Acetolactate synthase and application thereof

Abstract

The invention relates to acetolactate synthase and application thereof, and belongs to the technical field of enzyme engineering and metabolic engineering. Wild acetolactate synthase from Corynebacterium glutamicum ATCC13032 is taken as a basis, error-prone PCR is performed on a coding gene ilvBN of the wild acetolactate synthase, acetolactate synthase mutants IlvBNM1 and IlvBNM2 are obtained through screening, the feedback inhibition effects of L-valine, L-leucine and L-isoleucine on the mutants are relieved, and the activity of the mutants is improved. Under the condition that the concentrations of the L-valine, the L-leucine and the L-isoleucine are respectively 0-30 mmol / L, the enzyme activity is not obviously changed, and the method can be widely applied to synthesis of the L-valine, the L-leucine and the L-isoleucine.

Description

Technical Field

[0001] This invention relates to an acetolactate synthase and its applications, belonging to the fields of enzyme engineering and metabolic engineering technology. Background Technology

[0002] L-valine, L-leucine, and L-isoleucine are collectively known as branched-chain amino acids, which are essential amino acids for vertebrates. Branched-chain amino acids have functions such as promoting protein synthesis, regulating blood sugar, strengthening mitochondrial function, and repairing muscle tissue, and are widely used in medicine, food, agriculture, and other fields.

[0003] Currently, branched-chain amino acids are mainly synthesized through microbial fermentation. This method relies heavily on microbial metabolic pathways, and key enzymes within these pathways are crucial for efficient synthesis. Research has found that acetolactate synthase is the rate-limiting enzyme in branched-chain amino acid synthesis, and its activity is subject to feedback inhibition by the branched-chain amino acids themselves. Acetolactate synthase can catalyze the synthesis of acetolactate from two molecules of pyruvate, leading to the synthesis of L-valine and L-leucine; it can also catalyze the synthesis of acetylhydroxybutyrate from one molecule of pyruvate and α-ketobutyrate, leading to the synthesis of L-isoleucine.

[0004] Acetolactate synthase (IlvBN) consists of the catalytic subunit IlvB and the regulatory subunit IlvBN. Intracellular branched amino acids bind to the regulatory subunit, altering their spatial structure and thus inhibiting acetolactate synthase activity, affecting the synthesis of branched amino acids. Therefore, relieving the feedback inhibition of branched amino acids on acetolactate synthase is beneficial for its efficient synthesis. Summary of the Invention

[0005] To overcome the shortcomings of current wild-type acetolactate synthase which is inhibited by branched-chain amino acids, this invention provides an acetolactate synthase mutant that relieves feedback inhibition by branched-chain amino acids, its encoding gene, and its applications.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] One of the technical solutions provided by the present invention is an acetolactate synthase mutant, which is obtained by G20D and S23F, or G20E and S23F mutations in the regulatory subunit IlvN of wild-type acetolactate synthase IlvBN; the amino acid sequence of the regulatory subunit IlvN of wild-type acetolactate synthase IlvBN is shown in SEQ ID NO.2; Furthermore, the acetolactate synthase mutant is IlvBN. M1 It consists of the catalytic subunit IlvB and the regulatory subunit IlvN. M1 Composition: The amino acid sequence of the catalytic subunit IlvB is shown in SEQ ID NO.1, and the regulatory subunit IlvN... M1The amino acid sequence is shown in SEQ ID NO.3; Furthermore, the acetolactate synthase mutant is IlvBN. M2 It consists of the catalytic subunit IlvB and the regulatory subunit IlvN. M2 Composition: The amino acid sequence of the catalytic subunit IlvB is shown in SEQ ID NO.1, and the regulatory subunit IlvN... M2 The amino acid sequence is shown in SEQ ID NO.4.

[0008] This invention also provides the encoding gene of the acetolactate synthase mutant described in one of the technical solutions; Furthermore, the acetolactate synthase mutant IlvBN M1 The gene encoding the catalytic subunit IlvB is ilvB The nucleotide sequence is shown in SEQ ID NO.5, and the regulatory subunit is IlvN. M1 The encoding gene is ilvN M1 The nucleotide sequence is shown in SEQ ID NO. 7; Furthermore, the acetolactate synthase mutant IlvBN M2 The gene encoding the catalytic subunit IlvB is ilvB The nucleotide sequence is shown in SEQ ID NO.5, and the regulatory subunit is IlvN. M2 The encoding gene is ilvN M2 The nucleotide sequence is shown in SEQ ID NO. 8.

