An acetolactate synthase mutant and its application in the preparation of L-isoleucine

Abstract

The present invention provides an acetolactate synthase mutant and its application in the preparation of L-isoleucine. A highly active acetolactate synthase mutant is obtained by performing point mutations on the amino acid sequence of wild-type acetolactate synthase, including the 9th position and / or the 12th position. The mutant, the nucleic acid molecule encoding the mutant, the expression cassette containing the nucleic acid molecule, the recombinant vector or the genetically engineered bacterium are used for the production of L-isoleucine, and the yield of L-isoleucine is significantly improved, laying a good foundation for the large-scale industrial production of L-isoleucine.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to an acetolactate synthase mutant and application thereof in the preparation of L-isoleucine. Background Art

[0002] L-isoleucine is an essential amino acid for the human body, and is also a glucogenic and ketogenic amino acid. It can be decomposed and converted into glucose more quickly. It is the most effective branched-chain amino acid and can effectively prevent muscle loss. L-isoleucine is a raw material for synthesizing hormones, enzymes, etc. It can promote protein production and inhibit its decomposition. It plays an important role in the body's life activities and has been widely used in food, biomedicine, cosmetics and other fields.

[0003] The production methods of L-isoleucine mainly include extraction, chemical synthesis, enzyme catalysis, and microbial fermentation. At present, the fermentation method is mainly used in industrial production. In the fermentation method for producing L-isoleucine, acetohydroxy acid synthase (EC2.2.1.6; acetohydroxy acid synthase, AHAS), also known as acetolactate synthase, is the second rate-limiting enzyme in the synthesis of L-isoleucine and the first common enzyme in the synthesis of branched-chain amino acids. The enzyme can catalyze two parallel reactions: ① It can catalyze the decarboxylation of 1 mol of pyruvate and 1 mol of 2-butyric acid to synthesize 1 mol of 2-acetyl-2-hydroxybutyrate (acetohydroxybutyrate), which is the precursor of L-isoleucine synthesis; ② It can catalyze the decarboxylation of 2 mol of pyruvate to synthesize 1 mol of acetolactate (acetolactate), which is the precursor of L-leucine and L-valine synthesis. In E. coli, AHAS has three isoenzymes, namely AHASⅠ, AHASⅡ and AHASⅢ. Among them, AHAS I has no substrate preference, and AHAS II and AHASIII both prefer 2-ketobutyric acid. Since the target product of this study is L-isoleucine, AHASIII, which prefers 2-ketobutyric acid, was selected as the research object. AHASⅢ is encoded by the ilvIH operon, which is composed of ilvI encoding the large subunit (catalytic subunit) and ilvH encoding the small subunit (control subunit). The large and small subunits need to be combined to maintain the activity of the whole enzyme, and are feedback inhibited by L-isoleucine, with an IC50 of 3.1mmol·L -1 Therefore, people hope to improve the phenotypic activity of the ilvH gene through the study of the ilvH structure, thereby improving the effect of relieving the feedback inhibition of L-isoleucine. Summary of the invention

[0004] The object of the present invention is to introduce mutation sites for relieving feedback inhibition into ilvH through the study of the structure of the ilvH gene, so as to relieve the feedback inhibition of L-isoleucine on AHAS, and to improve the yield of L-isoleucine in the host bacterium by transferring the mutated ilvH gene into the host bacterium.

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

[0006] In the first aspect, the present invention provides a mutant acetolactate synthase, which is a point mutation including the 9th position and / or the 12th position based on the amino acid sequence of acetolactate synthase shown in SEQ ID NO:1.

