An RthB protein mutant and its application in isoleucine production

Abstract

The present invention discloses an RthB protein mutant and its application in isoleucine production. The RthB protein mutant is obtained by performing single-site mutations or multi-site mutations at the 16th, 21st, 117th, and 156th positions of the amino acid sequence shown in SEQ ID NO:1. By mutating and modifying the RthB protein, the L-isoleucine fermentation yield of recombinant microorganisms expressing the mutant protein is increased, and the threonine content in the fermentation broth is decreased, thereby improving the yield and purity of isoleucine.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an RthB protein mutant and its application in isoleucine production. Background Art

[0002] L-isoleucine belongs to the three-branched chain amino acids and is also one of the eight essential amino acids in the human body. It plays a particularly important role in human life metabolism. If isoleucine is lacking in the human body for a long time, it will affect the physiological functions of the body, leading to metabolic disorders, decreased body resistance, etc. Also, due to its special structure and function, it is widely used in the fields of food, medicine, health products, cosmetics, etc.

[0003] The main methods for producing L-isoleucine at home and abroad are mainly microbial fermentation methods. The main advantages of this method are low requirements for equipment, relatively cheap raw materials, mild reaction conditions, and convenient for large-scale production, etc. Currently, commonly used L-isoleucine-producing bacteria include Corynebacterium crenatum, Corynebacterium glutamicum, Brevibacterium flavum, Escherichia coli, Brevibacterium lactofermentum, etc. The fermentation of these strains does not require the addition of precursor substances, and the microorganisms directly use their own metabolism to synthesize the required amino acids. It has been found that too high intracellular amino acid concentration will restrict the final yield of high-yield strains. For example, too high intracellular isoleucine concentration will have a negative feedback effect on the related synthase in its metabolic pathway, and reducing its intracellular isoleucine concentration can not only weaken the negative feedback effect, but also increase the yield of isoleucine. At the same time, when producing isoleucine by fermentation, threonine, as an intermediate product in the isoleucine synthesis pathway, will appear in the fermentation broth, and this by-product will become an impurity in the isoleucine product. The RthB family (homoserine / threonine tolerance family) transporter has the ability to transport threonine extracellularly. There is a prediction by bioinformatics methods that the protein encoded by NCgl2566 in the RthB family can participate in the secretion and transport of L-leucine. L-isoleucine and L-leucine have the same chemical formula, similar molecular size and shape, and are both branched-chain amino acids. Therefore, it is speculated that the transporters for transporting L-leucine and L-isoleucine may have similar spatial structures. Therefore, by modifying the protein encoded by the above NCgl2566 to change the transporter from excreting threonine to excreting isoleucine, it will weaken the feedback inhibition of isoleucine and reduce the content of miscellaneous acids in isoleucine, thereby increasing the yield and purity of isoleucine. Summary of the Invention

[0004] The object of the present invention is to modify the protein encoded by the NCgl2566 gene in the RthB transporter protein family to weaken the feedback inhibition of isoleucine and / or reduce the content of miscellaneous acids in isoleucine, thereby increasing the yield and purity of isoleucine.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In the first aspect, the present invention provides an RthB protein mutant, which is obtained by single-site mutation or multi-site mutation at the 16th, 21st, 117th, and 156th positions of the amino acid sequence shown in SEQ ID NO: 1.

[0007] In one embodiment of the present invention, the RthB protein mutant has undergone mutation at one or more of the following sites (1)-(4):

[0008] (1) The alanine at the 16th position of the amino acid sequence shown in SEQ ID NO: 1 is mutated to arginine, i.e., A16R, and the mutated amino acid sequence is SEQ ID NO: 3, and the nucleotide sequence is as shown in SEQ ID NO: 4;

[0009] (2) The glycine at the 21st position of the amino acid sequence shown in SEQ ID NO: 1 is mutated to aspartic acid, i.e., G21D, and the mutated amino acid sequence is SEQ ID NO: 5. The nucleotide sequence is as shown in SEQ ID NO: 6;

[0010] (3) The asparagine at the 117th position of the amino acid sequence shown in SEQ ID NO: 1 is mutated to tyrosine (N117Y, and the mutated amino acid sequence is SEQ ID NO: 7. The nucleotide sequence is as shown in SEQ ID NO: 8), or mutated to tryptophan (N117W, and the mutated amino acid sequence is SEQ ID NO: 9. The nucleotide sequence is as shown in SEQ ID NO: 10);

[0011] (4) The serine at the 156th position of the amino acid sequence shown in SEQ ID NO: 1 is mutated to phenylalanine (S156F, and the mutated amino acid sequence is SEQ ID NO: 11, and the nucleotide sequence is as shown in SEQ ID NO: 12), or mutated to leucine (S156L, and the mutated amino acid sequence is SEQ ID NO: 13, and the nucleotide sequence is as shown in SEQ ID NO: 14), or mutated to tyrosine (S156Y, and the mutated amino acid sequence is SEQ ID NO: 15, and the nucleotide sequence is as shown in SEQ ID NO: 16).