[0009] The second technical solution provided by the present invention is a recombinant vector or recombinant strain containing the coding gene of the acetolactate synthase mutant described in the first technical solution; Furthermore, the expression vectors used in the recombinant vector include, but are not limited to: pSTV28, pWSK29, pTrc99a, pET28a, etc.; pSTV28 plasmid is preferred. Furthermore, the host cells used in the recombinant strain include, but are not limited to: Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, etc.; Escherichia coli is preferred.

[0010] The third technical solution provided by the present invention is the application of the recombinant vector or recombinant strain described in the second technical solution, particularly its application in the preparation of the acetyllactate synthase mutant described in the first technical solution.

[0011] The fourth technical solution provided by this invention is the application of the acetolactate synthase mutant described in the first technical solution or the recombinant vector or recombinant strain described in the second technical solution in the catalytic synthesis of branched-chain amino acids; particularly in the catalytic synthesis of L-valine or L-leucine from pyruvate; or in the catalytic synthesis of L-isoleucine from pyruvate and α-ketobutyrate. The branched-chain amino acids include L-valine, L-leucine, and L-isoleucine; Furthermore, one of the technical solutions describes the application of the acetolactate synthase mutant in relieving feedback inhibition of L-valine, L-leucine, and L-isoleucine.

[0012] Beneficial effects: The acetyllactate synthase mutant IlvBN provided by this invention M1 and IlvBN M2 It exhibits the following characteristics: the feedback inhibition effects of L-valine, L-leucine, and L-isoleucine were eliminated; the enzyme activity showed no significant change under conditions where the concentrations of L-valine, L-leucine, and L-isoleucine were 0-30 mmol / L; and the acetolactate synthase mutant IlvBN under conditions where the concentrations of L-valine, L-leucine, and L-isoleucine were 0-30 mmol / L... M1 and IlvBN M2 The enzyme activity was not significantly reduced compared with wild-type acetyllactate synthase IlvBN at L-valine, L-leucine, and L-isoleucine concentrations of 0 mmol / L. Figure 1 , 2 The mutant can be widely used in the synthesis of L-valine, L-leucine, and L-isoleucine. Attached Figure Description

[0013] Figure 1 Wild-type acetolactate synthase IlvBN and acetolactate synthase mutant IlvBN M1 and IlvBN M2 Comparison of enzyme activities in the absence of branched-chain amino acids.

[0014] Figure 2 Branched-chain amino acid pairs for wild-type acetolactate synthase IlvBN and acetolactate synthase mutant IlvBN M1 and IlvBN M2 Effect of activity In the figure, Figure A represents L-valine; Figure B represents L-leucine; and Figure C represents L-isoleucine. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this patent clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of this patent and are not intended to limit the scope of the invention.

[0016] This invention uses Corynebacterium glutamicum ( Corynebacterium glutamicum Based on the wild-type acetolactate synthase of ATCC13032, the gene encoding it was determined. ilvBN Error-prone PCR amplification was performed, and the amplification product was then ligated into the pSTV28 plasmid and transformed into E. coli. E. coli K-12 DH5α was used to obtain a transformant library. The transformants were plated on a solid basal medium containing 50 mg / L 2-hydroxy-3-methylbutyric acid (2-H2-methylbutyric acid), and 50 large colonies were selected and named V-1–V-50. These strains were then inoculated into a liquid medium containing 50 mg / L 2-H2-methylbutyric acid (5-H2-methylbutyric acid), and eight strains with the highest biomass were screened (V-8, V-12, V-16, V-19, V-26, V-33, V-38, and V-42).

[0017] Plasmids were extracted from the above-mentioned strains, and the gene encoding acetolactate synthase was amplified by PCR using designed primers. After the PCR product was recovered, it was sequenced, and it was found that the acetolactate synthase encoded by the mutant gene had a G20D / S23F or G20E / S23F mutation relative to the regulatory subunit of wild-type acetolactate synthase.

[0018] Based on this, the enzymatic properties of the acetolactate synthase mutants obtained through screening were determined, confirming that they relieved the feedback inhibition effect of branched-chain amino acids while maintaining enzyme activity. These mutants were then used in the production of branched-chain amino acids.

