Formaldehyde-lyase mutants and their use in the synthesis of 1,3-dihydroxyacetone

Abstract

The present application relates to formaldehyde lyase mutants and their application in the synthesis of 1,3-dihydroxyacetone. The present application discloses a formaldehyde lyase mutant obtained by molecular modification of the formaldehyde lyase of Polymorphobacter arshaanensis, a nucleic acid encoding the formaldehyde lyase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, preparation of the mutant enzyme catalyst, and application of the mutant enzyme catalyst in the synthesis of DHA. Compared with the optimal formaldehyde lyase FLS-M3 reported in the literature, the formaldehyde lyase mutant provided by the present application has a higher heterologous expression level, has a higher yield in catalyzing the condensation of formaldehyde to synthesize DHA, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a formaldehyde lyase mutant and its application in synthesis of 1,3-dihydroxyacetone. Specifically, the present application provides a formaldehyde lyase mutant from Polymorphobacter arshaanensis with the ability to catalyze formaldehyde to synthesize 1,3-dihydroxyacetone, a nucleic acid encoding the formaldehyde lyase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, preparation of the mutant enzyme catalyst, and application of the mutant enzyme catalyst in synthesis of 1,3-dihydroxyacetone. BACKGROUND

[0002] 1,3-dihydroxyacetone is the simplest ketose, which contains three functional groups (one carbonyl and two hydroxyl groups), has active chemical properties, can widely participate in condensation, addition and other reactions, is an important three-carbon platform compound, and is often used as a key synthetic building block of fine chemicals, drugs and the like. In addition, DHA is one of the intermediate products of sugar metabolism, plays an important role in the process of sugar metabolism, and is related to the ability of the body to promote fat consumption and increase protein content.

[0003] According to the published literature and patents, the current production methods of DHA mainly include chemical synthesis and glycerol fermentation. There are mainly two methods for chemical synthesis of DHA: oxidation of glycerol and formaldehyde polysaccharide. The first method uses strong oxidants (such as hydrogen peroxide or sodium chlorite) to oxidize glycerol, and this method often generates a mixture of DHA and by-products, and high yield and purity can be achieved by optimizing the reaction conditions. However, this method has the disadvantages of using toxic reagents and generating hazardous waste. The formaldehyde polysaccharide method for synthesizing DHA can be traced back to the 1980s, which uses formaldehyde as a raw material due to its low price and wide source, and generates high-value chemical DHA by condensing three molecules of formaldehyde, and is a relatively green and high-atom economy synthesis method, but the research progress is relatively slow and is still in the laboratory stage. Compared with the disadvantages of using toxic reagents and generating hazardous waste in the chemical synthesis method, the microbial fermentation method uses high-activity microbial strains to convert glycerol to DHA, which is also the main method for industrial production of DHA, but still has problems such as high cost, environmental pollution and complex subsequent separation and purification process.

[0004] There are few reports on the synthesis of DHA from formaldehyde by in vitro enzymatic method. In 2015, Justin B. Siegel et al. designed a benzaldehyde lyase by computer and obtained the first formaldehyde lyase FLS with DHA synthesis activity (Proceedings of the National Academy of Sciences of the United States of America. 2015, 112(12), 3704-3709). However, the activity of FLS is low, and the catalytic efficiency is only 4.7M -1 s -1 , and the product selectivity is not ideal. Subsequently, in 2018, Ma Yanhe et al. of the Institute of Industrial Microbiology of the Chinese Academy of Sciences improved FLS through three rounds of error-prone PCR (Science, 2021, 373, 1523-1527), and obtained the mutant FLS-M3 with the highest catalytic efficiency, which improved the catalytic efficiency from the original 4.7M -1 s -1 to 10.1M -1 S -1 , and the mutant is also the most widely used formaldehyde lyase. In the patent (201510446357.7), Jiang Huifeng molecularly modified benzoylformic acid decarboxylase and obtained a formaldehyde lyase BFD with the catalytic activity of formaldehyde to synthesize 1,3-dihydroxyacetone. Subsequently, Zhu Lei in 2020 further molecularly modified BFD and greatly improved its substrate tolerance to FALD (Green Chemistry, 2020, 22(20):6809-6814). However, the substrate affinity of the enzyme is poor and the catalytic efficiency is much lower than that of FLS-M3, only 1.57M -1 s -1 .

