L-threonine aldolase mutant and application thereof

Abstract

The invention discloses an L-threonine aldolase mutant and an application of the L-threonine aldolase mutant. The L-threonine aldolase mutant is named as LTAM101, and the amino acid sequence of the L-threonine aldolase mutant is as shown in SEQ ID NO. 2. The genetically engineered bacterium containing the LTAM101 recombinant expression plasmid is applied to fermentation preparation of glycine. The glycine is produced by taking xylose as a substrate through a simple shake flask fermentation process under an aerobic condition, the glycine yield of a strain R2 containing wild type LTA expression plasmids is 446.8 mg / L, and the glycine yield of a strain R3 containing mutant LTAM101 expression plasmids is 809.7 mg / L, which is 81.2% higher than that of R2. According to the mutant LTAM101, the yield of glycine is remarkably increased by improving the enzyme catalytic activity, the production cost can be reduced, the production efficiency can be improved, an efficient technical path is provided for biosynthesis of glycine, and biotransformation of numerous high-added-value products with glycine as a synthesis intermediate is promoted.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering technology and application, and particularly relates to an L-threonine aldolase mutant and application thereof. Background Art

[0002] Glycine, also known as aminoacetic acid, is a widely used fine chemical intermediate in many industries including pesticides, feed, and food. 1 In the pesticide field, it can be used to produce plant growth regulators and organophosphorus herbicides; in the feed field, it is mainly used as an additive in animal feed, providing important nutrients and extending the shelf life of feed; in the food field, it can be used as a food preservative and flavoring agent. 1 .

[0003] There are three glycine biosynthesis pathways in nature, namely the serine pathway, the glyoxylate pathway, and the threonine pathway. Among them, the main problems of the serine pathway are carbon atom waste and complex C1 metabolism regulation. 2 The glyoxylate cycle may be activated or enhanced under certain anaerobic conditions. For example, in Corynebacterium glutamicum, although the glyoxylate cycle maintains a certain level of activity and flux under aerobic conditions, its activity may be lower than that under anaerobic conditions. 3 .

[0004] The threonine synthesis pathway can be roughly divided into three parts. The glucose entering the cell is converted into L-threonine through the glycolysis pathway, the tricarboxylic acid cycle (TCA cycle) and the aspartate family amino acid metabolism pathway. L-threonine is then catalyzed by L-threonine aldolase to produce glycine. 4 .

[0005] Threonine aldolase (TA) is a glycine-dependent aldolase that catalyzes reversible alcohol-aldehyde condensation reactions and is an important biocatalyst for the formation of CC bonds. 5 Threonine aldolase uses glycine as a donor substrate and 5'-pyridoxal phosphate (PLP) as a cofactor to catalyze the synthesis of b-hydroxy-a-amino acids and their derivatives from glycine and various aldehyde compounds; or reversely catalyzes the cleavage of threonine into glycine and acetaldehyde. 6 According to Threonine C α Threonine aldolases can be divided into two categories based on their stereoselectivity: L-TA and D-TA. 7 Among them, L-TA is based on its β The stereoselectivity of L-TA can be further divided into three categories: (1) catalyzing only L-threonine; (2) catalyzing only L-allo-threonine; (3) catalyzing both L-threonine and L-allo-threonine. This type of L-TA is a low-specificity L-TA.

[0006] L-TA is mainly present in Escherichia coli 8 , Aeromonas jandaei 9 , Streptomyces coelicolor 10 and Pseudomonas putida 11 etc., and it is a key enzyme for synthesizing glycine in the threonine pathway. Although L-TA existing in the natural state undertakes certain metabolic functions in organisms, its catalytic activity is insufficient in industrial application scenarios and difficult to meet the requirements of high-efficiency and large-scale production. Limited by the insufficient catalytic activity of natural L-TA, during the current process of using this enzyme for glycine synthesis, there is generally a problem of low yield, which not only increases the production cost but also restricts the extensive application and in-depth development of glycine as an important chemical raw material in multiple fields such as medicine, food, and agriculture.

