Glutamate oxidase mutants with improved enzyme activity and their use in the production of alpha-ketoglutarate

Abstract

The application belongs to the field of bioengineering, and specifically discloses a glutamate oxidase mutant with improved enzyme activity. The application carries out site-directed mutation and saturation mutation on a glutamate oxidase encoding gene from Streptomyces mobaraensis, wherein the mutations are C536R, P532R, Q378D, V548E and G236D. After induced purification, the optimal glutamate oxidase mutant has a specific enzyme activity of 1.69 U / mg, which is about 30% higher than that of the wild type strain, indicating that the amino acid residue at position 378 has a great influence on the catalytic action and stability of the enzyme. In addition, the application also discloses a construction method of the glutamate oxidase mutant with improved enzyme activity and application thereof in the production of amino acid derivatives such as alpha-ketoglutaric acid. Therefore, the beneficial mutant provided by the application can lay a good foundation for the industrialized fermentation production of alpha-ketoglutaric acid and other products.

Description

Technical Field

[0001] The invention relates to the construction and application of a glutamate oxidase mutant with improved enzyme activity, and belongs to the technical field of genetic engineering. Background Art

[0002] α-Ketoglutarate (α-KG) is an important organic acid and a key intermediate in the tricarboxylic acid cycle and amino acid metabolism. As a precursor of various amino acids and heterocyclic compounds, it plays a crucial role in cellular metabolism, serving as a source of L-glutamine, L-glutamic acid, and L-proline, as well as a variety of metabolic intermediates. It not only participates in the synthesis and metabolism of amino acids, vitamins, and organic acids, but also plays a vital role in protein synthesis, skeletal muscle development, and metabolism. Therefore, it is used to reduce body loss in postoperative and chronic patients. α-KG also has anti-cyanide properties, scavenges free radicals, and slows aging. Clinically, it is used to reduce uremia in hemodialysis patients, promote wound healing, and treat chronic renal failure in hemodialysis patients. Due to its important physiological effects and excellent chemical properties, α-KG is widely used in food, medicine, cosmetics, and other fields, and therefore has broad application prospects.

[0003] Currently, there are three methods for producing α-KG: chemical synthesis, microbial fermentation, and bioconversion. The disadvantages of chemical production of α-KG are harsh conditions, high energy consumption, and low yields. The synthesis process uses large amounts of hazardous reagents such as cyanide, heavy metal ions, strong acids, and strong bases, often posing serious food safety risks and environmental pollution issues. Fermentation production of α-KG offers advantages such as abundant raw materials, low costs, and high yields. However, due to the excessive production of byproducts such as pyruvate, fumarate, and malate during the fermentation process, it also faces disadvantages such as a lack of bacterial strain resources, difficulty in isolation and purification, and a long production cycle. Currently, research on the use of Loa lipolytica for microbial fermentation of α-KG has been extensive, but its industrial application is limited by its long fermentation cycle, low acid production rate, high byproduct count, operational difficulties, high cost, and limited supply of n-alkanes. Bioconversion, on the other hand, offers advantages such as ease of operation, mild conditions, high raw material utilization, and high conversion rates. This method has the advantages of easy separation and purification, and has important application needs in significantly reducing production costs and solving the problem of glutamic acid overcapacity. Therefore, it is widely used in the production of α-KG.

[0004] L-glutamate oxidase (LGOX) is a flavin protease that uses flavin adenine dinucleotide as a cofactor. It exhibits high substrate stereoisomer selectivity, high catalytic efficiency, and mild reaction conditions. It can oxidize L-glutamate to α-KG, hydrogen peroxide, and ammonia without the addition of exogenous cofactors. L-glutamate oxidase's strict substrate specificity for L-glutamate and its ability to catalyze glutamate without the addition of any exogenous cofactors make it a highly advantageous catalyst for α-KG production. Numerous L-glutamate oxidases have been isolated from Streptomyces, but most studies have focused on their protein structure and mechanism of action. Industrial applications of L-glutamate oxidases for α-KG biocatalysis remain relatively limited. Therefore, obtaining glutamate oxidases with excellent catalytic performance is crucial for achieving high α-KG yields. Improving the catalytic activity of glutamate oxidases through random and site-directed mutagenesis is crucial for enhancing their industrial application. Summary of the Invention

[0005] The primary objective of the present invention is to provide a glutamate oxidase mutant with improved catalytic performance, thereby facilitating the production of metabolites such as α-KG. Another technical problem to be solved by the present invention is to provide a method for constructing and screening the aforementioned glutamate oxidase mutant.