[0019] 1. The nomenclature of amino acids and DNA nucleic acid sequences of this invention The IUPAC nomenclature, a widely accepted system for naming amino acid residues, is used, employing single-letter / three-letter codes. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0020] 2. Identification of the acetolactate synthase mutant of the present invention The mutated amino acid in the acetolactate synthase mutant is represented by "original amino acid + position + substituted amino acid". For example, G20D indicates that the amino acid at position 20 is replaced by Asp by Gly in the wild-type acetolactate synthase. The position number corresponds to the amino acid sequence number of the regulatory subunit IlvN of the wild-type acetolactate synthase in SEQ ID NO.2.

[0021] In this invention, IlvBN represents wild-type acetolactate synthase, IlvB represents the catalytic subunit of wild-type acetolactate synthase, and IlvN represents the regulatory subunit of wild-type acetolactate synthase.

[0022] In this invention, ilvBN The gene encoding wild-type acetolactate synthase IlvBN. ilvB The gene encoding the wild-type acetolactate synthase catalytic subunit IlvB. ilvN The gene encoding the wild-type acetolactate synthase regulatory subunit IlvN; ilvN M1 Represents the acetolactate synthase mutant IlvBN M1 Regulatory subunit IlvN M1 The encoding gene, ilvN M2 Represents the acetolactate synthase mutant IlvBN M2 Regulatory subunit IlvN M2 The gene that encodes it.

[0023] In this invention, the acetolactate synthase mutant IlvBN M1 Composed of catalytic subunit IlvB and regulatory subunit IlvN M1 The composition, the amino acid sequence of the catalytic subunit is shown in SEQ ID NO.1, and the regulatory subunit IlvN M1 It was obtained by mutations of G20D and S23F in the wild-type acetolactate synthase regulatory subunit IlvN shown in SEQ ID NO.2. M1 The amino acid sequence is shown in SEQ ID NO.3.

[0024] In this invention, the acetolactate synthase mutant IlvBN M2 Composed of catalytic subunit IlvB and regulatory subunit IlvN M2 The composition, the amino acid sequence of the catalytic subunit is shown in SEQ ID NO.1, and the regulatory subunit IlvN M2 It was obtained by mutations of G20E and S23F in the wild-type acetolactate synthase regulatory subunit IlvN shown in SEQ ID NO.2. M2 The amino acid sequence is shown in SEQ ID NO.4.

[0025] The details are shown in the table below:

[0026] In this invention, the source is C. glutamicumThe wild-type acetolactate synthase IlvBN of ATCC13032 is composed of a catalytic subunit IlvB and a regulatory subunit IlvN. The amino acid sequence of the catalytic subunit IlvB is shown in SEQ ID NO.1, and the amino acid sequence of the regulatory subunit IlvN is shown in SEQ ID NO.2. In this invention, the acetolactate synthase mutant IlvBN M1 Composed of catalytic subunit IlvB and regulatory subunit IlvN M1 Composition: The amino acid sequence of the catalytic subunit IlvB is shown in SEQ ID NO.1, and the regulatory subunit IlvN... M1 The amino acid sequence is shown in SEQ ID NO.3; In this invention, the acetolactate synthase mutant IlvBN M2 Composed of catalytic subunit IlvB and regulatory subunit IlvN M2 Composition: The amino acid sequence of the catalytic subunit IlvB is shown in SEQ ID NO.1, and the regulatory subunit IlvN... M2 The amino acid sequence is shown in SEQ ID NO.4; SEQ ID NO.1:MNVAASQQPTPATVASRGRSAAPERMTGAKAIVRSLEELNADIVFGIPGGAVLPVYDPLYSSTKVRHVLVRHEQGAGHAATGYAQVTGRVGVCIATSGPGATNLVTPIADANLDSVPMVAITGQVGSGLLGTDAFQEADIRGITMPVTKHNFMVTNPNDIPQALAEAFHLAITGRPGPVLVDIPKDVQNAELDFVWPPKIDLPGYRPVSTPHARQIEQAVKLIGEAKKPVLYVGGGVIKADAHEELRAFAEYTGIPVVTTLMALGTFPESHELHMGMPGMHGTVSAVGALQRSDLLIAIGSRFDDRVTGDVDTFAPDAKIIHADIDPAEIGKIKQVEVPIVGDAREVLARLLETTKASKAETEDISEWVDYLKGLKARFPRGYDEQPGDLLAPQFVIETLSKEVGPDAIYCAGVGQHQMWAAQFVDFEKPRTWLNSGGLGTMGYAVPAALGAKAGAPDKEVWAIDGDGCFQMTNQELTTAAVEGFPIKIALINNGNLGMVRQWQTLFYEGRYSNTKLRNQGEYMPDFVTLSEGLGCVAIRVTKAEEVLPAIQKAREINDRPVVIDFIVGEDAQVWPMVSAGSSNSDIQYALGLRPFFDGDESAAEDPADIHEAVSDIDAAVESTEA SEQ ID NO.2:MANSDVTRHILSVLVQDVDGIISRVSGMFTRRAFNLVSLVSAKTETHGINRITVVVDADELNIEQITKQLNKLIPVLKVVRLDEETTIARAIMLVKVSADSTNRPQIVDAANIFRARVVDVAPDSVVIESTGTPGKLRALLDVMEPFGIRELIQSGQIALNRGPKTMAPAKI SEQ ID NO.3: MANSDVTRHILSVLVQDVDDIIFRVSGMFTRRAFNLVSLVSAKTETHGINRITVVVDADELNIEQITKQLNKLIPVLKVVRLDEETTIARAIMLVKVSADSTNRPQIVDAANIFRARVVDVAPDSVVIESTGTPGKLRALLDVMEPFGIRELIQSGQIALNRGPKTMAPAKI SEQ ID NO.4: MANSDVTRHILSVLVQDVDEIIFRVSGMFTRRAFNLVSLVSAKTETHGINRITVVVDADELNIEQITKQLNKLIPVLKVVRLDEETTIARAIMLVKVSADSTNRPQIVDAANIFRARVVDVAPDSVVIESTGTPGKLRALLDVMEPFGIRELIQSGQIALNRGPKTMAPAKI 3. Assay of acetyllactate synthase activity (1) Definition of specific enzyme activity: The amount of sodium pyruvate consumed per minute per milligram of total protein containing acetolactate synthase (nmol).

[0027] (2) Enzyme activity assay: 100 μL of crude enzyme solution was added to 800 μL of 100 mmol / L potassium phosphate buffer (pH 7.8, containing 10 mmol / L sodium pyruvate, 1 mmol / L MgCl2, and 0.2 mmol / L thiamine pyrophosphate), and reacted at 37℃ for 1 h. 100 μL of 3 mol / L H2SO4 was added to terminate the enzymatic reaction. After the reaction was completed, the amount of sodium pyruvate consumed was determined by high performance liquid chromatography.

[0028] (3) The detection method of sodium pyruvate is as follows: After centrifuging the reaction solution at 8000×g for 10min, the supernatant is taken. The detection conditions are: chromatographic column REzex RoA-organic Acid H+, mobile phase 5 mmol / L H2SO4, flow rate 0.5mL / min, column temperature 30℃, detection wavelength 215nm, and injection volume 20μL.

[0029] (4) Formula for calculating specific enzyme activity: Specific enzyme activity = (ΔC) Pyr × V_total) / (t × C) TP × V TP ) C Pyr : The decrease in sodium pyruvate concentration (nmol / L); Vtotal: Total volume of the reaction solution (L); t represents the reaction time: 60 min; C TP Total protein concentration in crude enzyme solution (mg / L); V TP : Crude enzyme solution volume (L).

[0030] 4. Some primers and sequences involved in the embodiments of the present invention Table 1. List of primer sequences used in the embodiments of the present invention

[0031] The present invention will be further explained and illustrated below through specific embodiments.

[0032] Example 1: The gene encoding acetolactate synthase that relieves branched-chain amino acid feedback inhibition ilvBN M1 and ilvBN M2 The acquisition wild-type Corynebacterium glutamicum C. glutamicum Using the ATCC13032 genome as a template, error-prone PCR (ready-to-use error-prone PCR kit, Beijing Tianenze Gene Technology Co., Ltd.) was performed using primers ilvBN-1 / ilvBN-2 for amplification. ilvBN Mutant library.