[0012] In one embodiment of the present invention, the RthB protein mutant is one of the following:

[0013] (1) The alanine at position 16 of the amino acid sequence shown in SEQ ID NO:1 is mutated to arginine (A16R), and the mutated amino acid sequence is SEQ ID NO:3, and the nucleotide sequence is as shown in SEQ ID NO:4;

[0014] (2) The glycine at position 21 of the amino acid sequence shown in SEQ ID NO:1 is mutated to aspartic acid (G21D), and the mutated amino acid sequence is SEQ ID NO:5, and the nucleotide sequence is as shown in SEQ ID NO:6;

[0015] (3) The asparagine at position 117 of the amino acid sequence shown in SEQ ID NO:1 is mutated to tyrosine (N117Y), and the mutated amino acid sequence is SEQ ID NO:7, and the nucleotide sequence is as shown in SEQ ID NO:8;

[0016] (4) The asparagine at position 117 of the amino acid sequence shown in SEQ ID NO:1 is mutated to tryptophan (N117W), and the mutated amino acid sequence is SEQ ID NO:9, and the nucleotide sequence is as shown in SEQ ID NO:10;

[0017] (5) The serine at position 156 of the amino acid sequence shown in SEQ ID NO:1 is mutated to phenylalanine (S156F), and the mutated amino acid sequence is SEQ ID NO:11, and the nucleotide sequence is as shown in SEQ ID NO:12;

[0018] (6) The serine at position 156 of the amino acid sequence shown in SEQ ID NO:1 is mutated to leucine (S156L), and the mutated amino acid sequence is SEQ ID NO:13, and the nucleotide sequence is as shown in SEQ ID NO:14;

[0019] (7) The serine at position 156 of the amino acid sequence shown in SEQ ID NO:1 is mutated to tyrosine (S156Y), and the mutated amino acid sequence is SEQ ID NO:15, and the nucleotide sequence is as shown in SEQ ID NO:16.

[0020] The above RthB protein mutants are converted from excreting L-threonine to excreting L-isoleucine.

[0021] In a second aspect, the present invention provides a nucleic acid molecule encoding the above RthB protein mutant.

[0022] In one embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:4.

[0023] In one embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 6.

[0024] In one embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 8.

[0025] In one embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.

[0026] In a third aspect, the present invention provides a recombinant vector containing a nucleic acid molecule encoding the above-mentioned RthB protein mutant.

[0027] In a fourth aspect, the present invention provides a recombinant microorganism expressing the above-mentioned RthB protein mutant.

[0028] Furthermore, the recombinant microorganism contains a nucleic acid molecule or a recombinant vector encoding the above-mentioned RthB protein mutant.

[0029] Furthermore, the recombinant microorganism is a prokaryotic microorganism or a eukaryotic microorganism expressing the above-mentioned RthB protein mutant.

[0030] In one embodiment of the present invention, the prokaryotic microorganism is Corynebacterium or its subspecies, or Escherichia coli.

[0031] In one embodiment of the present invention, the prokaryotic microorganism is Corynebacterium crenatum, Corynebacterium glutamicum, Brevibacterium flavum, Escherichia coli, or Brevibacterium lactofermentum, preferably Corynebacterium glutamicum.

[0032] In one embodiment of the present invention, the recombinant microorganism is a bacterium obtained by introducing the recombinant plasmid pK18mobsacB - mutant RthB into Corynebacterium glutamicum ATCC14308.

[0033] Compared with the recombinant microorganism expressing the RthB protein, the recombinant microorganism expressing the above-mentioned RthB protein mutant has increased L - isoleucine production and decreased content of by - product L - threonine.

[0034] In a fifth aspect, the present invention provides the use of the above-mentioned RthB protein mutant, the nucleic acid molecule encoding the RthB protein mutant, or the recombinant vector containing the nucleic acid molecule in the preparation of isoleucine - producing bacteria.

[0035] In a sixth aspect, the present invention provides the use of the above-mentioned RthB protein mutant, a nucleic acid molecule encoding the RthB protein mutant, a recombinant vector containing the nucleic acid molecule, or a recombinant microorganism expressing the above-mentioned RthB protein mutant in any one of the following (a1)-(a4):

[0036] (a1) Use in the production of isoleucine;

[0037] (a2) Use in increasing the yield of isoleucine;

[0038] (a3) Use in reducing the accumulation of threonine in the isoleucine fermentation broth;

[0039] (a4) Use in increasing the yield of isoleucine and reducing the accumulation of threonine.

[0040] In a seventh aspect, the present invention provides a method for increasing the yield of isoleucine. The strain used contains an RthB protein mutant, and the RthB protein mutant is obtained by single-site mutation or multi-site mutation at positions 16, 21, 117, and 156 of the amino acid sequence shown in SEQ ID NO:1.

[0041] In one embodiment of the present invention, the RthB protein mutant has undergone mutation at one or more of the following sites (1)-(4):

[0042] (1) Alanine at position 16 of the amino acid sequence shown in SEQ ID NO:1 is mutated to arginine;

[0043] (2) Glycine at position 21 of the amino acid sequence shown in SEQ ID NO:1 is mutated to aspartic acid;

[0044] (3) Asparagine at position 117 of the amino acid sequence shown in SEQ ID NO:1 is mutated to tyrosine, or mutated to tryptophan;

[0045] (4) Serine at position 156 of the amino acid sequence shown in SEQ ID NO:1 is mutated to phenylalanine, or mutated to leucine, or mutated to tyrosine.