[0005] Although some progress has been made in the study of formaldehyde lyase, the existing formaldehyde lyases are few in species and poor in catalytic performance, and there is still a big gap from practical application. Therefore, it is urgent to obtain a new type of formaldehyde lyase with better catalytic performance for efficient synthesis of 1,3-dihydroxyacetone. SUMMARY

[0006] In view of the application defects of low catalytic efficiency of formaldehyde lyase in the synthesis of 1,3-dihydroxyacetone from formaldehyde by biological catalysis, the present application provides a formaldehyde lyase mutant and its application in the synthesis of 1,3-dihydroxyacetone.

[0007] Specifically, the present application provides a formaldehyde-lyase mutant with significantly improved catalytic performance, a nucleic acid encoding the formaldehyde-lyase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, a formaldehyde-lyase mutant catalyst, and the application of the formaldehyde-lyase mutant catalyst in catalyzing the synthesis of 1,3-dihydroxyacetone from formaldehyde by condensation.

[0008] Compared with the optimal formaldehyde-lyase FLS-M3 reported in the literature, the formaldehyde-lyase mutant provided by the present application has a higher heterologous expression level and a higher efficiency in catalyzing the condensation of formaldehyde to generate DHA.

[0009] The object of the present application can be achieved by the following technical solutions:

[0010] According to one of the technical solutions, the present application provides a formaldehyde-lyase mutant with significantly improved catalytic performance.

[0011] The present application clones a formaldehyde-lyase from Polymorphobacter arshaanensis by a gene database mining method, which is named PaFLS, and the nucleotide sequence of the edited gene is shown in SEQ ID No. 1, and the amino acid sequence of the formaldehyde-lyase PaFLS is shown in SEQ ID No. 2. Then, the PaFLS is molecularly modified by semi-rational design strategies such as site-directed saturation mutation and combinatorial mutation, and a formaldehyde-lyase mutant with significantly improved 1,3-dihydroxyacetone synthesis activity is obtained.

[0012] The formaldehyde-lyase mutant is a protein corresponding to a new amino acid sequence formed by replacing one or more amino acid residues at positions 31, 77, 287, 396, 421, 480, 481, 482 and 571 of the amino acid sequence shown in SEQ ID No. 2 with other amino acid residues, and the protein exhibits significantly improved catalytic activity for catalyzing the condensation of formaldehyde to synthesize 1,3-dihydroxyacetone.

[0013] Further, the formaldehyde-lyase mutant provided by the present application with high catalytic activity for catalyzing the condensation of formaldehyde to synthesize 1,3-dihydroxyacetone is a protein with the following amino acid sequence:

[0014] (1) replacing the glycine at position 31 of the amino acid sequence shown in SEQ ID No. 2 with serine, which is named PaFLSG31S ;

[0015] (2) Replace the threonine at position 77 of the amino acid sequence shown as SEQ ID No. 2 with cysteine, named PaFLS T77C ;

[0016] (3) Replace the methionine at position 287 of the amino acid sequence shown as SEQ ID No. 2 with cysteine, named PaFLS M287C ;

[0017] (4) Replace the glycine at position 396 of the amino acid sequence shown as SEQ ID No. 2 with asparagine, named PaFLS G396N ;

[0018] (5) Replace the glycine at position 421 of the amino acid sequence shown as SEQ ID No. 2 with asparagine, named PaFLS G421N ;

[0019] (6) Replace the glycine at position 421 of the amino acid sequence shown as SEQ ID No. 2 with tryptophan, named PaFLS G421W ;

[0020] (7) Replace the glycine at position 421 of the amino acid sequence shown as SEQ ID No. 2 with tyrosine, named PaFLS G421Y ;

[0021] (8) Replace the methionine at position 480 of the amino acid sequence shown as SEQ ID No. 2 with leucine, named PaFLS M480L ;

[0022] (9) Replace the serine at position 481 of the amino acid sequence shown as SEQ ID No. 2 with glycine, named PaFLS S481G ;

[0023] (10) Replace the tyrosine at position 571 of the amino acid sequence shown as SEQ ID No. 2 with phenylalanine, named PaFLS Y571F ;

[0024] (11) Replace the methionine at position 287 of the amino acid sequence shown as SEQ ID No. 2 with cysteine, and the glycine at position 421 with tryptophan, named PaFLS M287C / G421W ;

[0025] (12) Replace the glycine at position 421 of the amino acid sequence shown as SEQ ID No. 2 with tryptophan, and the methionine at position 480 with leucine, named PaFLSG421W / M480L ;