[0007] In addition, as a key organic synthesis intermediate, glycine often relies on the catalytic action of L-TA during the biotransformation process of participating in many high-value-added products (such as amino acid derivatives, pharmaceutical intermediates, pesticides, and fine chemicals, etc.). However, due to the inherent defect of low catalytic activity of natural L-TA, the conversion efficiency of glycine is low, the reaction process is slow, and the yield and purity of the target product are difficult to meet the requirements of industrial production. This bottleneck not only restricts the application potential of glycine as a substrate in the field of biocatalysis but also restricts the industrialization process of related green manufacturing technologies. Therefore, it is urgent to develop L-TA mutants with high catalytic activity through means such as enzyme engineering modification, directed evolution, or protein design to break through the existing technical limitations and promote the innovation and development of glycine biotransformation technology. Summary of the Invention

[0008] To overcome the deficiency of low catalytic activity of natural L-TA in the prior art, the main object of the present invention is to provide an L-threonine aldolase mutant and its application. Through directed evolution technology, the problems of low glycine conversion efficiency, sluggish reaction kinetics, and difficult yield of the target product to meet the requirements of industrial production caused by the low catalytic activity of natural L-TA are solved.

[0009] To achieve the aforementioned invention object, the technical solutions adopted by the present invention include:

[0010] An L-threonine aldolase mutant, named LTAM101, whose amino acid sequence is shown in SEQ ID NO.2.

[0011] A gene encoding the L-threonine aldolase mutant, whose nucleotide sequence is shown in SEQ ID NO.39.

[0012] A recombinant expression plasmid containing the encoding gene, and the recombinant expression plasmid is named pEC-aspC-LTAM101, and its nucleotide sequence is shown in SEQ ID NO.38.

[0013] A genetically engineered bacterium R3 containing the recombinant expression plasmid.

[0014] Use of the genetically engineered bacterium in the fermentation preparation of glycine.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] The present invention provides an L-threonine aldolase mutant LTAM101 and a genetically engineered bacterium containing a recombinant expression plasmid of LTAM101. Under aerobic conditions, glycine is produced by a simple shake-flask fermentation process using xylose as a substrate. The glycine yield of strain R2 containing the wild-type LTA expression plasmid is 446.8 mg / L, and the glycine yield of strain R3 containing the mutant LTAM101 expression plasmid is 809.7 mg / L, which is 81.2% higher than that of R2. The mutant LTAM101 significantly increases the glycine yield by enhancing the enzyme catalytic activity, can reduce the production cost and improve the production efficiency, provides an efficient technical route for the biosynthesis of glycine, and helps to promote the bioconversion of many high-value-added products with glycine as a synthetic intermediate. Description of the Drawings

[0017] Figure 1 It is the plasmid map of the mutant plasmid pEC-BS294-LTA1;

[0018] Figure 2 It is the plasmid map of the expression plasmid pEC-aspC-LTA1;

[0019] Figure 3 It is the plasmid map of the expression plasmid pEC-aspC-LTAM101;

[0020] Figure 4 It is a schematic diagram of the yields of the genetically engineered bacteria R1, RM101, R2, and R3 in shake-flask fermentation for the production of glycine. Detailed Embodiments

[0021] The following examples are exemplary descriptions of the main experimental evidences, rather than limiting the core content and application scope of the present invention disclosed by the quantity of evidences. It should be noted that in all these drawings and corresponding descriptions, only the concepts, principles and representative experimental evidences of the disclosed embodiments of the present invention are shown exemplarily. When the evidence chain is complete, it is not necessary to show all the specific detailed information and extended details of each example listed in the present invention.

[0022] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present invention pertains. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0023] The original strain Corynebacterium glutamicum ATCC13032 used in the present invention was sourced from ATCC (The Global Bioresource Center, http: / / www.atcc.org / ) and purchased in October 2012.

[0024] The starting strain used in the present invention is the Corynebacterium glutamicum recombinant strain Cev-18-5, and the construction method of Cev-18-5 has been described in detail in the authorized patent "Corynebacterium glutamicum for Producing Succinic Acid by Fermenting Xylose and Its Use" (Patent No.: CN201810541718.X).

[0025] The genotype of the Corynebacterium glutamicum recombinant strain Cev-18-5 is C.glutamicum ATCC13032ΔldhAΔackA-ptaΔpqoΔcatPsodpycPsodppc,Psod talPsodtktPtufaraE(del21bp),ackA-pta::PsodxylAB:C131T,NCgl2538:C331T.