[0007] In one embodiment of the present invention, the mutant acetolactate synthase is any one of the following:

[0008] (1) Leucine at the 9th position of the amino acid sequence of acetolactate synthase shown in SEQ ID NO:1 is mutated to glutamic acid, and the mutated amino acid sequence is as shown in SEQ ID NO:2;

[0009] (2) Glutamic acid at the 12th position of the amino acid sequence of acetolactate synthase shown in SEQ ID NO:1 is mutated to asparagine, and the mutated amino acid sequence is as shown in SEQ ID NO:4;

[0010] (3) Leucine at the 9th position of the amino acid sequence of acetolactate synthase shown in SEQ ID NO:1 is mutated to glutamic acid, and glutamic acid at the 12th position is mutated to asparagine, and the mutated amino acid sequence is as shown in SEQ ID NO:5.

[0011] Compared with the wild-type acetolactate synthase (the amino acid sequence is as shown in SEQ ID NO:1), the fermentation yield of L-isoleucine of the above mutant acetolactate synthase (the amino acid sequence is as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5) is increased by more than 21%.

[0012] In the second aspect, the present invention provides a nucleic acid molecule encoding the mutant acetolactate synthase.

[0013] In the third aspect, the present invention provides an expression cassette containing the nucleic acid molecule.

[0014] In the fourth aspect, the present invention provides a recombinant vector containing the nucleic acid molecule.

[0015] In the fifth aspect, the present invention provides a genetically engineered bacterium expressing the mutant acetolactate synthase.

[0016] In one embodiment of the present invention, the genetically engineered bacterium expressing the acetolactate synthase mutant contains the above nucleic acid molecule, expression cassette, or recombinant vector.

[0017] As described above for the genetically engineered bacterium, the ilvH gene of the present invention can be inserted into the vector DNA and then introduced into the host. The ilvH gene of the invention can be retained in the host as plasmid-like extrachromosomal DNA, or the above gene can be incorporated into the chromosome of the host microorganism by methods such as transduction, transposon, Mu phage, or homologous recombination. To effectively express the above gene, the ilvH of the present invention can be placed under the control of promoters such as lac, trp, PL, and tac that function in microorganisms.

[0018] In one embodiment of the present invention, the following modification is also carried out on the genetically engineered bacterium: knocking out the threonine dehydrogenase gene tdh in the strain.

[0019] In a sixth aspect, the present invention provides the application of the acetolactate synthase mutant, the nucleic acid molecule encoding the acetolactate synthase mutant, the expression cassette containing the nucleic acid molecule, the recombinant vector containing the nucleic acid molecule, or the genetically engineered bacterium expressing the acetolactate synthase mutant in the production of L-isoleucine.

[0020] In a seventh aspect, the present invention provides the application of the acetolactate synthase mutant, the nucleic acid molecule encoding the acetolactate synthase mutant, the expression cassette containing the nucleic acid molecule, the recombinant vector containing the nucleic acid molecule, or the genetically engineered bacterium expressing the acetolactate synthase mutant in the preparation of acetolactate synthase.

[0021] In an eighth aspect, the present invention provides a method for producing L-isoleucine, characterized in that: the production method is any one of the following:

[0022] (1) Fermentatively culturing the above-mentioned genetically engineered bacterium expressing the acetolactate synthase mutant to obtain L-isoleucine;

[0023] (2) Using acetolactate synthase as a catalyst to produce L-isoleucine by enzymatic method; the acetolactate synthase comprises the above-mentioned acetolactate synthase mutant.

[0024] In the enzymatic production of L-isoleucine, acetolactate synthase acts as a catalyst to catalyze the decarboxylation of pyruvate and 2-ketobutyrate to synthesize 2-acetyl-2-hydroxybutyrate (a precursor for L-isoleucine synthesis), and is then used for the production of L-isoleucine.

[0025] Compared with the prior art, the present invention has the following beneficial technical effects: Based on the wild-type acetolactate synthase, an acetolactate synthase mutant is obtained by site-directed mutagenesis technology. The L-isoleucine fermentation yield of the recombinant engineering bacteria expressing the acetolactate synthase mutant can reach 13.9 g / L, which is more than 124.2% higher than that of the wild-type acetolactate synthase, laying a good foundation for the large-scale industrial production of L-isoleucine. Detailed implementation manners

[0026] The present invention will be further described below in conjunction with specific embodiments, but any embodiment or its combination should not be construed as a limitation on the protection scope or implementation manner of the present invention. Any method transformation made by those of ordinary skill in the art based on these implementation manners is included in the protection scope of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, 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); the reagents and materials used, unless otherwise specified, can all be obtained from commercial channels.