[0006] The present invention is achieved through the following technical approach: using the wild-type glutamate oxidase gene from Streptomyces mobaraensis as a template, primers are designed for site-directed saturation mutagenesis, and a mutant library is obtained through site-directed mutagenesis. Subsequently, a recombinant plasmid library is constructed using pET21b as a backbone, and the recombinant plasmid library is transformed and introduced into Escherichia coli BL21 (DE3). A high-throughput screening system for the mutant library is established. Through primary screening in 96-well plates and secondary screening in test tubes and shake flasks, glutamate oxidase mutants with enhanced enzyme activity are finally screened.

[0007] A glutamate oxidase mutant is obtained by having one or both of the mutations P532R or Q378D in the wild-type glutamate oxidase, wherein the amino acid sequence of the wild-type glutamate oxidase is shown in SEQ ID NO: 3. The specific enzyme activity of the mutant can be increased by about 30% compared with the wild-type enzyme, for example.

[0008] The wild-type glutamate oxidase gene coding sequence provided by the present invention is shown as SEQ ID NO: 1, and the coding gene of the glutamate oxidase mutant, preferably, has a nucleotide sequence as shown in SEQ ID NO: 2.

[0009] The present invention further provides a recombinant expression vector containing the gene, preferably, a prokaryotic expression vector, including but not limited to pET21b and pXMJ19, etc. The present invention also provides a recombinant cell containing the encoding gene or the recombinant expression vector, preferably, a prokaryotic cell, including but not limited to Escherichia coli and Corynebacterium glutamicum, etc.

[0010] Finally, the present invention also provides the use of the mutant or its encoding gene in the biosynthesis of α-KG. Specifically, the method includes culturing recombinant cells containing the encoding gene to produce α-KG, and collecting the produced α-KG. Optionally, the method also includes purifying the produced α-KG.

[0011] The present invention has the beneficial effect of obtaining L-glutamate oxidase mutants with enhanced catalytic activity through directed evolution and screening of L-glutamate oxidase from Streptomyces moyuanensis. Therefore, the glutamate oxidase mutants provided by the present invention can lay a good foundation for the efficient and rapid fermentation production of α-KG and have promising prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] 图1 Schematic diagram of α-KG concentration determination based on color change. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific examples and with reference to the accompanying drawings, but it should not be understood as limiting the present invention. The experimental methods used in the examples are conventional methods well known to those skilled in the art unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0014] Example 1 Construction of glutamate oxidase mutants

[0015] L-glutamate oxidase (LGOX) catalyzes the conversion of L-glutamate to α-KG. It is a flavoenzyme that uses flavin adenine dinucleotide (FAD) as a coenzyme. However, because it contains a non-covalently bound FAD, it can catalyze the reaction without the addition of an exogenous FAD cofactor. Studies have found that this enzyme is primarily found in Streptomyces, but the enzyme production capacity varies greatly. Discovering and identifying glutamate oxidases with high activity or substrate specificity, and obtaining enzyme catalytic systems that meet the requirements of physiological research and production applications, will provide favorable conditions for the industrial production of α-KG.

[0016] Protein language models have been proven to be effective in generating functional proteins and facilitating the optimization of a given protein. ESM (Evolutionary Scale Modeling) is a method that uses deep learning technology to predict protein structure and function, which can easily predict and generate proteins [Meier J, et al. Language models enable zero-shot prediction of the effects of mutations on protein function. Advances in neural information processing systems, 2021, 34: 29287-29303.]. The core idea of ESM is to regard protein sequence as a kind of language, and each amino acid as a character, and then use an autoregressive neural network Transformer to learn the statistical rules of this language, by learning the evolutionary rules and sequence-structure-function relationships of proteins on a large-scale protein sequence database. The advantage of ESM is that it can extract rich information from protein sequences to predict the mutation effects of proteins, i.e. the influence of changes in single or multiple amino acids in the protein sequence on the structure and function of the protein, such as stability, activity, affinity, etc. In this invention, the above protein language model and machine learning algorithm are used to predict the potential mutation sites and effects in the LGOX protein (sequence shown as SEQ ID NO: 3) derived from Streptomyces mobaraensis glutamate oxidase, and the top five mutation sites with the highest scores are selected for subsequent experimental verification, i.e. C536R, P532R, Q378D, V548E and G236D.