[0033] Error-prone PCR conditions are: 95℃ pre-denaturation for 5 min; 95℃ for 30 s, 54℃ for 30 s, 72℃ for 3 min, 40 cycles; 72℃ for 10 min.

[0034] use Eco pSTV28 was digested with RI enzyme and recovered, then combined with the above-mentioned recombinant kit ClonExpress II One Step Cloning Kit (Nanjing Novizan Medical Technology Co., Ltd.) ilvBN Mutant library recombination ligation and transformation to E. coli After revival, DH5α competent cells were plated onto solid selection medium containing 100 μg / mL chloramphenicol and incubated at 37°C. The next day, 50 larger single colonies were randomly selected from the plates and transferred to 96-well cell culture plates containing 100 μg / mL chloramphenicol liquid selection medium, and cultured at 37°C with shaking for 24 h.

[0035] OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 600 Select OD 600 The top 8 strains were used to extract plasmids for analysis. ilvBNThe mutants were sequenced. Two types of mutants were identified, with the regulatory subunit amino acid sequences shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. That is, relative to those from... C. glutamicum ATCC13032 wild-type acetolactate synthase, these mutants have the following amino acid mutation: IlvBN M1 (G20D / S23F) and IlvBN M2 (G20E / S23F), whose encoding genes are named respectively. ilvBN M1 and ilvBN M2 The plasmids containing the above genes were named pST- ilvBN M1 and pST- ilvBN M2 Will contain wild type ilvBN The gene plasmid is named pST- ilvBN .

[0036] Solid screening medium: glucose 10 g / L, MgSO4 0.24 g / L, KH2PO4 2.5 g / L, (NH4)2SO4 5 g / L, FeSO4 2 g / L, 2-hydroxy-3-methylbutyric acid 50 mg / L, agar 20 g / L, deionized water 1000 mL, pH 6.5-7.0.

[0037] Liquid screening medium: glucose 10 g / L, MgSO4 0.24 g / L, KH2PO4 2.5 g / L, (NH4)2SO4 5 g / L, FeSO4 2 g / L, 2-hydroxy-3-methylbutyric acid 50 mg / L, deionized water 1000 mL, pH 6.5-7.0.

[0038] The principles of the above solid-state and liquid-state screening are as follows: branched-chain amino acids bind to acetolactate synthase, altering its spatial structure and thus inhibiting its activity. Since the binding of branched-chain amino acids to acetolactate synthase is reversible, this inhibition is also reversible and has no lethal effect on bacterial cells. Therefore, it cannot be used to screen for acetolactate synthase mutants.

[0039] 2-Hydroxy-3-methylbutyric acid (2-HMA) is a structural analog of branched-chain amino acids and can also bind to acetolactate synthase (ASY), but this binding is irreversible and lethal to bacterial cells. Therefore, recombinants expressing wild-type or non-positively mutated ASY do not grow in media containing 2-HMA. Some acetolactate synthases mutate and do not bind to 2-HMA, thus strains containing these mutants can grow in media containing 2-HMA. Since 2-HMA is a structural analog of branched-chain amino acids, this means that these mutants also do not bind to branched-chain amino acids, thereby relieving the feedback inhibition of branched-chain amino acids. Using this principle, resistant bacterial colonies are first obtained in a solid medium containing 2-HMA (initial screening), and then these colonies are transferred to a liquid medium containing 2-HMA. Growth is rapid (OD). 600 The presence of high OD values ​​in strains indicates strong resistance to 2-hydroxy-3-methylbutyric acid stress; therefore, strains with high OD values ​​were selected. 600 High-strength strains (secondary screening).

[0040] Example 2: Enzymatic Characteristics Analysis of Acetolactate Synthase 1. Preparation of crude enzyme solution Cultured in LB liquid medium containing pST- ilvBN pST- ilvBN M1 and ilvBN M2 of E. coli After 12 hours of DH5α culture, 20 mL of the culture was centrifuged at 10,000 g for 1 min at 4°C to collect the bacterial cells. The precipitate was washed three times with 1 mL of buffer (200 mmol / L Tris-HCl, pH 8.1) and then resuspended in 1 mL of buffer. The bacterial suspension was then sonicated using an ultrasonic homogenizer under the following conditions: 350 W power, 5 s working time, 10 s interval, 5 cycles, operated on ice. The homogenate was centrifuged at 8,000 g at 4°C, and the supernatant was collected as IlvBN. M1 and IlvBN M2 The total protein concentration of the crude enzyme solution was determined using the BCA Protein Quantification Kit (Nanjing Novizan Medical Technology Co., Ltd.).

