α-Keto acid decarboxylase mutants and their applications

Abstract

The present invention relates to a class of α-keto acid decarboxylase mutants with significantly improved enzyme activity and their applications. The mutants of the present invention contain amino acid mutations at positions 286, 402, and / or 461 of the amino acid sequence, and the amino acid sequence of the α-keto acid decarboxylase is as shown in SEQ ID NO: 1. Based on homology modeling and molecular docking methods, the present invention analyzed the key amino acid residues within the radius of the active pocket, and on the basis of virtual screening, experimental verification was carried out. By using single-point mutation and combinatorial mutation techniques, a class of mutants with improved catalytic efficiency was obtained. The research results show that compared with the wild type, the yield of 1,2,4-butanetriol catalyzed by the mutants of the present invention has been greatly improved, which is of great significance for reducing the cost of industrial scale production of 1,2,4-butanetriol.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and specifically relates to a class of α-keto acid decarboxylase mutants with improved enzyme activity and their applications. Background Art

[0002] As an unnatural polyol, 1,2,4-butanetriol (abbreviated as BT) has great application value in civil, military, and pharmaceutical fields. It can be used to prepare anti-tumor, anti-viral, anti-AIDS drugs, platelet activating factor, cholesterol-lowering drugs, and drugs for treating skin diseases. In the military field, 1,2,4-butanetriol can be used to synthesize the high-energy propellant material butanetriol trinitrate (abbreviated as BTTN).

[0003] The chemical production of butanetriol has disadvantages such as harsh synthesis conditions, high cost, complex purification process, and serious environmental pollution. Therefore, the biological synthesis of BT has received extensive attention, but there is no natural biosynthetic pathway for BT in nature. Currently, researchers have successfully constructed a synthetic pathway for producing BT by fermenting xylose. However, the yield is low and cannot meet the industrial production of BT.

[0004] The third step in the synthetic pathway, namely the decarboxylation reaction of 3-deoxy-D-glycero-pentoketonic acid, is a key step. α-Keto acid decarboxylase is the key enzyme for the reaction. Using protein engineering means to improve the activity of α-keto acid decarboxylase is of great significance for increasing the yield of BT and reducing production costs. Summary of the Invention

[0005] The present invention takes the branched-chain α-keto acid decarboxylase KDCA derived from Lactococcus lactis as the research object. Through homology modeling and molecular docking techniques, the key amino acid residues within the range of the active pocket radius were analyzed. By virtual screening, hot-spot amino acids capable of improving enzyme catalytic activity were discovered, and site-directed mutagenesis was performed on them to obtain a series of α-keto acid decarboxylase mutants, which can be used as biocatalysts to achieve the efficient preparation of BT.

[0006] The specific technical solution of the present invention is as follows:

[0007] An α-keto acid decarboxylase mutant, wherein the mutant contains one or more mutations of the 286th, 402nd, and 461st amino acids in the amino acid sequence of α-keto acid decarboxylase, and the amino acid sequence of the α-keto acid decarboxylase is as shown in SEQ ID NO: 1.

[0008] The mutation at the 286th position is: serine is mutated to valine, arginine, proline, asparagine, leucine, lysine, isoleucine, histidine, glycine, phenylalanine, glutamic acid, aspartic acid or cysteine, preferably leucine.

[0009] The mutation at the 402nd position is: glycine is mutated to tyrosine, valine, serine, arginine, proline, asparagine, methionine, leucine, lysine, isoleucine, histidine, phenylalanine, glutamic acid, aspartic acid or cysteine, preferably aspartic acid or proline.

[0010] The mutation at the 461st position is: valine is mutated to tryptophan, serine, arginine, glutamine, proline, leucine, lysine, isoleucine, glutamic acid, aspartic acid, cysteine or alanine, preferably lysine or arginine.

[0011] Preferably, the α-keto acid decarboxylase mutant of the present invention is a single-site mutation, where serine at the 286th position is mutated to leucine (S286L); or, glycine at the 402nd position is mutated to proline (G402P); or valine at the 461st position is mutated to lysine or arginine (V461K, V461R).

[0012] Preferably, the α-keto acid decarboxylase mutant of the present invention is a double-site mutation, where serine at the 286th position is mutated to leucine and glycine at the 402nd position is mutated to proline.