[0028] The media and their formulations involved in the following examples are as follows:

[0029] LB liquid medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the solvent is deionized water, pH 7;

[0030] LB plate: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 20 g / L, the solvent is deionized water, pH 7;

[0031] Seed medium: Glucose 20 g / L, corn steep liquor dry powder 10 g / L, KH2PO4 8.8 g / L, (NH4)2SO4 2.5 g / L, MgSO4·7H2O 2 g / L, the solvent is water.

[0032] Fermentation medium: The difference from the seed medium is only that the glucose concentration is 50 g / L, and 15 g / L of threonine is additionally added.

[0033] The strain, plasmid and primer information involved in the following examples are as follows:

[0034] Table 1 Strains and plasmids used in the present invention

[0035]

[0036]

[0037] Table 2 Primers used in the present invention

[0038]

[0039]

[0040] Example 1: Bioinformatics analysis of the ilvH gene structure - AutoDock Vina molecular docking

[0041] AHASⅢ is composed of a catalytic subunit encoded by ilvI and a regulatory subunit encoded by ilvH. Using isoleucine as a substrate, molecular docking was performed with the ilvH domain in AutoDock Vina to observe their interactions. L-isoleucine formed hydrogen bonds with the residues at positions L9, N11, and E12 of ilvH, and the above residues are key residues of the active pocket. Therefore, using the ilvH gene as a template, homologous sequences were obtained through BLAST in NCBI, and multiple sequence alignments were performed through Muscle in MEGA11 to analyze its Seqlogo. The results showed that the above residues are non-fully conserved residues, and point mutations can be performed according to Seqlogo.

[0042] Example 2: Construction of recombinant bacteria containing the ilvH gene

[0043] Using Escherichia coli CGMCC No.19458 (see the strain Sval065 in patent CN202010401422.5) as the initial bacterium, the gene encoding threonine dehydrogenase tdh was knocked out to prevent the consumption of threonine in the medium to produce by-product glycine, and the gene encoding acetolactate synthase (ilvH or ilvH mutant) was introduced to catalyze the decarboxylation of pyruvate and 2-ketobutyrate to synthesize 2-acetyl-2-hydroxybutyrate, the precursor of L-isoleucine, thereby increasing the yield of L-isoleucine.

[0044] Example 3: Knockout of the threonine dehydrogenase gene tdh

[0045] Starting from Escherichia coli CGMCC No.19458, the threonine dehydrogenase gene tdh was knocked out by a two-step homologous recombination method. The specific steps are as follows:

[0046] In the first step, using the pRE112 plasmid DNA as a template, a 3569bp DNA fragment I was amplified using the primers tdh-cs-up / tdh-cs-down for the first step of homologous recombination.

[0047] The amplification system is as follows: 10 μl of Phusion 5X buffer (New England Biolabs), 1 μl of dNTP (10 mM for each dNTP), 20 ng of DNA template, 2 μl of each primer (10 μM), 0.5 μl of Phusion High-Fidelity DNA polymerase (2.5 U / μl), and 33.5 μl of distilled water, with a total volume of 50 μl.

[0048] The amplification conditions are as follows: pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 56°C for 10 seconds, extension at 72°C for 4 minutes (30 cycles); extension at 72°C for 10 minutes (1 cycle).

[0049] The above DNA fragment I was used for the first homologous recombination: First, the pKD46 plasmid - (purchased from the E. coli Stock Center of Yale University, USA, CGSC #7739) was transformed into E. coli CGMCC No. 19458 (Sval065-pKD46) by electroporation, and then DNA fragment I was electroporated into E. coli CGMCC No. 19458 carrying pKD46.