[0041] 2. Enzyme activity assay IlvBN, IlvBN M1 and IlvBN M2The enzyme activity was determined as follows: 100 μL of crude enzyme solution was added to 800 μL of 100 mmol / L potassium phosphate buffer (pH 7.8, containing 10 mmol / L sodium pyruvate, 1 mmol / L MgCl2, and 0.2 mmol / L thiamine pyrophosphate), and the reaction was carried out at 37℃ for 1 h. 100 μL of 3 mol / L H2SO4 was then added to terminate the enzymatic reaction. After the reaction was complete, the amount of sodium pyruvate consumed was determined by high-performance liquid chromatography (HPLC). The specific enzyme activity was then calculated using the aforementioned method.

[0042] The results are as follows Figure 1 As shown, IlvBN, IlvBN M1 and IlvBN M2 The specific enzyme activities were 6.1, 6.2, and 5.9 nmol / (min·mg total protein), respectively, indicating that in the absence of branched-chain amino acids, the mutant IlvBN... M1 and IlvBN M2 There was no significant difference in enzyme activity compared to wild-type IlvBN.

[0043] 3. Branched-chain amino acids on IlvBN, IlvBN M1 and IlvBN M2 The effect of enzyme activity Branched chain amino acids for IlvBN, IlvBN M1 and IlvBN M2 The method for determining the effect of enzyme activity is as follows: Take 100 μL of acetyllactate synthase IlvBN or its mutant IlvBN respectively M1 and IlvBN M2 The crude enzyme solution was added to 800 μL of 100 mmol / L potassium phosphate buffer (pH 7.8, containing 10 mmol / L sodium pyruvate, 1 mmol / L MgCl2, and 0.2 mmol / L thiamine pyrophosphate). Then, 0, 5, 10, 15, 20, 25, and 30 mmol / L of branched-chain amino acids (including L-valine, L-leucine, and L-isoleucine) were added to the above reaction solution. The reaction was incubated at 37°C for 1 h, and 100 μL of 3 mol / L H2SO4 was added to terminate the enzymatic reaction. After the reaction was complete, the consumption of sodium pyruvate was determined by high-performance liquid chromatography (HPLC). This was to investigate the effects of IlvBN and IlvBN. M1 and IlvBN M2 To relieve the feedback inhibition of branched-chain amino acids.

[0044] IlvBN and IlvBN were added at a concentration of 0 with branched-chain amino acids. M1 and IlvBN M2IlvBN and IlvBN were defined as having specific enzyme activities of 100% and the concentrations of the remaining branched chain amino acids, respectively. M1 and IlvBN M2 The relative enzyme activity is the enzyme activity compared to its 100% enzyme activity. The results are as follows: Figure 2 As shown, the relative enzyme activity of IlvBN decreases rapidly with increasing branched-chain amino acid concentration, and is almost non-existent when the branched-chain amino acid concentration is above 10 mmol / L, indicating that this wild-type enzyme is subject to feedback inhibition by branched-chain amino acids; while the mutant IlvBN M1 and IlvBN M2 The relative activity of the compound did not change significantly with the increase of branched-chain amino acid concentration, indicating that it relieved the feedback inhibition effect of branched-chain amino acids.

[0045] Based on the above results, the acetolactate synthase mutant IlvBN M1 and IlvBN M2 The feedback inhibition effect of branched-chain amino acids was eliminated. Furthermore, in the absence of branched-chain amino acids, the enzyme activity of the mutants was not significantly reduced compared to the wild-type IlvBN.

[0046] Example 3: IlvBN M1 and IlvBN M2 Applications in L-valine synthesis (1) Apply pST-ilvBN and pST-ilvBN respectively M1 and pST-ilvBN M2 Transformed into E. coli E. coli W3110 was used to obtain recombinant strains Val-1 (control), Val-2, and Val-3.

[0047] (2) Val-1, Val-2 and Val-3 were inoculated into 30 mL of basic culture medium and cultured at 37℃ and 220 rpm for 24 h.