[0026] The pUC18-BSTG plasmid used in the present invention has been described in detail in the patent "Construction Method and Application of Glycine Riboswitch Gene Regulation Circuit" (Application No.: CN202111609842.3).

[0027] The wild-type L-threonine aldolase (LTA) used in the present invention is derived from Pseudomonas putida KT2440. For specific information, see ATCC (The Global Bioresource Center, http: / / www.atcc.org / ). Its amino acid sequence is shown in SEQ ID NO.1, and the plasmid pUC18-LTA1 containing this gene was obtained by entrusting Genewiz (https: / / www.genewiz.com.cn / ) for synthesis.

[0028] The glycine-activated riboswitch BS2-94 used in the present invention is a mutant derived from the natural glycine-activated riboswitch of Bacillus subtilis, and its specific sequence is shown in SEQ ID NO.3. The related plasmid pUC18-M137-BS294 was obtained by commissioning Genewiz, Inc. for synthesis (https: / / www.genewiz.com.cn / ).

[0029] The aspartate aminotransferase (AspC) used in the present invention is derived from Escherichia coli K-12 MG1655. For specific information, see ATCC (The Global Bioresource Center, http: / / www.atcc.org / ). Its specific sequence is shown in SEQ ID NO.4. The related plasmid pUC18-aspC was obtained by commissioning Genewiz, Inc. for synthesis (https: / / www.genewiz.com.cn / ).

[0030] The tetracycline resistance gene (tetA) used in the present invention has a specific sequence shown in SEQ ID NO.5.

[0031] The constitutive promoter M1-37 used in the present invention has a specific sequence shown in SEQ ID NO.6.

[0032] Both the above-mentioned tetracycline resistance gene (tetA) and constitutive promoter M1-37 are referred to in the literature Development and characterization of a glycine biosensor system for fine-tuned metabolic regulation in Escherichia coli.

[0033] The constitutive promoter sod used in the present invention is derived from Corynebacterium glutamicum ATCC13032, and its specific sequence is shown in SEQ ID NO.7.

[0034] The plasmid pEC-XK99E was obtained by commissioning Genewiz, Inc. for synthesis (https: / / www.genewiz.com.cn / ).

[0035] The primers used were obtained by commissioning Genewiz, Inc. for synthesis (https: / / www.genewiz.com.cn / ).

[0036] The seamless cloning reagents used were purchased from Abbkine (https: / / abclonal.com.cn / ).

[0037] Other biochemical reagents used were purchased from Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0038] E. coli DH5α competent cells were prepared by the conventional CaCl2 method.

[0039] Corynebacterium glutamicum Cev-18-5 competent cells were prepared by conventional methods.

[0040] LB liquid medium: yeast extract powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L. 2% agar powder was added to LB solid medium.

[0041] BHI liquid medium: brain heart infusion broth powder 74 g / L. 2% agar powder was added to BHI solid medium.

[0042] CGIII medium: yeast extract powder 10 g / L, tryptone 10 g / L, MOPS 21 g / L, NaCl 2.5 g / L. The pH was adjusted to 7 with 5 M NaOH aqueous solution.

[0043] CGXII medium: (NH4)2SO4 20 g / L, Urea 5 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, MOPS 21 g / L, MgSO4·7H2O 0.25 g / L, 1 mL of 10 g / L CaCl2 solution. The pH was adjusted to 7 with 5 M NaOH aqueous solution.

[0044] The concentration of the antibiotic was: kanamycin 10 μg / mL.

[0045] Detection method for glycine: 100 μL of the sample was placed in a brown centrifuge tube. Then, 100 μL of sodium bicarbonate solution and 100 μL of 2,4-dinitrofluorobenzene acetonitrile solution were added successively. After mixing, it was placed in a 60 °C water bath in the dark for 1 h, and then cooled to room temperature. 700 μL of 0.05 mol / L phosphate buffer solution (pH = 7.0) was added, and centrifuged at 12000 rpm for 30 min. The supernatant was taken for detection.

[0046] Mobile phase A solution: 0.02 mol / L NaAc-HAc buffer solution. Weighed 1.64 g of anhydrous sodium acetate solid, added 800 mL of distilled water to dissolve, adjusted the pH to about 6.2 with HAc, and made up the volume to 1000 mL.