[0026] (13) the glycine at position 421 of the amino acid sequence shown as SEQ ID No. 2 is replaced by tryptophan, the serine at position 481 is replaced by glycine, and is named as PaFLS G421W / S481G ;

[0027] (14) the glycine at position 421 of the amino acid sequence shown as SEQ ID No. 2 is replaced by tryptophan, the tyrosine at position 571 is replaced by phenylalanine, and is named as PaFLS G421W / Y571F ;

[0028] (15) the glycine at position 421 of the amino acid sequence shown as SEQ ID No. 2 is replaced by tryptophan, the isoleucine at position 482 is replaced by arginine, and is named as PaFLS G421W / I482R ;

[0029] (16) the glycine at position 421 of the amino acid sequence shown as SEQ ID No. 2 is replaced by tryptophan, the isoleucine at position 482 is replaced by phenylalanine, and is named as PaFLS G421W / I482F ;

[0030] (17) the glycine at position 421 of the amino acid sequence shown as SEQ ID No. 2 is replaced by tryptophan, the isoleucine at position 482 is replaced by glutamine, and is named as PaFLS G421W / I482Q ;

[0031] (18) the glycine at position 421 of the amino acid sequence shown as SEQ ID No. 2 is replaced by tryptophan, the isoleucine at position 482 is replaced by alanine, and is named as PaFLS G421W / I482A ;

[0032] (19) the glycine at position 421 of the amino acid sequence shown as SEQ ID No. 2 is replaced by tryptophan, the methionine at position 480 is replaced by leucine, and the isoleucine at position 482 is replaced by alanine, and is named as PaFLS G421W / M480L / I482A .

[0033] The second technical solution of the present application provides a nucleic acid encoding the formaldehyde-lyase mutant as described in the first technical solution of the present application. The nucleic acid encodes the formaldehyde-lyase mutant as described in the first technical solution, which is obtained by cloning the gene sequence of the series of formaldehyde-lyase mutants as described in the first technical solution through genetic engineering technology, or is obtained by synthesizing the nucleic acid encoding the formaldehyde-lyase mutant as described in the first technical solution through the method of artificial full-sequence synthesis.

[0034] The third aspect of the present application provides a recombinant expression vector comprising the nucleic acid as described in the second aspect of the present application. The recombinant expression vector can be constructed by linking the nucleic acid encoding the formaldehyde lyase mutant as described in the present application to various suitable vectors by conventional methods in the art. The vector can be any conventional vector in the art, as long as the recombinant expression vector can be normally replicated in the corresponding expression host and the formaldehyde lyase mutant can be expressed. The formaldehyde lyase mutant gene can be operably linked downstream of a suitable regulatory sequence in the vector to achieve constitutive or inducible expression of the formaldehyde lyase mutant. For the E. coli host, the plasmid vector is preferably pET-28a(+) plasmid.

[0035] The fourth aspect of the present application provides a recombinant expression transformant comprising the formaldehyde lyase mutant gene as described in the first aspect of the present application, the nucleic acid as described in the second aspect of the present application, or the recombinant expression plasmid as described in the third aspect of the present application. The recombinant expression transformant can be prepared by transforming the recombinant expression vector that has been constructed into a host cell to prepare the recombinant expression transformant by conventional techniques in the art. The host cell can be any conventional host cell in the art, as long as the recombinant expression vector can be stably replicated by itself and the formaldehyde lyase mutant protein can be effectively expressed after induction by an inducer. The present application preferably uses E. coli as the host cell, and more preferably uses E. coli BL21(DE3) for efficient expression of the formaldehyde lyase mutant as described in the present application.

[0036] The fifth aspect of the present application provides a recombinant formaldehyde lyase mutant catalyst, which is in any of the following forms:

[0037] (1) culturing the recombinant expression transformant as described in the present application to isolate transformant cells containing the formaldehyde lyase mutant;

[0038] (2) culturing the recombinant expression transformant as described in the present application to isolate transformant cells containing the formaldehyde lyase mutant, crushing the transformant cells containing the formaldehyde lyase mutant to obtain a cell crushing solution;

[0039] (3) culturing the recombinant expression transformant as described in the present application to isolate transformant cells containing the formaldehyde lyase mutant, crushing the transformant cells containing the formaldehyde lyase mutant to obtain a cell crushing solution, and freeze-drying the cell crushing solution of the formaldehyde lyase mutant to obtain a freeze-dried enzyme powder.