[0013] Another object of the present invention is to provide a DNA molecule that encodes the α-keto acid decarboxylase mutant of the present invention.

[0014] Another object of the present invention is to provide an expression vector for the α-keto acid decarboxylase mutant, which expresses the α-keto acid decarboxylase mutant as described in the present invention. The expression vector contains a DNA molecule encoding the α-keto acid decarboxylase mutant of the present invention.

[0015] The expression vector is a plasmid, phage, virus or host cell.

[0016] The host cell is a prokaryotic cell or a eukaryotic cell, and can be Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma, preferably Escherichia coli.

[0017] Another object of the present invention is to provide the application of the α-keto acid decarboxylase mutant, DNA molecule or expression vector of the α-keto acid decarboxylase mutant of the present invention in the synthesis of 1,2,4-butanetriol.

[0018] Advantages of the present invention:

[0019] The present invention takes the branched-chain α-keto acid decarboxylase KDCA derived from Lactococcus lactis obtained by previous screening as the research object. Through homology modeling and molecular docking techniques, the key amino acid residues within the range of the active pocket radius are analyzed, and hot-spot amino acids capable of enhancing the enzyme catalytic activity are discovered through virtual screening. Saturated mutations are performed on sites S286, V461, G402, and Q377, and it is found that the enzyme activities of single-point mutations S286L, G402D, G402P, V461K, and V461R are all higher than that of the wild type. Among them, the enzyme activities of single mutants V461K and V461R are increased more significantly, being 2.5 times and 2.2 times that of the original enzyme KDCA, respectively. Then, combined mutations are performed on the high-quality single mutants, and it is found that the combined mutant S286L / G402P has a relatively high enzyme activity, which is 1.8 times that of the original enzyme KDCA. Within the range, the key amino acid residues are analyzed, and hot-spot amino acids capable of enhancing the enzyme catalytic activity are discovered through virtual screening. Saturated mutations are performed on sites S286, V461, G402, and Q377, and it is found that the enzyme activities of single-point mutations S286L, G402D, G402P, V461K, and V461R are all higher than that of the wild type. Among them, the enzyme activities of single mutants V461K and V461R are increased more significantly, being 2.5 times and 2.2 times that of the original enzyme KDCA, respectively. Then, combined mutations are performed on the high-quality single mutants, and it is found that the combined mutant S286L / G402P has a relatively high enzyme activity, which is 1.8 times that of the original enzyme KDCA.

[0020] The present invention uses xylose as the substrate and successfully constructs an in vitro catalytic system for butanetriol. The abilities of wild-type KDCA and the dominant mutant enzyme V461K to synthesize butanetriol are compared. It is found that the BT production of the mutant enzyme V461K in cell-free catalysis for 48 h is 6.5 g / L, which is 1.9 times that of the wild type. This result indicates that the α-keto acid decarboxylase mutant described in the present invention has higher catalytic activity than the original enzyme, can fully meet the requirements of industrial applications, and its application in catalytic reactions can improve the production efficiency of the product 1, 2, 4-butanetriol while reducing the production cost. Description of the Drawings

[0021] The drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0022] Figure 1 Interaction of the substrate 3-deoxy-D-glycero-pentoketonic acid (DGP) in KDCA.

[0023] Figure 2 Virtual screening of key amino acids of KDCA.

[0024] Figure 3 SDS-PAGE electrophoresis patterns of KDCA and mutants.

[0025] Figure 4 Relative enzyme activity graphs of saturated mutations at sites S286, V461, G402, Q377 and the original enzyme KDCA.

[0026] Figure 5 Relative enzyme activity of the combined mutant strain and the original enzyme KDCA.