[0050] The electroporation conditions are as follows: Sval065-pKD46 was prepared as competent cells by the CaCl2 method; then 50 μl of Sval065-pKD46 competent cells were placed on ice, 50 ng of DNA fragment I was added, and the mixture was placed on ice for 2 minutes and then transferred to a 0.2 cm Bio-Rad electroporation cuvette. Using a MicroPulser (Bio-Rad) electroporator, the electroporation parameters were a voltage of 2.5 kV. Immediately after electroporation, 1 ml of LB medium was transferred to the electroporation cuvette, pipetted 5 times and then transferred to a test tube, incubated at 75 rpm and 30°C for 2 hours. 200 μl of the bacterial solution was spread on an LB plate containing ampicillin (final concentration 100 μg / ml) and chloramphenicol (final concentration 34 μg / ml), and after overnight culture at 30°C, single colonies were selected for PCR verification. The primers used were XZ-tdh-up / XZ-tdh-down, and the correct colony amplification product was a 4449 bp fragment. One correct single colony was selected and named mIVL001.

[0051] In the second step, using the genomic DNA of E. coli CGMCC No. 19458 as a template, a 532 bp DNA fragment II was amplified with primers XZ-tdh-up / tdh-del-down for the second homologous recombination.

[0052] The electroporation conditions are as follows: The pKD46 plasmid was transformed into mIVL001 (mIVL001-pKD46) by electroporation, and mIVL001-pKD46

[0053] First, prepare electrocompetent cells of mIVL001 carrying the pKD46 plasmid; place 50 μl of electrocompetent cells on ice, add 50 ng of DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV voltage. Immediately after electroporation, transfer 1 ml of LB liquid medium to the electroporation cuvette, pipette 5 times and then transfer to a test tube, incubate at 75 rpm and 30 °C for 4 hours. Transfer the bacterial solution to LB liquid medium without sodium chloride containing 10% sucrose (50 ml of medium in a 250 ml flask), culture for 24 hours and then streak on an LB plate containing 6% sucrose and no sodium chloride. After PCR verification, the primers used are XZ-tdh-up / tdh-del-down, and the correct colony amplification product is a 532 bp fragment. Select a correct single colony and name it mIVL002.

[0054] Example 4: Mutation of the acetolactate synthase gene ilvH

[0055] Introduce mutations into the ilvH gene by a two-step homologous recombination method to relieve the feedback inhibition of L-isoleucine. The specific steps are as follows:

[0056] In the first step, using pRE112 plasmid DNA as a template, amplify a 3539 bp DNA fragment I with primers ilvH-mut-cat-up / ilvH-mut-cat-down for the first-step homologous recombination.

[0057] The amplification system is: 10 μl of Phusion 5X buffer (New England Biolabs), 1 μl of dNTP (10 mM each of dNTP), 20 ng of DNA template, 2 μl each of primers (10 μM), 0.5 μl of Phusion High-Fidelity DNA polymerase (2.5 U / μl), 33.5 μl of distilled water, with a total volume of 50 μl.

[0058] The amplification conditions are: pre-denaturation at 98 °C for 2 minutes (1 cycle); denaturation at 98 °C for 10 seconds, annealing at 56 °C for 10 seconds, extension at 72 °C for 4 minutes (30 cycles); extension at 72 °C for 10 minutes (1 cycle).

[0059] Use the above DNA fragment I for the first homologous recombination: First, transform the plasmid pKD46 (purchased from the E. coli collection center of CGSC, Yale University, USA, CGSC#7739) into E. coli mIVL002 by electroporation (designated as mIVL002-pKD46), and then electroporate DNA fragment I into E. coli mIVL002 carrying pKD46.