[0046] In one embodiment of the present invention, the RthB protein mutant is one of the following:

[0047] (1) Alanine at position 16 of the amino acid sequence shown in SEQ ID NO:1 is mutated to arginine (A16R), and the mutated amino acid sequence is SEQ ID NO:3, and the nucleotide sequence is as shown in SEQ ID NO:4;

[0048] (2) The glycine at position 21 of the amino acid sequence shown in SEQ ID NO:1 was mutated to aspartic acid (G21D), and the mutated amino acid sequence is SEQ ID NO:5, and the nucleotide sequence is as shown in SEQ ID NO:6;

[0049] (3) The asparagine at position 117 of the amino acid sequence shown in SEQ ID NO:1 was mutated to tyrosine (N117Y), and the mutated amino acid sequence is SEQ ID NO:7, and the nucleotide sequence is as shown in SEQ ID NO:8;

[0050] (4) The asparagine at position 117 of the amino acid sequence shown in SEQ ID NO:1 was mutated to tryptophan (N117W), and the mutated amino acid sequence is SEQ ID NO:9, and the nucleotide sequence is as shown in SEQ ID NO:10;

[0051] (5) The serine at position 156 of the amino acid sequence shown in SEQ ID NO:1 was mutated to phenylalanine (S156F), and the mutated amino acid sequence is SEQ ID NO:11, and the nucleotide sequence is as shown in SEQ ID NO:12;

[0052] (6) The serine at position 156 of the amino acid sequence shown in SEQ ID NO:1 was mutated to leucine (S156L), and the mutated amino acid sequence is SEQ ID NO:13, and the nucleotide sequence is as shown in SEQ ID NO:14;

[0053] (7) The serine at position 156 of the amino acid sequence shown in SEQ ID NO:1 was mutated to tyrosine (S156Y), and the mutated amino acid sequence is SEQ ID NO:15, and the nucleotide sequence is as shown in SEQ ID NO:16.

[0054] Compared with the prior art, the present invention has the following beneficial technical effects:

[0055] 1. The present invention provides an RthB protein mutant and its coding gene. The mutant is obtained by site-directed mutagenesis (single-site or multi-site mutagenesis among A16R, G21D, N117Y / N117W, S156F / S156L / S156Y) of NCgl2566 encoding the RthB protein derived from Corynebacterium glutamicum ATCC 14308. It is used to construct an isoleucine-producing bacterium, and the L-isoleucine fermentation yield of the isoleucine-producing bacterium is increased and the threonine content in the fermentation broth is decreased, thereby improving the yield and purity of isoleucine.

[0056] 2. The present invention provides a method for increasing the production of L-isoleucine. By expressing the RthB protein mutant in a microorganism and fermenting and culturing the recombinant microorganism, the fermentation yield of L-isoleucine can be increased. Compared with the recombinant microorganism expressing the RthB protein, the recombinant microorganism expressing the above RthB protein mutant can significantly increase the yield of L-isoleucine while reducing the content of L-threonine by-product. Among them, the single mutation of N117W in the amino acid sequence of the RthB protein has the most significant effect. Its fermentation yield of L-isoleucine is increased by 47.18%, the conversion rate is increased by 29.48%, and the fermentation by-product L-threonine is reduced by 96.73%, which has great industrial application value. Detailed implementation manners

[0057] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0058] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions, such as "Molecular Cloning: A Laboratory Manual" (J. Sambrook, D.W. Russell, translated by Huang Peitang, Wang Jiaxi, Zhu Houchu, etc. 3rd edition, Beijing: Science Press, 2002).

[0059] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0060] 1. The sources or formulations of the kits and culture media involved in the following embodiments are as follows:

[0061] TAKARA Max DNA Polymerase kit, purchased from TaKaRa, product number R045A.

[0062] ClonExpress II One Step Cloning Kit, purchased from Novoprotein, product number C112-02.

[0063] LBB solid medium (1L): Trypton 5g, NaCl 5g, Yeast Extract 2.5g, Brain Heart Infusion 18.5g, made up to 1L with ultrapure water; 2% Agar is added to each solid medium, and the sterilization conditions are 121 °C for 20 min.

[0064] LBB Liquid Medium (1L): 5g of Trypton, 5g of NaCl, 2.5g of Yeast Extract, 18.5g of Brain Heart Infusion. Make up to 1L with ultrapure water. Sterilization condition: 121°C, 20 min.

[0065] Corynebacterium glutamicum Competent Medium (1L): 10g of Trypton, 10g of NaCl, 5g of Yeast Extract, 30g of Glycine, 1g of Tween 80. Make up to 1L with ultrapure water. Aliquot into shake flasks (30 / 250 mL). Sterilization condition: 121°C, 20 min.