[0040] The culture method and condition of the recombinant expression transformant are conventional methods and conditions in the art, and different preferred culture methods and conditions are used for recombinant expression transformants constructed using different hosts, as long as the recombinant expression transformant can grow and efficiently produce the formaldehyde lyase mutant of the present application.

[0041] For the recombinant E. coli, the preferred culture medium is LB medium: 10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of NaCl, pH 6.5-7.0. The preferred culture method is as follows: the recombinant E. coli as described above is inoculated into LB medium containing kanamycin and cultured at 37℃ with 180 rpm shaking overnight. The LB medium (containing kanamycin) is inoculated at 1% (v / v) and cultured at 37℃ with 180 rpm shaking, and when the OD of the culture solution reaches 0.6-0.8, 0.2 mM of isopropyl-β-D-thiogalactoside (IPTG) is added as an inducer to induce at 16℃ for 16-24 h. The culture solution is centrifuged, and the cells are washed twice with normal saline to obtain the recombinant expression transformant cells. The harvested recombinant cells are stored in a freezer at -80℃, or the harvested recombinant cells are suspended in 5-10 times the volume (v / w) of buffer, sonicated, and the supernatant is collected by centrifugation to obtain the cell lysate of the recombinant formaldehyde lyase mutant. The collected cell lysate is frozen at -80℃, and then low-temperature dried using a vacuum freeze dryer to obtain the freeze-dried enzyme powder of the recombinant formaldehyde lyase mutant. The obtained freeze-dried enzyme powder is stored in a refrigerator at 4℃ and can be conveniently used.

[0042] Sixth, a purification method of the formaldehyde lyase mutant is provided. The cell lysate of the recombinant formaldehyde lyase mutant obtained in the technical solution five is further purified. The following is the formula of the protein purification buffer: Buffer A: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole; Buffer B: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole; Buffer C: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT. First, the cell lysate of the recombinant formaldehyde lyase mutant obtained in the technical solution five is loaded onto a nickel column equilibrated with A liquid, then the impurities are eluted with 10% B liquid, and finally the target protein is eluted with 50% B liquid, and the purity of the target protein is verified by SDS-PAGE, and the eluate containing the target protein is collected. The ultrafiltration tube (10 kDa) is used for ultrafiltration and concentration, and then the C liquid is used for replacement twice to remove the imidazole in the protein solution. The purified protein is subjected to activity and reaction effect determination, then is divided, is quickly frozen in liquid nitrogen, and is stored at -80℃ for standby use.

[0043] The seventh technical solution of the present invention provides a method for determining the activity of formaldehyde lyase mutants. Specifically, a 1 mL reaction system contains 50 mM FALD, 5 mM TPP, 0.5 mM MgSO4, 0.1 mg formaldehyde lyase, and 50 mM potassium phosphate buffer (pH 8.0). After reacting for 45 minutes at 30 ° C and 800 rpm, an equal volume of 500 mM sulfuric acid is added to terminate the reaction. The supernatant is centrifuged and filtered with an aqueous nylon membrane. The product 1,3-dihydroxyacetone is detected by high performance liquid chromatography. The specific analysis conditions are as follows: using a BioRad Aminex HPX-87H column (300 × 7.8 mm), an injection volume of 10 μL, a column temperature of 35 ° C, isocratic elution, a flow rate of 0.5 mL / min, and a UV detection wavelength of 210 nm. Mobile phase: 10 mM dilute sulfuric acid. The enzyme activity unit (U) of formaldehyde lyase is defined as the amount of enzyme required to catalyze FALD to produce 1 μmol DHA per minute under the above reaction conditions.

[0044] The eighth technical solution of the present invention provides the use of the formaldehyde lyase mutant as described in the first technical solution or the recombinant formaldehyde lyase mutant catalyst as described in the fifth technical solution in the enzymatic reaction catalyzing the condensation of formaldehyde to prepare 1,3-dihydroxyacetone.

[0045] The mutant formaldehyde lyase catalyzes the condensation of formaldehyde to synthesize 1,3-dihydroxyacetone. Figure 1 In one embodiment of the present invention, in the application, the concentration of formaldehyde is 50-100 mM, the amount of thiamine pyrophosphate (TPP) in the reaction solution is 0.1-1.5 mM, and the amount of MgSO4 is 5-15 mM.