[0027] Figure 6 Yields of DCA and mutants in cell-free catalysis to produce BT. Detailed implementation manners

[0028] To enable those skilled in the art to better understand the solution of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the present embodiment is only used to explain the present invention, rather than limiting the scope of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0029] Example 1: Establishment of key amino acids in the active architecture of α-keto acid decarboxylase KDCA

[0030] For the biosynthesis of 1,2,4-butanetriol, xylose is mostly used as a substrate. Xylose is dehydrogenated by xylose dehydrogenase XYLB to generate xylonic acid, and then under the action of xylonic acid dehydratase XYLD, branched-chain α-keto acid decarboxylase KDCA and alcohol dehydrogenase ADHP, it is finally converted into BT. By calculating the Gibbs free energy, it is determined that the decarboxylase is the key enzyme for the reaction. Therefore, the present invention improves the yield of 1,2,4-butanetriol by molecularly modifying α-keto acid decarboxylase KDCA. First, the tertiary structure of 3-deoxy-D-glyceropentanose acid (DGP) is docked into the active center of KDCA ( Figure 1 ), and the docking free energy of DGP in KDCA is -5.5 kcal / mol. The structure of branched-chain α-keto acid decarboxylase KDCA is a dimer, and the two subunit chains are named chain A and chain B respectively. The substrate binding pocket is near the interface between the subunits. The amino acid residues that interact with the substrate DGP within the range of

[0031]

[0032]

[0033] To study the effects of mutations of key amino acids on the enzyme and the substrate, the MOE software is used to perform virtual saturation mutations on the amino acid residues that interact with DGP within the above-mentioned substrate radius range, and analyze the changes in the affinity between the mutants and the substrate. Since two histidines (His112 and His113), one aspartic acid (Asp26) and one glutamate (Glu462) near the thiazole ring are highly conserved amino acids related to catalysis, mutations are not considered for the time being. The selection of hot-spot amino acid sites needs to be conducive to substrate binding (dAffinity < 0). The key sites found by virtual screening are as Figure 2As shown, in order to obtain mutant enzymes with high catalytic activity, subsequent saturation mutagenesis was performed on the hot-spot amino acid sites S286, Q377, G402, and V461.

[0034] Example 2 Construction of the KDCA Mutant Library of α-Keto Acid Decarboxylase

[0035] As described in Example 1, saturation mutagenesis was performed on α-keto acid decarboxylase KDCA at the positions of S286, Q377, G402, and V461, and the excellent mutants S286L, Q377M, G402P, and V461K obtained in the single-site saturation mutagenesis were subjected to combinatorial mutagenesis in order to obtain mutant strains with higher catalytic efficiency. Taking S286 as an example, its construction method is as follows, and the primers used are shown in the following table:

[0036]

[0037]

[0038] Using the pET-22b(+) plasmid carrying the α-keto acid decarboxylase KDCA gene sequence (SEQ ID NO: 1) as a template, and referring to the Vazyme biological products and operation manual, the site-directed mutation sequence was amplified by whole plasmid using the mutant primer pair. The PCR product was digested with Dpn I. After the template digestion was completed, it was transformed into the Escherichia coli competent cell E. coli BL21(DE3) by heat shock method and spread on an LB agar plate containing 100 μg / ml kanamycin sulfate, and cultured overnight at 37°C in an inverted position. The mutation results were verified by sequence determination by Anhui General Biology Company. Taking the S286 site as an example, the mutant library of S286 was constructed by using the conventional PCR technique to introduce mutations by whole plasmid amplification using the α-keto acid decarboxylase KDCA expression vector as a template, and the constructed S286 mutant library was verified to be successful by sequencing.

[0039] Example 3 Fermentation of the Recombinant α-Keto Acid Decarboxylase KDCA Mutant in Escherichia coli

[0040] The respective S286 mutants constructed in Example 2 and the original enzyme KDCA were separately inoculated into 50 mL of LB liquid medium containing 100 μg / mL kanamycin sulfate and cultured overnight at 37°C and 180 rpm; the seed solution was inoculated into fresh 50 mL of LB liquid medium at an inoculation amount of 2%, and cultured at 37°C and 180 rpm until the OD 600 reached 0.6 - 1.0, then taken out and cooled in an ice-water bath for 5 min, and the inducer IPTG (isopropyl-β-D-thiogalactoside) (final concentration 0.1 mmol / L) was added, and induced expression was carried out at 16°C and 150 rpm for 24 h.