[0017] According to the sequence design primer LGOX-For / LGOX-Rev, the codon-optimized glutamate oxidase from Streptomyces mobaraensis synthesized by Jinshui Company was used as a template to obtain the wild-type glutamate oxidase gene fragment. After double digestion with NdeI / XhoI, it was subcloned into the pET21b plasmid backbone to obtain the recombinant plasmid pET21b-LGOX. Using site-directed mutagenesis strategy, according to the above amino acid sites to be mutated, mutation primers were designed, and the recombinant plasmid pET21b-LGOX was used as a template to obtain a plasmid vector containing the mutation site by PCR method, and then transformed into E. coli expression strain BL21(DE3).

[0018] Example 2 Activity determination of glutamate oxidase mutants

[0019] The recombinant E. coli containing the pET21-LGOX, pET21-LGOX-C536R, pET21-LGOX-P532R, pET21-LGOX-Q378D, pET21-LGOX-V548E and pET21-LGOX-G236D plasmids obtained above were induced to express proteins. The specific method was as follows: single clones were picked and inoculated into 100 mL LB liquid medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl) and cultured overnight. Subsequently, according to the starting OD 600 ≈0.1. Transfer the seed solution to 100 mL of LB liquid medium and culture until the bacterial concentration reaches OD 600 When the protein reaches approximately 0.6-0.8, IPTG is added to a final concentration of 0.4-0.6 mM to induce expression of the target protein. The culture temperature is adjusted to 25-30°C and induction culture is continued for 16-20 hours. After induction is complete, the bacterial culture is homogenized by high-pressure homogenization and purified using a standard laboratory Ni-NTA nickel column method to obtain pure glutamate oxidase protein. Protein concentration is determined using the Bradford method, which uses Coomassie Brilliant Blue G-250 as a colorimetric assay and bovine serum albumin as a standard.

[0020] Enzyme activity was determined by performing an enzymatic reaction using pure enzyme purified using a Ni-NTA nickel column. The total reaction volume was 1 mL, consisting of 600 μL of 100 mM sodium glutamate substrate, 300 μL of ddH2O, and approximately 50 μL of enzyme solution of appropriate concentration. The mixture was incubated at 37°C for 30 min and terminated by boiling in a boiling water bath for 10 min. After completion of the reaction, 20 μL of the enzymatic reaction solution was added to 400 μL of 2 mM 2,4-dinitrophenylhydrazine solution. The reaction was incubated at 37°C for 20 min, followed by the addition of 1 mL of 1 M sodium hydroxide solution. The colorimetric solution was diluted 3-10 times and the absorbance was measured (detection wavelength 390 nm). Sample concentrations were calculated by plotting a standard curve. Enzyme activity was defined as 1 unit of enzyme required to generate 1 μmol of α-KG per minute.

[0021] The results showed that compared with the wild-type natural enzyme, the activity of the most preferred glutamate oxidase mutant LGOX-Q378D reached 1.69 U / mg, which was 30% higher than that of the wild-type bacteria. The activity of the second preferred mutant glutamate oxidase LGOX-P532R was 14% higher than that of the wild-type bacteria. However, the catalytic activities of other glutamate oxidase mutants were weaker than those of the natural enzyme to varying degrees.

[0022] Example 3 Construction and screening of glutamate oxidase Q378D site saturation mutations

[0023] Given the important contribution of amino acid residue 378 of glutamate oxidase to the catalytic activity of the enzyme, a site-directed saturation mutagenesis strategy was adopted for the most preferred glutamate oxidase mutant LGOX-Q378D in Example 2. Random degenerate primers Q378-For (cgccatgttNNNgtgagcccgctgatgagctatg) and Q378-Rev (gcgggctcacNNNaacatggcgcagaccggtaaac) were designed, where N represents any of the four bases A, C, G, or T. A recombinant plasmid library containing saturation mutations at this site was obtained by PCR. A recombinant plasmid library containing site-specific saturation mutations was introduced into the E. coli expression strain BL21(DE3) for screening. The following steps were performed: transformants were picked with a sterile toothpick and added to a 96-well plate containing 200 μL of LB medium. After incubation at 37°C and 800 rpm / min for 6-8 hours, 100 μL of the culture was transferred to a 96-deep-well plate containing 600 μL of LB medium. Induction was then added with 0.4 mM isopropylthiogalactopyranoside (IPTG) and the culture was induced overnight at 16°C and 800 rpm / min for 14-16 hours. After induction, the 96-deep-well plate was centrifuged at 4000 rpm / min for 10 minutes to harvest the cells, and the supernatant was discarded. After resuspension, 600 μL of 1 M sodium glutamate substrate was added to each well using a dispenser. The cells were then incubated on a plate shaker at 37°C for 2 hours for whole-cell catalysis. α-KG readily reacts with 2,4-dinitrophenylhydrazine to produce a reddish-brown phenylhydrazone nitroquinone, which can be used to screen for superior glutamate oxidase mutants based on their color depth. In this example, after multiple rounds of saturation mutagenesis and screening, no mutant superior to LGOX-Q378D was found, suggesting that mutating glutamine at position 378 of the glutamate oxidase encoding gene sequence to aspartic acid is the optimal approach.