[0046] In one embodiment of the present invention, the enzymatic reaction is carried out in potassium phosphate buffer at pH 8.0 at 30°C and 800 rpm. The reaction system comprises a final concentration of 100 mM formaldehyde, 0.3 mM TPP, 10 mM MgSO₄, and 2 mg / mL of the formaldehyde lyase mutant described in Technical Solution 1 or the recombinant formaldehyde lyase mutant described in Technical Solution 5. The reaction time is determined by the complete conversion of the substrate or the cessation of product concentration increase. During the reaction, 100-200 μL of the reaction solution is sampled intermittently and terminated with an equal volume of 500 mM H₂SO₄. The reaction is then centrifuged at 12,000 rpm for 5 minutes. The supernatant is removed and filtered through an aqueous nylon membrane. The product, 1,3-dihydroxyacetone, is detected by high-performance liquid chromatography.

[0047] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following aspects:

[0048] The application provides a formaldehyde lyase mutant with better catalytic performance by semi-rational design, site-directed mutation and combined mutation, which can efficiently catalyze formaldehyde condensation to generate 1,3-dihydroxyacetone. Compared with the optimal formaldehyde lyase FLS-M3 reported in the literature, the formaldehyde lyase mutant provided by the application has the advantages of good protein heterologous expression effect, high yield and the like, and has a good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 A reaction formula of the formaldehyde lyase mutant catalyzing formaldehyde condensation to synthesize 1,3-dihydroxyacetone;

[0050] Figure 2 SDS-PAGE protein electrophoretograms of PaFLS and FLS-M3, wherein S represents cell crushing supernatant, and P represents cell crushing precipitate;

[0051] Figure 3 An enzymatic reaction kinetics curve diagram of PaFLS parent and mutant;

[0052] Figure 4 A catalytic reaction effect diagram of PaFLS mutant and FLS-M3. DETAILED DESCRIPTION

[0053] The application will be described in detail below in combination with the drawings and specific embodiments.

[0054] The reaction or detection conditions described in the content of the application can be combined or changed according to the common sense in the art, and can be verified by experiments.

[0055] The technical solutions and technical effects in the application will be described clearly and completely in combination with specific embodiments below, but the protection scope of the application is not limited to these embodiments, and any change or equivalent replacement without departing from the concept of the application is included in the protection scope of the application.

[0056] The material sources in the following embodiments are as follows:

[0057] The recombinant plasmid pET28a-PaFLS contains the nucleic acid sequence shown in the sequence table SEQ ID No. 1, and is constructed by the inventors.

[0058] The empty plasmid vector pET-28a is purchased from Novagen Company.

[0059] The E. coli BL21 (DE3) competent cell, 2xTaq PCR MasterMix and agarose gel DNA recovery kit are all purchased from Beijing Tiangen Biotech Co., Ltd.

[0060] The restriction enzyme Dpn I is a commercially available product of New England Biolabs (NEB).

[0061] mM in the examples is a shorthand for mmol / L.

[0062] Unless otherwise indicated, the following examples were carried out according to conventional methods and conditions in the art, or according to the manufacturer's instructions for the kits.

[0063] Example 1 Rational design screening for mutants with improved formaldehyde lyase activity

[0064] The structure of PaFLS protein was predicted by AlphaFold2, the chemical structure of TPP-GALD intermediate complex was drawn by software ChemDraw, and energy minimization was performed in Discovery Studio. Based on the structure model of PaFLS, the semi-flexible docking method in Autodock4.0 was used to dock the TPP-GALD intermediate complex to the active site of the structure model, with the C2 atom of coenzyme TPP as the docking center. Hydrogen was added to the ligand and protein and its Gasteiger charge was calculated, and then Lamarckian genetic algorithm was used for docking simulation to screen the structure model with the closest distance to the active center and the highest docking energy.

[0065] The pET28a-PaFLS was used as a template for site-directed saturation mutation of Leu at position 29, His at position 30, Gly at position 31, Gly at position 32, His at position 33, Thr at position 77, Pro at position 80, Gin at position 117, Met at position 287, Gly at position 396, Glu at position 397, Gly at position 421, Leu at position 423, Ala at position 450, Phe at position 453, Trp at position 478, Gly at position 479, Met at position 480, Ser at position 481, Ile at position 482, Thr at position 497, and Tyr at position 571 as shown in SEQ ID No. 2. First, a mutation primer containing a mutation site was designed. The mutation primer containing a mutation site required by the present application is a commonly used primer in the art, which only needs to be mutated to other 19 amino acid residues by general genetic engineering technology, and the nucleic acid mutation codon is the preferred codon of Escherichia coli.