[0066] LS Medium (Electroporation Recovery Medium) (1L): 5g of Trypton, 5g of NaCl, 2.5g of Yeast Extract, 18.5g of Brain Heart Infusion, 91g of Sorbitol. Make up to 1L with ultrapure water. Sterilization condition: 121°C, 20 min.

[0067] Antibiotic and Sucrose Solution:

[0068] 1000×Kanamycin Sulfate (Kan) Stock Solution 0.03 g / mL;

[0069] 10×Sucrose (S) Stock Solution 1 g / mL. Sterilization condition: 115°C, 15 min.

[0070] Seed Medium: 25 g / L of Glucose Monohydrate, 0.5 g / L of Ammonium Sulfate, 0.5 g / L of Magnesium Sulfate, 1.25 g / L of Urea, 40 g / L of Corn Steep Liquor, 1 g / L of Potassium Dihydrogen Phosphate. Solvent is water, pH 7.2.

[0071] Fermentation Medium: 120 g / L of Glucose, 40 g / L of Ammonium Sulfate, 10 g / L of Corn Steep Liquor, 1 g / L of Potassium Dihydrogen Phosphate, 0.5 g / L of Magnesium Sulfate, 0.5 g / L of Ferrous Sulfate, 20 g / L of Calcium Carbonate. Solvent is water, pH 7.2.

[0072] 2. The primer, plasmid and strain information involved in the following examples are as follows:

[0073] The primer information used in the present invention is shown in Table 1.

[0074] Table 1

[0075]

[0076]

[0077] The plasmids constructed in the present invention are shown in Table 2.

[0078] Table 2

[0079]

[0080]

[0081] The strains constructed in the present invention are shown in Table 3.

[0082] Table 3

[0083]

[0084] 3. Method for constructing an editing plasmid containing an RthB protein mutant

[0085] (1) Amplify the coding gene Ncgl2566 of the RthB protein (including 300 bp upstream and downstream thereof) from the genomic DNA of Corynebacterium glutamicum ATCC14308. The primers used are P2566-F and P2566-R. Connect the amplified Ncgl2566 fragment to the multiple cloning site of plasmid pK18mobsacB by one-step cloning to construct the editing plasmid pK18mobsacB-RthB.

[0086] (2) Using plasmid pK18mobsacB-RthB as a template, perform PCR amplification with mutant primers and the TAKARA MaxDNA Polymerase kit to obtain the mutated linear plasmid.

[0087] PCR amplification system (total volume 50 μL): PrimeSTAR Max Premix (2X) 25 μL, template 10 ng, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, supplement ddH2O to 50 μL;

[0088] PCR program: Pre-denaturation at 98 °C for 5 min; denaturation at 98 °C for 10 s, annealing at 68 °C for 15 s, extension at 72 °C for 2 min, cycle 10 times, and reduce the annealing temperature by 1 °C for each cycle; denaturation at 98 °C for 10 s, annealing at 63 °C for 15 s, extension at 72 °C for 2 min, cycle 30 times; final extension at 72 °C for 5 min.

[0089] (3) Plasmid template digestion and recovery of the target product:

[0090] Add 1 μL of Thermo Scientific DpnI restriction endonuclease to the above 50 μL PCR reaction product, react at 37 °C for 1 - 2 h to digest the plasmid template; then perform agarose gel electrophoresis on the digestion product and recover the target fragment from the gel.

[0091] (4) Circularize and recover the linear plasmid fragment using Exnase II enzyme:

[0092] Use the ClonExpress II One Step Cloning Kit to catalyze the high-efficiency recombination of the linear plasmid to be mutated at the target site to obtain a circular mutated plasmid; Reaction system: 7 μL of gel-extracted product, 2 μL of 5×CE II buffer, 1 μL of Exnase II; Mix well and react at 37 °C for 30 min.

[0093] 4. Preparation and electrotransformation of Corynebacterium glutamicum competent cells

[0094] (1) Pick a loop of bacteria from the Corynebacterium glutamicum plate cultured by streaking and inoculate it into the LBB liquid medium. Incubate overnight at 37 °C and 200 r / min on a shaker.

[0095] (2) Prepare 10% glycerol and pre-cool it on ice.

[0096] (3) Transfer the activated bacteria to the Corynebacterium glutamicum competent cell medium, and adjust the initial OD of the bacterial liquid 600 to reach 0.4, and culture at 37 °C and 200 r / min until OD562 reaches 0.8.

[0097] (4) Place the cultured bacterial liquid and a sterilized 50 mL centrifuge tube on ice for pre-cooling for 30 min.

[0098] (5) Transfer the pre-cooled bacterial liquid to a centrifuge tube and centrifuge at 4 °C and 4000 r / min for 10 min.

[0099] (6) Discard the supernatant, gently resuspend the pellet with 10% glycerol, and centrifuge the resuspended solution at 4 °C and 4000 r / min for 10 min.

[0100] (7) Repeat step (6) twice.

[0101] (8) Discard the supernatant, gently resuspend the pellet according to the amount of adding 400 μL of glycerol per 50 mL of bacterial liquid, and aliquot 80 μL into each 1.5 mL EPP tube and store at -80 °C.

