GenBank登录号为KM817194.1，所述葡萄糖脱氢酶的编码基因起始密码子与质粒上对应核糖体结合位点之间的连接碱基长度为8～10bp。
  7. 如权利要求5所述的基因工程菌，其特征在于，所述甲酸脱氢酶的编码基因序列如SEQ ID No.3所示，甲酸脱氢酶突变体是将第224的组氨酸突变为谷氨酰胺。
  8. 如权利要求1所述草铵膦脱氢酶突变体、如权利要求2所述基因或如权利要求3～7所述基因工程菌在制备L-草铵膦中的应用。
  9. 一种L-草铵膦的制备方法，其特征在于，以2-羰基-4-(羟基甲基膦酰基)丁酸为底物，葡萄糖或甲酸铵为辅助底物，在无机氨基供体、和辅酶循环系统存在的条件下，利用催化剂催化底物反应获得L-草铵膦；
    所述辅酶循环系统为葡萄糖脱氢酶循环系统或甲酸脱氢酶循环系统；所述催化剂为如权利要求5～7所述的基因工程菌、该基因工程菌的粗酶液或固定化的所述基因工程菌，
    当所述基因工程菌中含有所述葡萄糖脱氢酶的编码基因时，辅助底物为葡萄糖，辅酶循环系统为葡萄糖脱氢酶循环系统；当所述基因工程菌中含有所述甲酸脱氢酶的编码基因时，辅助底物为甲酸铵，辅酶循环系统为甲酸脱氢酶循环系统。
  10. 一种一锅法多酶同步定向进化方法，其特征在于，包括：
    (1)将草铵膦脱氢酶基因导入pETDuet-1质粒上的MCS1，葡萄糖脱氢酶或甲酸脱氢酶导入带有草铵膦脱氢酶载体pETDuet-1质粒上的MCS2上进行异源表达；
    (2)将步骤(1)克隆有双酶基因的序列利用易错PCR方法同时进行两个酶基因元件和连接元件的突变，并构建易错PCR基因文库进行筛选获得四个有益位点，其中三个位点是草铵膦脱氢酶的A164，R205和T332位点，另外一个有益位点是在葡萄糖脱氢酶基因和核糖体结合位点之间的连接碱基长度或者甲酸脱氢酶的H224位点；
    (3)将上述的草铵膦脱氢酶的A164，R205和T332位点进行定点饱和突变，得到的各双酶单菌落分别进行L-草铵膦制备实验筛选获得草铵膦转化率最高的突变体A164G_R205K_T332A；
    (4)在上述使L-草铵膦转化率最高的草铵膦脱氢酶突变体A164G_R205K_T332A偶联酶基础上，进一步定向进化葡萄糖脱氢酶或者甲酸脱氢酶使L-草铵膦制进一步提高，具体就是葡萄糖脱氢酶基因和核糖体结合位点之间的连接碱基长度进行优化或者甲酸脱氢酶的H224位点进行定点饱和突变，然后得到的各双酶单菌落分别进行L-草铵膦制备实验筛选获得草铵膦转化率最高的突变体。
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Families Citing this family (8)

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CN111621482B (zh) * 2020-06-30 2022-04-29 浙江工业大学 一种草铵膦脱氢酶突变体、基因工程菌及一锅法多酶同步定向进化方法
CN111876396B (zh) * 2020-07-07 2022-04-01 浙江工业大学 双辅酶依赖型草铵膦脱氢酶突变体及其在催化合成l-草铵膦中的应用
CN112908417A (zh) * 2020-12-30 2021-06-04 浙江工业大学 功能序列和结构模拟相结合的基因挖掘方法、nadh偏好型草铵膦脱氢酶突变体及应用
CN112779233B (zh) * 2021-01-22 2024-02-20 浙江工业大学 重组草铵膦脱氢酶、基因工程菌及其在制备l-草铵膦中的应用
CN113817694B (zh) * 2021-09-01 2023-07-28 浙江工业大学 草铵膦脱氢酶突变体、编码基因、基因工程菌及其应用
CN113897382B (zh) * 2021-09-01 2023-10-20 浙江工业大学 辅酶自足型大肠杆菌及其构建方法与应用
WO2023114930A1 (en) * 2021-12-15 2023-06-22 The George Washington University Compositions for and methods of improving directed evolution of biomolecules
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108588045A (zh) * 2018-03-09 2018-09-28 浙江大学 谷氨酸脱氢酶突变体及其在制备l-草铵膦中的应用
CN109609475A (zh) * 2018-12-28 2019-04-12 浙江工业大学 草铵膦脱氢酶突变体及其合成l-草铵膦的应用
WO2019192505A1 (zh) * 2018-04-03 2019-10-10 上海弈柯莱生物医药科技有限公司 一种l-谷氨酸脱氢酶突变体及其应用
CN110592036A (zh) * 2019-08-30 2019-12-20 浙江工业大学 一种草铵膦脱氢酶突变体及在氧化-还原多酶偶联生产l-草铵膦中的应用
CN110885803A (zh) * 2019-11-27 2020-03-17 浙江工业大学 重组草铵膦脱氢酶、基因工程菌及其在制备l-草铵膦中的应用
CN111621482A (zh) * 2020-06-30 2020-09-04 浙江工业大学 一种草铵膦脱氢酶突变体、基因工程菌及一锅法多酶同步定向进化方法