[0048] The basic culture medium consisted of: glucose 15 g / L, MgSO4 0.3 g / L, KH2PO4 2 g / L, (NH4)2SO4 4 g / L, MgSO4 1 g / L, FeSO4·7H2O 10 mg / L, MnSO4 10 mg / L, deionized water 1000 mL, and pH 6.5-7.0.

[0049] (3) Detection of L-valine in fermentation broth After centrifuging the fermentation broth at 8000 g for 10 min, the supernatant was collected and diluted with deionized water. The fermentation broth was then derivatized with 0.8% (V / V) 2,4-dinitrofluorobenzene. The L-valine content was determined by high performance liquid chromatography (HPLC) under the following conditions: Agilent C18 (150 mm × 4.6 mm, 5 μm), acetonitrile / sodium acetate binary gradient elution, column temperature 33℃, and detection wavelength 360 nm.

[0050] The L-valine yields of Val-1, Val-2, and Val-3 were 0.01 g / L, 5.42 g / L, and 5.78 g / L, respectively. This indicates that the acetolactate synthase mutant IlvBN obtained in this invention... M1 and IlvBN M2 The ability to synthesize L-valine was significantly improved compared to the wild type.

[0051] Example 4: IlvBN M1 and IlvBN M2 Application in L-Isoleucine Synthesis Threonine dehydratase (by) ilvA The enzyme (encoded in Val-1, Val-2, and Val-3) is a key enzyme in the synthesis of L-isoleucine and is subject to feedback inhibition by L-isoleucine. Therefore, expression of this enzyme in Val-1, Val-2, and Val-3 relieves the feedback inhibition of L-isoleucine. ilvA M (C1339T, G1341T, C1351G and T1352C) can synthesize L-isoleucine.

[0052] (1) with E. coli Using the W3110 genome as a reference, a design was created. ilvA M Amplification primers ilvA-1, ilvA-2, ilvA-3, and ilvA-4 were used. Primer ilvA-2 contained G1339A, C1341A, G1351C, and A1352G, while ilvA-3 contained C1339T, G1341T, C1351G, and T1352C. E. coli Using the W3110 genome as a template, amplification was performed using ilvA-1 / ilvA-2 and ilvA-3 / ilvA-4, respectively. ilvA M Upstream and downstream gene fragments were mixed in an equimolar ratio as an overlap PCR template, amplified using primers ilvA-1 and ilv4-4, and then recovered. Xba I and Bam The plasmid pWSK29 was ligated to the same digested pWSK29 plasmid using HI double digestion to obtain the recombinant plasmid pWSK-ilvA. M .

[0053] (2) Add pWSK-ilvA M The strains were transformed into Val-1, Val-2, and Val-3 obtained in Example 3, respectively, to obtain recombinant strains ILE-1 (control), ILE-2, and ILE-3.

[0054] (3) ILE-1, ILE-2 and ILE-3 were inoculated into 30 mL of basic culture medium and cultured at 37℃ and 220 rpm for 24 h.

[0055] The basic culture medium consisted of: glucose 15 g / L, MgSO4 0.3 g / L, KH2PO4 2 g / L, (NH4)2SO4 4 g / L, MgSO4 1 g / L, FeSO4·7H2O 10 mg / L, MnSO4 10 mg / L, deionized water 1000 mL, and pH 6.5-7.0.

[0056] (4) Detection of L-isoleucine in fermentation broth After centrifuging the fermentation broth at 8000 g for 10 min, the supernatant was collected and diluted with deionized water. The fermentation broth was then derivatized with 0.8% (V / V) 2,4-dinitrofluorobenzene. The L-isoleucine content was determined by high performance liquid chromatography (HPLC) under the following conditions: Agilent C18 (150 mm × 4.6 mm, 5 μm), acetonitrile / sodium acetate binary gradient elution, column temperature 33℃, and detection wavelength 360 nm.

[0057] The L-isoleucine yields of ILE-1, ILE-2, and ILE-3 were 0.02 g / L, 1.31 g / L, and 1.17 g / L, respectively. This indicates that the acetolactate synthase mutant IlvBN obtained in this invention... M1 and IlvBN M2 The ability to synthesize L-isoleucine was significantly improved compared to the wild type.