[0102] Electrotransformation procedure: Add the edited plasmid to the Corynebacterium glutamicum competent cells, gently mix well, then transfer the mixture to a 1 mm electroporation cuvette (pre-cooled on ice) and let it stand for 5 - 10 min. Apply an electric shock at 18 kV for 5 ms, incubate in a 46 °C water bath for 6 min, add 1 mL of pre-warmed LS medium, resuscitate at 30 °C and 100 r / min on a shaker for 2 h, and then spread on an LBB plate supplemented with Kan (30 μg / mL) and culture at 37 °C for 36 h.

[0103] 5. Construction of a recombinant strain containing the RthB protein mutant

[0104] The constructed editing plasmid was electrotransformed into the competent cells of the starting strain ATCC14308. The cells were recovered at 30 °C for 2 h and then collected. The cells were spread on LBB solid medium containing Kan (30 μg / mL) and cultured at 37 °C for about 36 h. The resulting transformants were the strains that had undergone the first homologous recombination. The corresponding transformants were selected and cultured in LBB liquid medium for 12 h. During this culture stage, a very small number of cells would undergo the second homologous recombination. The cells were diluted to 10 -5 or 10 -6 and then spread on LBB solid medium containing 10% sucrose. The resulting transformants were the strains that had undergone the second homologous recombination. Then, the transformants were randomly selected and spread on LBB solid medium containing 10% sucrose and LBB solid medium containing Kan (30 μg / mL) in turn for further screening. The transformants that could grow on the sucrose plate but not on the resistance plate were selected by mutual comparison for colony PCR identification and sequencing verification. The correctly identified strains were the recombinant strains containing the RthB protein mutant and could be preserved with 15% glycerol for future use.

[0105] 6. High-performance liquid chromatography detection method for the contents of L-threonine and L-isoleucine:

[0106] The amino acid yield was determined by HPLC derivatization method: The sample was diluted 20-fold with ultrapure water, filtered through a 0.22-μm aqueous filter membrane, and then analyzed by HPLC.

[0107] The chromatographic column was: ZORBAX Eclipse AAA (4.6 mm × 75 mm 3.5-Micron);

[0108] Mobile phase A: Weigh 6.24 g of sodium dihydrogen phosphate dihydrate, add 1000 mL of ultrapure water and stir until all the crystals are completely dissolved. Adjust the pH of the solution to 7.80 with sodium hydroxide and filter it through a 0.22-μm aqueous membrane.

[0109] Mobile phase B: Mix analytical grade acetonitrile, methanol and ultrapure water in a volume ratio of 45:45:10 and filter it through a 0.22-μm organic membrane.

[0110] Borax buffer solution: Weigh 19.06 g of borax and dissolve it in 1 L of pure water to obtain a 0.05 mol / L borax solution. Then adjust the pH to 9.5 with 2 mol / L sodium hydroxide and store it at 4 °C in the refrigerator;

[0111] Derivatizing agent: Weigh 0.343 g of o-phthalaldehyde and 0.1472 g of N-acetyl-L-cysteine, place them in a 25 mL volumetric flask, add 5 mL of absolute ethanol, dissolve them with ultrasonic waves, and then make up the volume to 25 mL with 0.05 M sodium borate solution. Filter through a membrane and store in the dark for later use.

[0112] High performance liquid chromatography detection method: Add 200 μL of borax buffer solution to the reaction flask, then add 20 μL of the test solution, and then add 20 μL of the derivatizing agent. The derivatization time is 1 min. Finally, add 360 μL of ultrapure water filtered through a 0.22 μm aqueous membrane.

[0113] The detection wavelength is 338 nm;

[0114] The flow rate of the mobile phase is 1.0 mL / min;

[0115] The column temperature of the chromatograph is 40 °C;

[0116] The injection volume is 5 μL.

[0117] The gradient elution conditions are as follows:

[0118]

[0119] Example 1. Selection of mutation sites of RthB protein

[0120] In the present invention, the key amino acid sites are determined by Autodock Vina docking. Sequence alignment and amino acid conservation analysis of the RthB protein are carried out, and then the RthB protein is subjected to single-point mutation modification. The results are shown in Table 4 below:

[0121] Table 4

[0122]

[0123]

[0124] As can be seen from Table 4, when the RthB protein and the RthB protein mutant are respectively docked with threonine (Thr) and isoleucine (Ile), any one of the amino acid mutations in A16R, G21D, N117Y, N117W, S156F, S156L, S156N, S156Y can reduce the affinity of the RthB protein for threonine and increase the affinity for isoleucine.

[0125] Example 2. Construction of pK18mobsacB-RthB plasmid

[0126] Amplify the gene Ncgl2566 encoding the RthB protein (including 300 bp of its upstream and downstream regions) from the genomic DNA of Corynebacterium glutamicum ATCC14308. The primers used are P2566-F and P2566-R, and the amplification conditions are as follows;

[0127] PCR amplification system (total volume 50 μL): PrimeSTAR Max Premix (2X) 25 μL, ATCC14308 genomic DNA 1 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, supplemented with ddH2O to 50 μL;

[0128] PCR program: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 1 min, 30 cycles, and final extension at 72°C for 5 min.