[0047] Mobile phase B solution: 100% pure acetonitrile.

[0048] The specific detection parameters are shown in Table 1:

[0049] Table 1

[0050]

[0051] Example 1: Construction of the high-throughput screening platform plasmid pEC-BS294-LTA1 and the LTA plasmid mutant library P1, including the following steps:

[0052] (1) Amplify the plasmid vector fragment using pEC-XK99E (SEQ ID NO.30) as a template, and the primers used are BSTA-F-1 (SEQ ID NO.8) / BSTA-F-2 (SEQ ID NO.9); amplify the M1-37 promoter and tetA gene fragments using the plasmid pUC18-BSTG (SEQ ID NO.31) as a template, and the primers used are M137-1 (SEQ ID NO.10) / M137-2 (SEQ ID NO.11) and TETA-1 (SEQ ID NO.12) / TETA-2 (SEQ ID NO.13); amplify the sod promoter from the Corynebacterium glutamicum ATCC 13032 genome, and the primers used are Sod-1 (SEQ ID NO.18) / Sod-2 (SEQ ID NO.19); amplify LTA1 using the plasmid pUC18-LTA1 (SEQ ID NO.32) as a template, and the primers used are LTA1-1 (SEQ ID NO.16) / LTA1-2 (SEQ IDNO.17); amplify aspC using the plasmid pUC18-aspC (SEQ ID NO.33) as a template, and the primers used are AspC-1 (SEQ IDNO.20) / AspC-2 (SEQ ID NO.21); amplify the riboswitch BS294 using the plasmid pUC18-M137-BS294 (SEQ ID NO.34) as a template, and the primers used are BS294-1 (SEQ ID NO.14) / BS294-2 (SEQ ID NO.15); as Figure 1 shown, obtain the high-throughput screening platform plasmid pEC-BS294-LTA1 (SEQ ID NO.35) by fusion PCR and seamless cloning;

[0053] (2) Use LTA1-1 (SEQ ID NO.16) / LTA1-2 (SEQ ID NO.17) as primers, amplify the LTA1 fragment using the plasmid pEC-BS294-LTA1 (SEQ ID NO.35) as a template for error-prone PCR, and the obtained PCR product is subjected to gel cutting and recovery and used as the template for the next round of error-prone PCR. A total of three rounds of error-prone PCR are performed to increase the mutation rate of the gene library;

[0054] (3) Amplify the vector fragment of the plasmid mutant library using plasmid pEC-BS294-LTA1 (SEQ ID NO.35) as a template, and the primers used are YC-F-1 (SEQ ID NO.22) / YC-F-2 (SEQ ID NO.23); obtain plasmid mutant library P1 through seamless cloning.

[0055] Example 2: Enrichment and screening of mutants in the LTA mutant library, including the following steps:

[0056] (1) Electrotransform plasmid pEC-BS294-LTA1 (SEQ ID NO.35) into the competent cells of Cev-18-5 to obtain strain R1. Culture R1 in a test tube containing 5 ml of BHI liquid medium for 12 h, and then dilute and spread it on BHI solid medium plates containing tetracycline at concentrations of 30, 40, 50, 60, 70, 80, 90, and 100 mg / L. Observe the growth of the strains after culturing for 24 h. When the tetracycline concentration of the solid BHI plate is set to 70 mg / L, the strain containing the wild type hardly grows. Therefore, the tetracycline concentration for subsequent screening is set to 70 mg / L;

[0057] (2) Electrotransform plasmid library P1 into the competent cells of Cev-18-5 to obtain strain library C1. Dilute the bacterial solution and spread it on several BHI solid plates containing 70 mg / L tetracycline. A total of 101 single colonies survived on all the plates;

[0058] (3) Inoculate the above 101 single colonies into 24-well plates containing CGXⅡ medium and culture for 72 h to detect the glycine yield. As Figure 4 shown, the R1 strain contains the wild-type L-threonine aldolase LTA1 screening plasmid, and its glycine yield is 117 mg / L; the RM101 strain contains the L-threonine aldolase mutant LTAM101 screening plasmid, and its glycine yield is the highest, reaching 223.8 mg / L, which is 91.3% higher than that of R1. Among them, the amino acid sequence of the L-threonine aldolase mutant LTAM101 is as shown in SEQ ID NO.2, and the gene nucleotide sequence encoding the L-threonine aldolase mutant LTAM101 is as shown in SEQ ID NO.39.