[0058] Example 5: IlvBN M1 and IlvBN M2 Applications in L-leucine synthesis Isopropyl malate synthase (by...) leuA The enzyme (encoded in Val-1, Val-2, and Val-3) is a key enzyme in the synthesis of L-leucine and is subject to feedback inhibition by L-leucine. Therefore, expression of this enzyme in Val-1, Val-2, and Val-3 relieves the feedback inhibition of L-leucine. leuA M (ZL201910820591.X) can synthesize L-leucine.

[0059] (1) with leuA MUsing leuA-1 / leuA-2 as templates, amplification was performed separately. leuA M Genes, after being recycled Xba I and Bam The plasmid pWSK29 was ligated to the same digested pWSK29 plasmid using HI double digestion to obtain the recombinant plasmid pWSK- leuA M .

[0060] (2) pWSK-leuA M The strains were transformed into Val-1, Val-2, and Val-3 obtained in Example 3, respectively, to obtain recombinant strains LEU-1 (control), LEU-2, and LEU-3.

[0061] (3) LEU-1, LEU-2 and LEU-3 were inoculated into 30 mL of basic culture medium and cultured at 37℃ and 220 rpm for 24 h.

[0062] The basic culture medium consisted of: glucose 15 g / L, MgSO4 0.3 g / L, KH2PO4 2 g / L, (NH4)2SO4 4 g / L, MgSO4 1 g / L, FeSO4·7H2O 10 mg / L, MnSO4 10 mg / L, deionized water 1000 mL, and pH 6.5-7.0.

[0063] (4) Detection of L-leucine in fermentation broth After centrifuging the fermentation broth at 8000 g for 10 min, the supernatant was collected and diluted with deionized water. The fermentation broth was then derivatized with 0.8% (V / V) 2,4-dinitrofluorobenzene. The L-leucine content was determined by high performance liquid chromatography (HPLC) under the following conditions: Agilent C18 (150 mm × 4.6 mm, 5 μm), acetonitrile / sodium acetate binary gradient elution, column temperature 33℃, and detection wavelength 360 nm.

[0064] The L-leucine yields of LEU-1, LEU-2, and LEU-3 were 0.07 g / L, 1.56 g / L, and 1.47 g / L, respectively. This indicates that the acetolactate synthase mutant IlvBN obtained in this invention... M1 and IlvBN M2 The ability to synthesize L-leucine was significantly improved compared to the wild type.

[0065] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. An acetolactate synthase mutant, characterized in that, The mutants were obtained by mutations of G20D and S23F, or G20E and S23F, in the regulatory subunit IlvN of wild-type acetolactate synthase IlvBN.

2. The acetolactate synthase mutant as described in claim 1, characterized in that, The acetyllactate synthase mutant is IlvBN. M1 It consists of the catalytic subunit IlvB and the regulatory subunit IlvN. M1 Composition: The amino acid sequence of the catalytic subunit IlvB is shown in SEQ ID NO.1, and the regulatory subunit IlvN... M1 The amino acid sequence is shown in SEQ ID NO.3; or, The acetyllactate synthase mutant is IlvBN. M2 It consists of the catalytic subunit IlvB and the regulatory subunit IlvN. M2 Composition: The amino acid sequence of the catalytic subunit IlvB is shown in SEQ ID NO.1, and the regulatory subunit IlvN... M2 The amino acid sequence is shown in SEQ ID NO.

4.

3. The encoding gene of the acetolactate synthase mutant according to claim 1.

4. A recombinant vector or recombinant strain containing the encoding gene of the acetolactate synthase mutant of claim 1.

5. The recombinant vector as described in claim 4, characterized in that, The recombinant vectors used include the expression vectors pSTV28, pWSK29, pTrc99a, and pET28a.

6. The recombinant strain according to claim 4, characterized in that, The host cells used in the recombinant strains include: Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and Saccharomyces cerevisiae.

7. The use of the recombinant vector or recombinant strain of claim 4 in the preparation of the acetolactate synthase mutant of claim 1.

8. The use of the acetolactate synthase mutant of claim 1 or the recombinant vector or recombinant strain of claim 4 in the catalytic synthesis of branched-chain amino acids.

9. The application as described in claim 8, characterized in that, The branched-chain amino acids include L-valine, L-leucine, or L-isoleucine.

10. The use of the acetolactate synthase mutant of claim 1 in relieving feedback inhibition of L-valine, L-leucine and L-isoleucine.

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