[0066] PCR reaction system (20 μL): 50-100 ng of template, 1 μL of each mutant primer (10 μM), 10 μL of PrimeStarmix, and sterilized double distilled water to make up to 20 μL. The template is pET28a-PaFLS (UniProt Accessions No: A0A4Y9ENV7_9SPHN of formaldehyde-lyase PaFLS gene). PCR reaction procedure: (1) denaturation at 95°C for 3 min; (2) denaturation at 98°C for 10 s; (3) annealing at 55°C for 5 s; (4) extension at 72°C for 9 min; steps (2)-(4) for a total of 25 cycles, and finally extension at 72°C for 10 min, and preservation at 4°C.

[0067] The amplified PCR product was digested with restriction endonuclease Dpn I at 37°C for 0.5-2 h, then transformed into E. coli BL21(DE3) competent cells, and uniformly coated on LB medium (peptone: 10 g / L, yeast extract: 5 g / L, sodium chloride: 10 g / L, agar powder: 20 g / L) containing 50 μg / mL kanamycin. After incubation at 37°C for 10-16 h, single colonies were selected, and E. coli BL21(DE3) recombinant strains containing mutant recombinant expression plasmids were obtained, which were sent to Shanghai Qianke Biological Technology Co., Ltd. for sequencing analysis. The sequencing results were compared with the wild-type formaldehyde-lyase gene sequence using SnapGene software, and the differences in gene sequences and corresponding amino acid sequences before and after mutation were confirmed.

[0068] Through screening, it was found that the formaldehyde lyase had the following mutants: (1) glycine at position 31 was replaced by serine, (2) threonine at position 77 was replaced by cysteine, (3) methionine at position 287 was replaced by cysteine, (4) glycine at position 396 was replaced by asparagine, (5) glycine at position 421 was replaced by asparagine, (6) glycine at position 421 was replaced by tryptophan, (7) glycine at position 421 was replaced by tyrosine, (8) methionine at position 480 was replaced by leucine, (9) serine at position 481 was replaced by glycine, (10) tyrosine at position 571 was replaced by phenylalanine, (11) methionine at position 287 and glycine at position 421 were replaced by cysteine ​​and tryptophan, respectively, (12) glycine at position 421 and methionine at position 480 were replaced by tryptophan and leucine, respectively. (13) Glycine at position 421 and serine at position 481 were replaced by tryptophan and glycine, respectively; (14) glycine at position 421 and tyrosine at position 571 were replaced by tryptophan and phenylalanine, respectively; (15) glycine at position 421 and isoleucine at position 482 were replaced by tryptophan and arginine, respectively; (16) glycine at position 421 and isoleucine at position 482 were replaced by tryptophan and phenylalanine, respectively; (17) glycine at position 421 and isoleucine at position 482 were replaced by tryptophan and glutamine, respectively; (18) glycine at position 421 and isoleucine at position 482 were replaced by tryptophan and alanine, respectively; (19) glycine at position 421, methionine at position 480 and isoleucine at position 482 were replaced by tryptophan, leucine and alanine, respectively; the obtained preferred mutants had significantly improved DHA synthesis activity.

[0069] Example 2 Purification of formaldehyde lyase mutants

[0070] The preferred mutant monoclonal clone obtained in Example 1 was picked into 4 mL LB medium (containing kanamycin) and cultured in a shaking incubator at 37°C and 180 rpm for 12-16 h. Subsequently, 100 mL LB medium (containing kanamycin) was inoculated at a 1% (v / v) inoculum and cultured in a shaking incubator at 37°C and 180 rpm. When the OD value of the culture solution reached 0. 600 When the pH reaches 0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) is added as an inducer at a final concentration of 0.2 mM and induced at 16°C for 16-24 hours. The culture medium is centrifuged at 10,000 rpm for 3 minutes, and the cells are washed twice with physiological saline to obtain recombinant expression transformant cells. The harvested recombinant transformant cells are then suspended in 10 mL of Buffer A, disrupted by ultrasound, and the supernatant is collected by centrifugation to obtain a cell lysate of the formaldehyde lyase mutant.