[0129] Using the ClonExpress II One Step Cloning Kit, ligate the amplified Ncgl2566 fragment and the pK18mobsacB plasmid by one-step cloning to construct the editing plasmid pK18mobsacB-RthB, and verify by sequencing. The verified plasmid pK18mobsacB-RthB is used for the construction of the following recombinant Corynebacterium glutamicum. The amino acid sequence of RthB is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2.

[0130] Example 3. Construction of recombinant Corynebacterium glutamicum mIH001 expressing the RthB*A16R protein mutant

[0131] 1. Construction of the circular mutant plasmid pK18mobsacB-RthB*A16R

[0132] (1) Using the pK18mobsacB-RthB plasmid as a template, A16R-F and A16R-R as mutant primers, perform PCR amplification using the TAKARA Max DNAPolymerase kit to obtain the mutated linear plasmid.

[0133] PCR amplification system (total volume 50 μL): PrimeSTAR Max Premix (2X) 25 μL, template 10 ng, A16R-F (10 μM) 1 μL, A16R-R (10 μM) 1 μL, supplemented with ddH2O to 50 μL;

[0134] PCR amplification program: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s, annealing at 68°C for 15 s, extension at 72°C for 2 min, 10 cycles, with the annealing temperature decreasing by 1°C for each cycle; denaturation at 98°C for 10 s, annealing at 63°C for 15 s, extension at 72°C for 2 min, 30 cycles; final extension at 72°C for 5 min.

[0135] (2) Plasmid template digestion and target product recovery:

[0136] Add 1 μL of Thermo Scientific DpnI restriction endonuclease to the above 50 μL of PCR reaction product, react at 37°C for 1 - 2 h to digest the plasmid template pK18mobsacB-RthB; then perform agarose gel electrophoresis on the digestion product, and recover the target fragment by gel extraction, namely the linear plasmid pK18mobsacB-RthB*A16R.

[0137] (3) Circularize the recovered linear plasmid fragment of pK18mobsacB-RthB*A16R using ExnaseⅡ enzyme:

[0138] Use the ClonExpress II One Step Cloning Kit to catalyze the high-efficiency recombination of the recovered linear plasmid pK18mobsacB-RthB*A16R at the site to be mutated to obtain the circularized mutant plasmid pK18mobsacB-RthB*A16R;

[0139] The reaction system and conditions are: 7 μL of gel extraction product, 2 μL of 5×CEⅡ buffer, 1 μL of ExnaseⅡ. Mix well and react at 37°C for 30 min.

[0140] 2. Transform the starting strain ATCC14308

[0141] Electrotransform the circularized mutant plasmid pK18mobsacB-RthB*A16R into the competent cells of the starting strain ATCC14308, resuscitate and culture at 30°C for 2 h to collect the cells, spread them on the LBB solid medium containing Kan (30 μg / mL), culture at 37°C for about 36 h, and the resulting transformants are the strains that have undergone the first homologous recombination. Select the corresponding transformants and culture them in the LBB liquid medium for 12 h. During this culture stage, a very small number of cells will undergo the second homologous recombination. Dilute the bacterial cells by 10 5After multiplying, it was coated on the LBB solid medium containing 10% sucrose, and the resulting transformants were the strains that had undergone secondary homologous recombination. Then, the transformants were randomly selected and successively coated on the LBB solid medium containing 10% sucrose and the LBB solid medium containing Kan (30 μg / mL) for re-screening. By mutual comparison, the transformants that could grow on the sucrose plate but not on the resistant plate were selected. Single colonies were picked for sequencing verification, and the correctly identified strain was the recombinant strain mIH001 containing the RthB*A16R protein mutant.

[0142] The RthB protein mutant RthB*A16R expressed by the recombinant strain mIH001 was a single mutation of A16R in the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence of this mutant was as shown in SEQ ID NO:3, and the nucleotide sequence was as shown in SEQ ID NO:4.

[0143] Example 4. Construction of recombinant Corynebacterium glutamicum mIH002, mIH003, mIH004, mIH005, mIH006, mIH007, mIH008 expressing RthB protein mutants

[0144] 1. Construction of recombinant Corynebacterium glutamicum mIH002 expressing the RthB*G21D protein mutant

[0145] Using the pK18mobsacB-RthB plasmid as a template and G21D-F and G21D-R as mutant primers, the editing plasmid pK18mobsacB-RthB*G21D was constructed. Through two rounds of homologous recombination, the recombinant strain mIH002 containing the RthB*G21D protein mutant was obtained (the specific plasmid construction and electrotransformation operation methods were the same as in Example 3);

[0146] The RthB protein mutant RthB*G21D expressed by the recombinant strain mIH002 was a single mutation of G21D in the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence of this mutant was as shown in SEQ ID NO:5, and the nucleotide sequence was as shown in SEQ ID NO:6.