[0041] Take the induced fermentation broth, centrifuge at 12,000 rpm for 20 min, discard the supernatant, then resuspend and wash the cells with 50 mM Na2HPO4-KH2PO4 (pH 6.5) buffer, centrifuge at 12,000 rpm for 20 min, discard the supernatant, resuspend with the buffer again, and then perform ultrasonic disruption. The disrupted solution was centrifuged at 12,000 rpm for 20 min, and the supernatant was taken for SDS-PAGE electrophoresis detection. The concentration of the stacking gel was 4%, and the concentration of the separating gel was 12.5%. The sample was mixed with the loading buffer in a ratio of 3:1, and subjected to a boiling water bath reaction for 5 min for loading and electrophoresis. The initial voltage of the electrophoresis apparatus was set at 120 V, and the voltage was increased to 230 V when the sample moved into the separating gel, and electrophoresis was terminated until the sample moved to the bottom of the electrophoresis tank.

[0042] The SDS-PAGE electrophoresis results of the crude enzyme solutions of the above KDCA mutants are as Figure 3 shown. The molecular weight of the target protein is 60.9 kDa, and obvious bands are observed at 60.9 kDa in each lane of the electrophoresis pattern, indicating that the target protein was successfully expressed in each mutant. Referring to the methods of Examples 2 and 3, saturated mutant libraries of Q377, G402, V461 and combined mutant libraries of S286L, Q377M, G402P, V461K were constructed respectively, and the corresponding mutants were purified.

[0043] Example 4 Enzyme Activity Assay of α-Ketoacid Decarboxylase

[0044] Xylonic acid is catalyzed by dehydratase to generate 3-deoxy-D-glycero-pentulosonic acid, and its derivatization product with semicarbazide hydrochloride reagent has an absorbance value at 250 nm (ε 250 = 10.2 mM -1 cm -1 ). The enzyme activity can be determined by the change in absorbance at 250 nm. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol of α-ketoacid per minute.

[0045] The reaction mixture contains 50 mM potassium phosphate buffer (pH 7.0), 1 mM MgCl2, 10 mM xylonic acid and an appropriate amount of xylonic acid dehydratase, and the reaction is initiated by adding the enzyme. The above reaction system was reacted at 25 °C for 30 min, then quenched by adding semicarbazide reagent, incubated at 30 °C for another 15 min, and the absorbance was measured at 250 nm after dilution by an appropriate multiple.

[0046] Since the direct substrate of decarboxylase, 3-deoxy-D-glycero-pentulosonic acid, is not easily obtained, the decarboxylation activity of KDCA was measured by an enzyme-linked method. 3,4-Dihydroxybutyraldehyde is dehydrogenated to generate BT under the catalysis of alcohol dehydrogenase, accompanied by the consumption of NADH, so the light absorption value of NADH at 340 nm (ε 340 = 6.22 mM-1 cm -1 ) The enzyme activity was measured by the change of -1 , and the measured enzyme activity represented the total activity of dehydratase and α-keto acid decarboxylase. One unit of enzyme activity was defined as the amount of consuming 1 μmol NADH per minute.

[0047] The enzyme activity assay system for decarboxylase was as follows: 10 mM xylonic acid, 1 mM MgSO4, 0.2 mM NADH, 0.15 mM ThDP, 10 U alcohol dehydrogenase, 5 μM xylonic acid dehydratase, and an appropriate amount of α-keto acid decarboxylase. Add 300 μL of the above system to the microplate, mix well, record the absorbance value at 340 nm every 1 min at 30 °C for a total of 10 min. Compare and analyze with the enzyme activity of the original enzyme KDCA ( Figure 4 ). In the saturation mutagenesis of S286 ( Figure 4 A), after replacing serine (S) with leucine (L), the enzyme activity of S286L increased to 1.6 times that of the wild type. In the saturation mutagenesis of site V461 ( Figure 4 B), after replacing valine (V) with lysine (K), the enzyme activity of V461K was much higher than that of the original enzyme, which was 2.5 times that of the original enzyme. Another better mutant was obtained by replacing valine (V) with arginine (R), and the enzyme activity of V461R was 2.2 times that of the original enzyme. In the saturation mutagenesis of Q377 site ( Figure 4 C), after replacing glutamine (Q) with methionine (M), the enzyme activity of Q377M increased to 1.4 times that of the wild type, and the enzyme activities of the other mutants remained at a level similar to or slightly lower than that of the original enzyme KDCA. In the saturation mutagenesis of G402 site ( Figure 4 D), after replacing glycine (G) with aspartic acid (D) and proline (P) respectively, better mutants G402D and G402P were obtained, and the enzyme activities increased by 51% and 65% respectively. Detect the enzyme activity of the combined mutant strains and compare and analyze it with the enzyme activity of KDCA ( Figure 5 ). The results showed that the enzyme activities of the double mutants were all better than that of the wild type KDCA. Among them, the enzyme activity of the double mutant S286L / G402P increased significantly, which was 1.8 times that of the original enzyme KDCA; while the enzyme activity of the mutant S286L / Q377M / G402P / V461K was slightly lower than that of the original enzyme.