[0071] The cell lysate of the obtained formaldehyde lyase mutant was further purified. The following is the protein purification buffer formula: Buffer A: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole; Buffer B: 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole; Buffer C: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT. The cell lysate of the formaldehyde lyase mutant was loaded onto a nickel column equilibrated with A, after loading, the impurities in the column were eluted with 10% B, then the target protein was eluted with 50% B, the purity of the target protein was verified by SDS-PAGE, and then the eluate containing the target protein was collected. Concentrate by ultrafiltration tube (10 kDa), then replace twice with C to remove imidazole in the protein solution. The purified protein was subjected to activity and reaction effect determination, then aliquoted, frozen in liquid nitrogen and stored at -80°C for standby.

[0072] Example 3 Activity determination of formaldehyde lyase mutant

[0073] Activity determination reaction system: 100 mM FALD, 5 mM TPP, 0.5 mM MgSO4, 0.1 mg pure enzyme, potassium phosphate buffer (50 mM, pH 8.0). After reaction at 30°C, 800 rpm for 45 min, the reaction was terminated by adding an equal volume of 500 mM sulfuric acid solution, the supernatant was obtained by centrifugation, and the concentration of DHA was detected by HPLC, with three parallel groups. 1 enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the condensation of formaldehyde to generate 1 μmol of DHA per minute.

[0074] The specific analysis conditions for detecting DHA by HPLC are as follows: BioRad Aminex HPX-87H column (300×7.8 mm) was used, injection volume was 10 μL, column temperature was 35°C, isocratic elution, flow rate was 0.5 mL / min, ultraviolet detection wavelength was 210 nm, and mobile phase was 10 mM dilute sulfuric acid.

[0075] Table 1 Specific activity of formaldehyde lyase parent and its mutants

[0076]

[0077]

[0078] Example 4 Kinetic parameter determination of formaldehyde lyase parent and its mutants

[0079] The initial reaction rate was measured when the substrate concentration was 5, 10, 20, 40, 50, 70, 80, 100 mM, respectively. The concentration of the substrate FALD (mM) was used as the horizontal coordinate, and the enzymatic reaction rate (pmol min -1 mg -1 ) was used as the vertical coordinate to draw the enzymatic reaction kinetics curve. The Michaelis equation was used to obtain the K m and V max values, so as to calculate k cat = 3V max / protein concentration and k cat / K m . The enzymatic reaction kinetics curves of the PaFLS parent and partial mutants are shown in Figure 2 , and the calculation results of K m , k cat and k cat / K m are shown in the following table.

[0080] Table 2 Kinetic constants of the PaFLS parent and mutants

[0081]

[0082] Example 5 PaFLS catalyzes the synthesis of 1,3-dihydroxyacetone from formaldehyde

[0083] In a 1 mL reaction system containing 50 mM FALD, 0.5 mM TPP, 5 mM MgSO4, 2 mg PaFLS pure enzyme, potassium phosphate buffer (50 mM, pH 8.0), after 12 h of reaction in a constant temperature shaker at 30°C and 800 rpm, an equal volume of dilute sulfuric acid (500 mM) was added to quench the reaction, and the substrate conversion rate was 7.9% measured by liquid chromatography.

[0084] Example 6 PaFLS G421W catalyzes the synthesis of 1,3-dihydroxyacetone from formaldehyde

[0085] In a 1 mL reaction system containing 100 mM FALD, 0.5 mM TPP, 5 mM MgSO4, 2 mg PaFLS G421W pure enzyme, potassium phosphate buffer (50 mM, pH 8.0), after 12 h of reaction in a constant temperature shaker at 30°C and 800 rpm, an equal volume of dilute sulfuric acid (500 mM) was added to quench the reaction, and the 1,3-dihydroxyacetone yield was 34.1% measured by liquid chromatography.

[0086] Example 7 PaFLS G421W / M480L catalyzes the synthesis of 1,3-dihydroxyacetone from formaldehyde

[0087] 100 mM FALD, 0.5 mM TPP, 5 mM MgS04, 2 mg PaFLS in 1 mL reaction system G421W / M480L Pure enzyme, potassium phosphate buffer (50 mM, pH 8.0), 30 °C, 800 rpm constant shaker for 12 h, equal volume of dilute sulfuric acid (500 mM) was added to quench the reaction, 1,3-dihydroxyacetone yield was 39.1% by liquid chromatography.

[0088] Example 8 PaFLS G421W / I482A Catalysis of formaldehyde to 1,3-dihydroxyacetone

[0089] 100 mM FALD, 0.5 mM TPP, 5 mM MgS04, 2 mg PaFLS in 1 mL reaction system G421W / I482A Pure enzyme, potassium phosphate buffer (50 mM, pH 8.0), 30 °C, 800 rpm constant shaker for 12 h, equal volume of dilute sulfuric acid (500 mM) was added to quench the reaction, 1,3-dihydroxyacetone yield was 54.3% by liquid chromatography.