[0147] 2. Construction of recombinant Corynebacterium glutamicum mIH003 expressing the RthB*N117Y protein mutant

[0148] Using the pK18mobsacB-RthB plasmid as a template and N117Y-F and N117Y-R as mutant primers, the editing plasmid pK18mobsacB-RthB*N117Y was constructed. Through two rounds of homologous recombination, the recombinant strain mIH003 containing the RthB*N117Y protein mutant was obtained (the specific plasmid construction and electrotransformation operation methods were the same as in Example 3);

[0149] The RthB protein mutant RthB*N117Y expressed by the recombinant strain mIH003 is a single N117Y mutation of the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence of this mutant is as shown in SEQ ID NO:7, and the nucleotide sequence is as shown in SEQ ID NO:8.

[0150] 3. Construction of recombinant Corynebacterium glutamicum mIH004 expressing the RthB*N117W protein mutant

[0151] Using the pK18mobsacB-RthB plasmid as a template and N117W-F and N117W-R as mutant primers, the editing plasmid pK18mobsacB-RthB*N117W was constructed. The recombinant strain mIH004 containing the RthB*N117W protein mutant was obtained through two rounds of homologous recombination (the specific plasmid construction and electrotransformation operation methods are the same as in Example 3);

[0152] The RthB protein mutant RthB*N117W expressed by the recombinant strain mIH004 is a single N117W mutation of the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence of this mutant is as shown in SEQ ID NO:9, and the nucleotide sequence is as shown in SEQ ID NO:10.

[0153] 4. Construction of recombinant Corynebacterium glutamicum mIH005 expressing the RthB*S156F protein mutant

[0154] Using the pK18mobsacB-RthB plasmid as a template and S156F-F and S156F-R as mutant primers, the editing plasmid pK18mobsacB-RthB*S156F was constructed. The recombinant strain mIH005 containing the RthB*S156F protein mutant was obtained through two rounds of homologous recombination (the specific plasmid construction and electrotransformation operation methods are the same as in Example 3);

[0155] The RthB protein mutant RthB*S156F expressed by the recombinant strain mIH005 is a single S156F mutation of the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence of this mutant is as shown in SEQ ID NO:11, and the nucleotide sequence is as shown in SEQ ID NO:12.

[0156] 5. Construction of recombinant Corynebacterium glutamicum mIH006 expressing the RthB*S156L protein mutant

[0157] Using the pK18mobsacB-RthB plasmid as a template and S156L-F and S156L-R as mutagenic primers, the editing plasmid pK18mobsacB-RthB*S156L was constructed. After two rounds of homologous recombination, the recombinant strain mIH006 containing the RthB*S156L protein mutant was obtained (the specific plasmid construction and electrotransformation operation methods are the same as in Example 3);

[0158] The RthB protein mutant RthB*S156L expressed by the recombinant strain mIH006 is a single S156L mutation of the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence of this mutant is as shown in SEQ ID NO:13, and the nucleotide sequence is as shown in SEQ ID NO:14.

[0159] 6. Construction of the recombinant Corynebacterium glutamicum mIH007 expressing the RthB*S156Y protein mutant

[0160] Using the pK18mobsacB-RthB plasmid as a template and S156Y-F and S156Y-R as mutagenic primers, the editing plasmid pK18mobsacB-RthB*S156Y was constructed. After two rounds of homologous recombination, the recombinant strain mIH007 containing the RthB*S156Y protein mutant was obtained (the specific plasmid construction and electrotransformation operation methods are the same as in Example 3);

[0161] The RthB protein mutant RthB*S156Y expressed by the recombinant strain mIH007 is a single S156Y mutation of the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence of this mutant is as shown in SEQ ID NO:15, and the nucleotide sequence is as shown in SEQ ID NO:16.

[0162] 7. Construction of the recombinant Corynebacterium glutamicum mIH008 expressing the RthB*S156N protein mutant

[0163] Using the pK18mobsacB-RthB plasmid as a template and S156N-F and S156N-R as mutagenic primers, the editing plasmid pK18mobsacB-RthB*S156N was constructed. After two rounds of homologous recombination, the recombinant strain mIH008 containing the RthB*S156N protein mutant was obtained (the specific plasmid construction and electrotransformation operation methods are the same as in Example 3);

[0164] The RthB protein mutant RthB*S156N expressed by the recombinant strain mIH008 is a single S156N mutation of the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence of this mutant is as shown in SEQ ID NO:17, and the nucleotide sequence is as shown in SEQ ID NO:18.

[0165] The above recombinant Corynebacterium glutamicum was preserved with 15% glycerol and stored in a -80°C ultra-low temperature freezer for future use.

[0166] Example 5: Fermentation of Recombinant Corynebacterium glutamicum to Produce L-Isoleucine

[0167] 1. Shake Flask Fermentation

[0168] Activation of the strain: Take out the strain preserved in a 15% glycerol tube in a -80°C ultra-low temperature freezer, quickly pick ice chips with a pipette tip onto the LBB solid medium, gently spread it and cover the entire plate, preferably without scratching the solid plate, and incubate it statically at 37°C. Pick monoclonal colonies of strains ATCC 14308, mIH001, mIH002, mIH003, mIH004, mIH005, mIH006, mIH007, mIH008 on the plate and inoculate them into the LBB liquid medium for activation, and incubate overnight at 37°C and 200 rpm. The activated strains were respectively subjected to seed culture and shake flask fermentation according to the following steps.