[0060] The electroporation conditions are as follows: Prepare mIVL002-pKD46 as competent cells by the CaCl2 method; then place 50 μl of competent cells on ice, add 50 ng of DNA fragment I, incubate on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB medium to the electroporation cuvette, pipette 5 times, and then transfer to a test tube. Incubate at 75 rpm and 30 °C for 2 hours. Take 200 μl of the bacterial solution and spread it on an LB plate containing ampicillin (final concentration 100 μg / ml) and chloramphenicol (final concentration 34 μg / ml). After overnight culture at 30 °C, pick single colonies for PCR verification. The primers used are XZ-ilvH-mut-up / XZ-ilvH-mut-down. The correct colony amplification product is a 4530 bp fragment. Select one correct single colony and name it mIVL003.

[0061] In the second step, using the DNA of wild-type E. coli ATCC 8739 as a template, amplify a 610 bp DNA fragment II with primers ilvH-mut-cs-F / ilvIH-R. The amplification system and conditions are the same as in the first step. DNA fragment II is used for the second homologous recombination. Electroporate DNA fragment II into strain mIVL003 and select a single colony expressing wild-type ilvH, named mIVL004(ilvH).

[0062] Using the DNA of wild-type E. coli ATCC 8739 as a template, amplify a 603 bp DNA fragment II with primers ilvH-mut-cs-F / ilvH-L9E-R. The amplification system and conditions are the same as in the first step. DNA fragment II is used for the second homologous recombination. Electroporate DNA fragment II into strain mIVL003 and select a single colony in which the 9th isoleucine of the wild-type ilvH gene has been successfully mutated to glutamic acid, named mIVL005(ilvH*L9E).

[0063] Using the DNA of wild-type Escherichia coli ATCC 8739 as a template, a 604-bp DNA fragment II was amplified with primers ilvH-mut-cs-F / ilvH-N11S-R. The amplification system and conditions were the same as in the first step. The DNA fragment II was used for the second homologous recombination. The DNA fragment II was electrotransformed into strain mIVL003, and a single colony (ilvH*N11S) in which the 11th asparagine of the wild-type ilvH gene was successfully mutated to serine was selected and named mIVL006.

[0064] Using the DNA of wild-type Escherichia coli ATCC 8739 as a template, a 612-bp DNA fragment II was amplified with primers ilvH-mut-cs-F / ilvH-E12N-R. The amplification system and conditions were the same as in the first step. The DNA fragment II was used for the second homologous recombination. The DNA fragment II was electrotransformed into strain mIVL003, and a single colony in which the 12th glutamate of the wild-type ilvH gene was successfully mutated to asparagine was selected and named mIVL007 (ilvH*E12N).

[0065] Using the DNA of wild-type Escherichia coli ATCC 8739 as a template, a 610-bp DNA fragment II was amplified with primers ilvH-mut-cs-F / L9E-E12N-R. The amplification system and conditions were the same as in the first step. The DNA fragment II was used for the second homologous recombination. The DNA fragment II was electrotransformed into strain mIVL003, and a single colony in which the 9th isoleucine of the wild-type ilvH gene was successfully mutated to glutamate and the 12th glutamate was successfully mutated to asparagine was selected and named mIVL008 (ilvH*L9E-E12N).

[0066] The electrotransformation conditions were as follows: First, prepare electrocompetent cells of mIVL003 carrying the pKD46 plasmid; place 50 μl of electrocompetent cells on ice, add 50 ng of DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2-cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electric shock parameter of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB medium to the electroporation cuvette and incubate at 30 °C for 4 hours. Transfer the bacterial solution to LB liquid medium without sodium chloride containing 10% sucrose (50 ml of medium in a 250-ml flask), and after culturing for 24 hours, streak on LB solid medium without sodium chloride containing 6% sucrose. After PCR verification, the primers used were XZ-ilvH-mut-up / XZ-ilvH-mut-down, and the correct colony amplification product was a 1553-bp fragment.