[0024] Example 4 Application of glutamate oxidase mutants in bioconversion production of α-KG

[0025] This example provides an application of a glutamate oxidase mutant in the production of α-KG. In the specific embodiment of this example, the preferred commercial expression vectors are pET21b and pXMJ19, and the preferred expression host bacteria are Escherichia ( Escherichia ) and Corynebacterium ( Corynebacterium ), more preferably Escherichia coli ( Escherichia coli ) and Corynebacterium glutamicum ( Corynebacterium glutamicum ), most preferably Escherichia coli BL21 (DE3) and Corynebacterium glutamicum ATCC 13032.

[0026] The pET21b-LGOX-Q378D recombinant plasmid was transformed into the Escherichia coli expression strain BL21(DE3), and α-KG-producing E. coli strains were obtained after plate resistance screening. Primers were designed based on the sequence, and the glutamate oxidase mutant gene LGOX-Q378D was subcloned into the pXMJ19 backbone using molecular assembly techniques such as the ClonExpress® II One Step Cloning Kit (Vazyme Biotech, China). This recombinant plasmid was then transformed into Corynebacterium glutamicum ATCC 13032, and α-KG-producing strains were obtained after plate resistance screening.

[0027] A fermentation tank was used to establish an α-KG bioconversion system. Monosodium glutamate monohydrate (MSG) substrate was added at a final concentration of 200 g / L, along with 20 g / L of bacterial cells. The fermentation speed was controlled at 300-500 r / min, the dissolved oxygen concentration was set at 10-30%, the catalytic reaction temperature was set at 30-37°C, and the reaction time was 20-40 hours. After the reaction, the components of the catalytic reaction liquid were quantitatively analyzed using liquid chromatography.

[0028] Liquid chromatography analysis revealed a relatively simple composition of the whole-cell conversion solution, with minimal residual substrate and impurities. Engineered strains constructed from mutant glutamate oxidases exhibited superior α-KG production capacity. Escherichia coli α-KG-producing strains achieved 142.2 g / L of α-KG after 32 hours of catalytic reaction, and Corynebacterium glutamicum α-KG-producing strains achieved 145.4 g / L after 24 hours of catalytic reaction. Compared with recombinant engineered strains containing unmutated glutamate oxidase genes, these strains offer advantages in terms of whole-cell catalytic time, product yield, and production capacity. This approach significantly reduces production costs and simplifies the process, demonstrating promising prospects for industrial application.

Claims

1. A glutamate oxidase mutant, characterized in that: The wild-type glutamate oxidase has only one mutation, Q378D or P532R, wherein the amino acid sequence of the wild-type glutamate oxidase is shown in SEQ ID NO:

3.

2. The gene encoding the glutamate oxidase mutant according to claim 1.

3. The coding gene according to claim 2, wherein Its nucleotide sequence is shown in SEQ ID NO:

2.

4. A recombinant expression vector containing the coding gene according to claim 2 or 3.

5. The recombinant expression vector according to claim 4, wherein It is a prokaryotic expression vector.

6. The recombinant expression vector according to claim 5, wherein The starting vector is pET21b or pXMJ19.

7. A recombinant host cell containing the encoding gene according to claim 2 or 3 or the recombinant expression vector according to claim 4.

8. The recombinant host cell according to claim 7, wherein It is a prokaryotic cell.

9. The recombinant host cell according to claim 8, wherein It is Escherichia coli or Corynebacterium glutamicum.

10. Use of the glutamate oxidase mutant or the gene encoding it according to claim 1 in the biosynthesis of α-ketoglutarate.

11. The use according to claim 10, characterized in that The method comprises the steps of culturing a recombinant host cell containing the encoding gene to produce alpha-ketoglutarate, and collecting the produced alpha-ketoglutarate.

12. The use according to claim 11, characterized in that The process also includes a step of purifying the produced α-ketoglutarate.

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