[0048] Example 5 Production of 1,2,4-butanetriol by cell-free catalytic system

[0049] Establish a cell-free catalytic synthesis system for 1,2,4-butanetriol: 50 mM phosphate buffer (pH 7.0), 20 g / L xylose, 0.5 mM ThDP, 10 mM MgCl2, 0.5 mM NAD +, 0.5 mM NADH, 0.2 mg / mL xylose dehydrogenase, 0.2 mg / mL xylonic acid dehydratase, 0.2 mg / mL branched-chain α-keto acid decarboxylase, 0.2 mg / mL alcohol dehydrogenase, and perform light-avoiding conversion at 30 °C and 150 rpm for 48 h.

[0050] By detecting xylose and 1,2,4-butanetriol standards, it was found that the retention times of the substrate xylose and the product 1,2,4-butanetriol were 13.118 min and 19.625 min, respectively. Under the conditions of 30 °C and 150 rpm, light-avoiding catalysis was carried out for 48 h, and 1,2,4-butanetriol was successfully detected in the catalytic solution by high-performance liquid chromatography (HPLC). The catalytic solution was detected by mass spectrometry, and the m / z value of the product was exactly the same as the theoretically calculated value of butanetriol, indicating that an in vitro synthesis system for butanetriol has been successfully constructed. Comparing the abilities of the original enzyme KDCA and the dominant mutant V461K to catalyze the synthesis of butanetriol ( Figure 6 ), it was found that the BT production of V461K in cell-free catalysis for 48 h was 6.5 g / L, which was 1.9 times that of the wild type. This result indicates that the mutant V461K has higher catalytic activity than the original enzyme KDCA, which will be beneficial to reducing the enzyme dosage in the production of 1,2,4-butanetriol, thereby reducing the overall production cost.

[0051] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. An α-keto acid decarboxylase mutant, characterized in that The mutant is such that the 461st position mutation in the amino acid sequence of the α-keto acid decarboxylase shown in SEQ ID NO: 1 is: valine is mutated to lysine or arginine.

2. An α-keto acid decarboxylase mutant, characterized in that The mutant is such that the valine at the 461st position in the amino acid sequence of the α-keto acid decarboxylase shown in SEQ ID NO: 1 is mutated to lysine and the serine at the 286th position is mutated to leucine; or, the mutant is such that the valine at the 461st position in the amino acid sequence of the α-keto acid decarboxylase shown in SEQ ID NO: 1 is mutated to lysine and the glycine at the 402nd position is mutated to proline.

3. A DNA molecule, characterized in that, The DNA molecule encodes the α-keto acid decarboxylase mutant according to claim 1 or 2.

4. An expression vector of an α-keto acid decarboxylase mutant, characterized in that Express the α-keto acid decarboxylase mutant according to claim 1 or 2.

5. The expression vector according to claim 4, characterized in that Express the DNA molecule according to claim 3.

6. The expression vector according to claim 4, wherein The expression vector is a plasmid, phage, or virus.

7. A host cell expressing an α-keto acid decarboxylase mutant, characterized in that The host cell is a prokaryotic cell or a eukaryotic cell.

8. The host cell according to claim 7, characterized in that The host cell is selected from Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus, or Trichoderma.

9. Use of the α-keto acid decarboxylase mutant according to any one of claims 1 or 2, the DNA molecule according to claim 3, or the expression vector of the α-keto acid decarboxylase mutant according to any one of claims 4 - 6, or the host cell expressing the α-keto acid decarboxylase mutant according to claim 7 or 8 in the catalytic synthesis of 1,2,4-butanetriol.

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