[0059] Example 3: Construction of the LTAM101 expression plasmid and the engineering bacteria containing the expression plasmid, including the following steps:

[0060] (1) The gene fragments of LTA1 and LTAM101 were amplified using plasmids pEC-BS294-LTA1 (SEQ ID NO.35) and pEC-LTAM101 (SEQ ID NO.36) as templates, respectively. The primers used were LTAM-1 (SEQ ID NO.24) / LTAM-2 (SEQ ID NO.25); the expression plasmid vector and the aspC gene fragment were amplified using plasmid pEC-BS294-LTA1 (SEQ ID NO.35) as a template, and the primers used were LTAM-F-1 (SEQ ID NO.26) / LTAM-F-2 (SEQ ID NO.27) and AspC-M-1 (SEQ ID NO.28) / AspC-M-2 (SEQ ID NO.29); as Figure 2 and 3 shown, the expression plasmids pEC-aspC-LTA1 (SEQ ID NO.37) and pEC-aspC-LTAM101 (SEQ ID NO.38) were obtained by seamless cloning;

[0061] (2) The constructed expression plasmids pEC-aspC-LTA1 (SEQ ID NO.37) and pEC-aspC-LTAM101 (SEQ ID NO.38) were electrotransformed into the competent cells of Cev-18-5 to obtain strains R2 and R3.

[0062] Example 4: Shake flask fermentation of genetically engineered bacteria R2 and R3 to produce glycine, including the following steps:

[0063] (1) The cryotubes storing strains R2 and R3 were taken out from the -80 °C refrigerator, and the bacterial liquid was picked up with an inoculation loop and streaked on a solid BHI medium containing kanamycin resistance, and cultured at 30 °C and 220 rpm for 12 h to obtain single colonies;

[0064] (2) The single colonies were picked into a test tube containing 5 mL of liquid BHI medium and cultured overnight at 30 °C and 220 rpm to obtain a primary seed solution;

[0065] (3) Kanamycin and xylose with a final concentration of 20 g / L were added to 250 mL of CGIII medium with a liquid loading of 50 mL, and 1 mL of the primary seed solution was transferred to the medium and cultured at 30 °C and 220 rpm for 12 h to obtain a secondary seed solution;

[0066] (4) 50 μL of Trace element, Biotin, kanamycin and xylose with a final concentration of 20 g / L were added to 250 mL of fermentation medium CGXII with a liquid loading of 50 mL, and an appropriate amount of the secondary seed solution was transferred to the medium to make the initial OD600 was 0.3, and the culture was carried out at 30 °C and 220 rpm for 60 h;

[0067] (5) Samples were taken regularly every 12 h to measure the OD of the bacteria 600 , sugar concentration and glycine production.

[0068] As Figure 4 shown, the R2 strain contains the wild-type L-threonine aldolase LTA1 expression plasmid, and its glycine production is 446.8 mg / L; the R3 strain contains the L-threonine aldolase mutant LTAM101 expression plasmid, and its glycine production reaches 809.7 mg / L, which is 81.2% higher than that of R2.

[0069] The content of the above embodiments only describes in detail a part of the specific implementation manners of the present invention, but is not limited to the embodiments currently disclosed in the present invention. In addition, the substantial content protected by the present invention is not limited to this. Without departing from the design scope of the present invention, any other modifications, equivalent replacements, improvements, etc. made according to the thinking principle and technical means of the present invention all belong to the protection scope of the present invention.

Claims

1. An L-threonine aldolase mutant, characterized in that, The L-threonine aldolase mutant is named LTAM101, and its amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding the L-threonine aldolase mutant according to claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

39.

3. The recombinant expression plasmid containing the coding gene described in claim 2, characterized in that, The recombinant expression plasmid is named pEC-aspC-LTAM101, and its nucleotide sequence is shown in SEQ ID NO.

38.

4. The genetically engineered bacterium R3 containing the recombinant expression plasmid described in claim 3.

5. Use of the genetically engineered bacterium described in claim 4 in the fermentative preparation of glycine.

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