[0169] Seed culture: Inoculate the activated bacterial liquid into a 500 mL baffled shake flask containing 50 mL of seed medium at an inoculation amount of 5%, and culture it at 37°C and 200 rpm for 18 h to obtain the seed culture solution.

[0170] Shake flask fermentation: Take 1 mL of the seed culture solution and measure the OD 562 ; According to the conversion of the initial OD 562 = 1.8 of the fermentation medium, transfer the seed culture solution to a 500 mL baffled shake flask containing 50 mL of fermentation medium, and culture it at 37°C and 200 rpm for 48 h to obtain the fermentation broth. No additional aeration was carried out during the culture process.

[0171] 2. Product Extraction and Detection

[0172] Take 1 mL of the fermentation broth, centrifuge it at 10000 g for 1 min, dilute the supernatant 20 times, filter it through a 0.22 μm filter head, and detect the concentration of the product L-isoleucine and the concentration of the by-product L-threonine in the filtrate by liquid phase according to the above high performance liquid chromatography detection method.

[0173] After detection, the results of L-isoleucine shake flask fermentation of the original strain ATCC 14308 and the modified recombinant Corynebacterium glutamicum mIH001, mIH002, mIH003, mIH004, mIH005, mIH006, mIH007, mIH008 are shown in Table 5.

[0174] Table 5

[0175]

[0176] As shown in Table 5, compared with the starting strain ATCC14308, the L-isoleucine production and conversion rates of the recombinant strains mIH001, mIH002, mIH003, mIH004, mIH005, mIH006, and mIH007 were all increased, and the by-product L-threonine of all mutant strains was reduced; among them, compared with the starting strain ATCC14308, the L-isoleucine production of the recombinant strain mIH004 expressing the RthB protein mutant RthB*N117W was increased by 47.18%, the conversion rate was increased by 29.48%, and the by-product L-threonine was reduced by 96.73%. The above results indicate that any of the following mutant modifications to the RthB protein, namely RthB*A16R, RthB*G21D, RthB*N117Y, RthB*N117W, RthB*S156F, RthB*S156L, RthB*S156Y, can not only increase the L-isoleucine production, but also reduce the production of the by-product L-threonine. It is speculated that the above mutations cause the RthB protein to change from originally transporting threonine outwards to transporting isoleucine outwards. On the one hand, it prevents threonine from leaking out of the cell, reducing the threonine content in the fermentation broth, and the threonine that has not leaked out can also continue to synthesize more isoleucine downstream; on the other hand, it excretes isoleucine, reducing the intracellular isoleucine concentration, and to a certain extent relieving the negative feedback inhibition of the related synthase on the metabolic pathway, thereby increasing the isoleucine production and purity.

[0177] The above is only the preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the idea of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and refinements made without departing from the principle of the present invention should be regarded as within the protection scope of the present invention.

Claims

1. An RthB protein mutant, which is obtained by performing any one of the following single mutations on the amino acid sequence shown in SEQ ID NO: 1: The asparagine at position 117 in the amino acid sequence shown in SEQ ID NO: 1 is mutated to tyrosine; The asparagine at position 117 in the amino acid sequence shown in SEQ ID NO: 1 is mutated to tryptophan; The serine at position 156 in the amino acid sequence shown in SEQ ID NO: 1 is mutated to phenylalanine; The serine at position 156 in the amino acid sequence shown in SEQ ID NO: 1 is mutated to leucine; The serine at position 156 in the amino acid sequence shown in SEQ ID NO: 1 is mutated to tyrosine.

2. A nucleic acid molecule encoding the RthB protein mutant according to claim 1.

3. A recombinant vector containing the nucleic acid molecule according to claim 2.

4. A recombinant microorganism expressing the RthB protein mutant according to claim 1.

5. The recombinant microorganism according to claim 4, wherein The recombinant microorganism contains the nucleic acid molecule according to claim 2, or the recombinant vector according to claim 3.

6. The recombinant microorganism according to claim 4, characterized in that, The recombinant microorganism uses corynebacterium or its subspecies, or Escherichia as the host.

7. The recombinant microorganism according to claim 6, characterized in that, The recombinant microorganism uses Corynebacterium crenatum, Corynebacterium glutamicum, Brevibacterium flavum, Escherichia coli, or Brevibacterium lactofermentum as the host.

8. Use of the RthB protein mutant according to claim 1, the nucleic acid molecule according to claim 2, or the recombinant vector according to claim 3 in the preparation of an isoleucine-producing bacterium.

9. Use of the RthB protein mutant according to claim 1, the nucleic acid molecule according to claim 2, the recombinant vector according to claim 3, or the recombinant microorganism according to any one of claims 4-7 in any one of the following (a1)-(a4): (a1) Use in isoleucine production; (a2) Use in increasing the yield of isoleucine; (a3) Use in reducing the accumulation amount of threonine in the isoleucine fermentation broth; (a4) Use in increasing the yield of isoleucine and reducing the accumulation amount of threonine.

10. A method for increasing the yield of isoleucine, characterized in that, The isoleucine-producing bacterium contains the RthB protein mutant according to claim 1.

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