[0067] Example 5: Fermentation production of L-isoleucine by recombinant strains

[0068] The recombinant strains mIVL002, mIVL004, mIVL005, mIVL006, mIVL007, and mIVL008 produce L-isoleucine through anaerobic fermentation, including the following steps:

[0069] (1) Seed culture: Inoculate fresh monoclonal colonies on an LB plate into a test tube containing 4 ml of seed medium, and culture overnight at 37°C with shaking at 250 rpm. Then, transfer the culture to a 250-ml Erlenmeyer flask containing 30 ml of seed medium at an inoculation amount of 2% (V / V), and culture with shaking at 37°C and 250 rpm for 12 hours to obtain a seed culture solution for inoculating the fermentation medium.

[0070] (2) Fermentation culture: The volume of the fermentation medium in a 500-ml anaerobic jar is 250 ml. Inoculate the seed culture solution into the fermentation medium at an inoculation amount with a final OD550 of 0.1, and ferment at 37°C and 150 rpm for 3 days to obtain a fermentation broth. The neutralizing agent is 5 M ammonia water to control the pH of the fermentation jar at 7.0. No gas is introduced during the culture process.

[0071] Analysis method: Use an Agilent (Agilent-1260) high-performance liquid chromatograph to measure the components in the fermentation broth after 3 days of fermentation. For isoleucine determination, use an amino acid analysis column ZORBAX Eclipse AAA 4.6 mm × 75 mm 3.5-Micron. Detection wavelength: 338 nm.

[0072] Repeat the experiment 3 times, and the average value of the analysis results is presented in Table 3 below.

[0073] Table 3 Comparison of L-isoleucine production capabilities

[0074]

[0075] As shown in Table 3, in the medium containing threonine, compared with the mIVL002 strain, the mIVL005, mIVL007, and mIVL008 strains containing the ilvH mutant all have increased isoleucine yields. In particular, the mIVL008 strain containing the ilvH*L9E-E12N mutant has a 37.6% increase in L-isoleucine production capacity. Compared with the mIVL004 strain containing the wild-type ilvH, its L-isoleucine production capacity has increased by 124.2%.

[0076] The above are only the preferred embodiments 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 concept 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 acetolactate synthase mutant, characterized in that, The acetolactate synthase mutant is any one of the following: (1) Mutating the leucine at position 9 of the amino acid sequence of acetolactate synthase shown in SEQ ID NO:1 to glutamic acid; (2) Mutating the glutamic acid at position 12 of the amino acid sequence of acetolactate synthase shown in SEQ ID NO:1 to asparagine; (3) Mutating the leucine at position 9 of the amino acid sequence of acetolactate synthase shown in SEQ ID NO:1 to glutamic acid and mutating the glutamic acid at position 12 to asparagine.

2. A nucleic acid molecule encoding the acetolactate synthase mutant according to claim 1.

3. An expression cassette containing the nucleic acid molecule according to claim 2.

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

5. A genetically engineered bacterium expressing the acetolactate synthase mutant according to claim 1.

6. The genetically engineered bacterium according to claim 5, characterized in that, The genetically engineered bacterium contains the nucleic acid molecule according to claim 2, the expression cassette according to claim 3, or the recombinant vector according to claim 4.

7. The genetically engineered bacterium according to claim 6, wherein The genetically engineered bacterium is further modified as follows: knocking out the threonine dehydrogenase gene tdh in the strain.

8. Use of the acetolactate synthase mutant according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette according to claim 3, the recombinant vector according to claim 4, or the genetically engineered bacterium according to any one of claims 5-7 in the production of L-isoleucine.

9. Use of the acetolactate synthase mutant according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette according to claim 3, the recombinant vector according to claim 4, or the genetically engineered bacterium according to any one of claims 5-7 in the preparation of acetolactate synthase.

10. A method for producing L-isoleucine, characterized in that: The production method is any one of the following: (1) Fermenting and culturing the genetically engineered bacterium according to any one of claims 5-7 to obtain L-isoleucine; (2) Using acetolactate synthase as a catalyst for enzymatic production of L-isoleucine; the acetolactate synthase contains the acetolactate synthase mutant according to claim 1.

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