Family Cites Families (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0022458D0 (en) * 2000-09-13 2000-11-01 Medical Res Council Directed evolution method
CN103275896B (zh) * 2013-05-27 2015-03-04 浙江工业大学 蜡状芽孢杆菌及其在微生物转化制备l-草铵膦中的应用
CN106811488A (zh) * 2015-12-02 2017-06-09 中国科学院大连化学物理研究所 一种生物法联产甘露醇与葡萄糖酸或葡萄糖酸盐的方法
CN107630052B (zh) * 2017-03-29 2018-05-29 武汉茵茂特生物技术有限公司 L-草铵膦的生物转化方法
CN106978368B (zh) * 2017-03-31 2020-04-21 浙江工业大学 解鸟氨酸拉乌尔菌及其应用
CN106978453B (zh) * 2017-03-31 2019-10-29 浙江大学 一种利用氨基酸脱氢酶制备l-草铵膦的方法
CA3058095A1 (en) * 2017-05-08 2018-11-15 Novozymes A/S Mannanase variants and polynucleotides encoding same
CN107384978A (zh) * 2017-07-15 2017-11-24 安徽工程大学 一种磁性固定化多酶体系一锅法制备葡萄糖酸的方法
CN108484665B (zh) * 2018-04-16 2020-06-23 浙江工业大学 一种从酶转化液中分离提取l-草铵膦的方法
US20210139542A1 (en) * 2018-05-18 2021-05-13 Chan Zuckerberg Biohub, Inc. Epitope restriction for antibody selection
CN108893455B (zh) * 2018-05-30 2020-07-28 浙江工业大学 一种转氨酶突变体及其生产l-草铵膦的应用
CN108660122B (zh) * 2018-05-30 2020-11-13 浙江工业大学 一种转氨酶、突变体及其生产l-草铵膦的应用
CN108676822B (zh) * 2018-05-31 2021-10-29 南京林业大学 一锅法酶水解-氧化纤维素制备葡萄糖酸的方法
KR20210040408A (ko) * 2018-07-30 2021-04-13 코덱시스, 인코포레이티드 조작된 글리코실트랜스퍼라제 및 스테비올 글리코시드 글루코실화 방법
CN109750009B (zh) * 2018-12-25 2019-12-17 浙江工业大学 一种草铵膦脱氢酶突变体及其应用
CN109609582B (zh) * 2019-01-17 2021-11-09 重庆惠健生物科技有限公司 一种微生物催化去消旋化制备l-草铵膦的方法
CN110791484B (zh) * 2019-08-15 2020-12-25 浙江工业大学 一种草铵膦脱氢酶突变体及其在生产l-草铵膦中的应用
CN111363775B (zh) * 2020-03-18 2022-08-05 浙江工业大学 一种生物酶法去消旋化制备l-草铵膦的方法、草铵膦脱氢酶突变体及应用
CN111321193B (zh) * 2020-03-18 2020-11-10 浙江工业大学 一种生物多酶偶联法氧化还原不对称制备l-草铵膦的方法
CN111876396B (zh) * 2020-07-07 2022-04-01 浙江工业大学 双辅酶依赖型草铵膦脱氢酶突变体及其在催化合成l-草铵膦中的应用
CN112908417A (zh) * 2020-12-30 2021-06-04 浙江工业大学 功能序列和结构模拟相结合的基因挖掘方法、nadh偏好型草铵膦脱氢酶突变体及应用
CN112779233B (zh) * 2021-01-22 2024-02-20 浙江工业大学 重组草铵膦脱氢酶、基因工程菌及其在制备l-草铵膦中的应用
CN112961181A (zh) * 2021-03-16 2021-06-15 北京鑫佰利科技发展有限公司 一种l-草铵膦的产品精制和酶的回收方法
CN112760353A (zh) * 2021-03-16 2021-05-07 北京鑫佰利科技发展有限公司 一种l-草铵膦的生产方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108588045A (zh) * 2018-03-09 2018-09-28 浙江大学 谷氨酸脱氢酶突变体及其在制备l-草铵膦中的应用
WO2019192505A1 (zh) * 2018-04-03 2019-10-10 上海弈柯莱生物医药科技有限公司 一种l-谷氨酸脱氢酶突变体及其应用
CN109609475A (zh) * 2018-12-28 2019-04-12 浙江工业大学 草铵膦脱氢酶突变体及其合成l-草铵膦的应用
CN110592036A (zh) * 2019-08-30 2019-12-20 浙江工业大学 一种草铵膦脱氢酶突变体及在氧化-还原多酶偶联生产l-草铵膦中的应用
CN110885803A (zh) * 2019-11-27 2020-03-17 浙江工业大学 重组草铵膦脱氢酶、基因工程菌及其在制备l-草铵膦中的应用
CN111621482A (zh) * 2020-06-30 2020-09-04 浙江工业大学 一种草铵膦脱氢酶突变体、基因工程菌及一锅法多酶同步定向进化方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHENG, F. ET AL.: "Tuning amino acid dehydrogenases with featured sequences for L-phosphinothricin synthesis by reductive amination", JOURNAL OF BIOTECHNOLOGY, vol. 312, 20 March 2020 (2020-03-20), XP086092028, DOI: 10.1016/j.jbiotec.2020.03.001 *
XU JIAN: "Directed Evolution of Enzymes for the Regulation of Selectivity and Catalytic Promiscuity", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, ENGINEERING SCIENCE & TECHNOLOGY I, no. 8, 15 August 2019 (2019-08-15), XP055883610 *
YIN, XJ. ET AL.: "Efficient Reductive Amination Process for Enantioselective Synthesis of L-Phosphinothricin Applying Engineered Glutamate Dehydrogenase", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 102, no. 10, 16 March 2018 (2018-03-16), XP036493130, DOI: 10.1007/s00253-018-8910-z *

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