[0090] Example 9 PaFLS G421W / I482A Catalysis of formaldehyde to 1,3-dihydroxyacetone

[0091] 100 mM FALD, 0.1 mM TPP, 15 mM MgS04, 2 mg PaFLS in 1 mL reaction system G421W / I482A Pure enzyme, potassium phosphate buffer (50 mM, pH 8.0), 30 °C, 800 rpm constant shaker for 12 h, equal volume of dilute sulfuric acid (500 mM) was added to quench the reaction, 1,3-dihydroxyacetone yield was 70.4% by liquid chromatography.

[0092] Example 10 PaFLS G421W / I482A Catalysis of formaldehyde to 1,3-dihydroxyacetone

[0093] 100 mM FALD, 0.3 mM TPP, 15 mM MgS04, 2 mg PaFLS in 1 mL reaction system G421W / I482A Pure enzyme, potassium phosphate buffer (50 mM, pH 8.0), 30 °C, 800 rpm constant shaker for 12 h, equal volume of dilute sulfuric acid (500 mM) was added to quench the reaction, 1,3-dihydroxyacetone yield was 73.5% by liquid chromatography.

[0094] Example 11 PaFLS G421W / I482A Catalysis of formaldehyde to 1,3-dihydroxyacetone

[0095] In 1 mL reaction system containing 100 mM FALD, 1.5 mM TPP, 15 mM MgSO4, 2 mg PaFLS G421W / I482A The reaction was quenched by adding equal volume of dilute sulfuric acid (500 mM) after 12 h reaction in 1 mL reaction system containing 100 mM FALD, 1.5 mM TPP, 15 mM MgSO4, 2 mg PaFLS in phosphate buffer (50 mM, pH 8.0) at 30°C with 800 rpm constant temperature shaker. The yield of 1,3-dihydroxyacetone was 75.7% measured by liquid chromatography.

[0096] Example 12 FLS-M3 catalyzed synthesis of 1,3-dihydroxyacetone from formaldehyde

[0097] The reaction was quenched by adding equal volume of dilute sulfuric acid (500 mM) after 12 h reaction in 1 mL reaction system containing 100 mM FALD, 0.5 mM TPP, 5 mM MgSO4, 2 mg FLS-M3 in phosphate buffer (50 mM, pH 8.0) at 30°C with 800 rpm constant temperature shaker. The yield of 1,3-dihydroxyacetone was 62.4% measured by liquid chromatography.

[0098] For convenience of observation, the experimental conditions and results of Examples 5-12 were summarized in Table 3. The results showed that the mutant PaFLS G421W / I482A The FLS-M3 catalyzed synthesis of 1,3-dihydroxyacetone from formaldehyde had the highest yield and the best effect.

[0099] Table 3 FLS catalyzed synthesis of 1,3-dihydroxyacetone from formaldehyde

[0100]

[0101] The above description of the examples is for the convenience of the ordinary skilled in the art to understand and use the invention. Those skilled in the art can easily make various modifications to these examples, and apply the general principles described herein to other examples without creative labor. Therefore, the present invention is not limited to the above examples, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the scope of protection of the present invention.

[0102] The sequence information involved in the present invention is as follows:

[0103] SEQ ID No. 1

[0104]

[0105] SEQ ID No. 2

[0106] MSSPEARYTGGDLLAQTLHDAGVTKIFALHGGHHEALFKGCIDQGIDLIDFRHEAAAGHAADAYARTTGKLGVCIITAGPGFTNAISAIANAQLDASPVLFLIGAPPLREVETNPLQGGIDQIAMARPAAKWALSIPSTERVRDLTAMAIRKAMTGRKGPVVLEIPIDILHMSVTGAQATPSAGLAVRPQPAPAPEEVAALAELLLRAERPVIVAGLESASAATAVALRALVAKLPLPVFAKPQAYGLLPAGHACDAGAAGNLAVLPIIGAGAPDLVILLGARLGLMLGGRSGALVPHDAHVVQIYSDASEIGRLRDIDLPIAADCAQTLTALTKALAAVDLPDTSAWTARAAGAKALAASAWPDAEVAGGIHPYHAAKAVANAAGQDAAYVFDGGESSSWGTATVAVDAPARVLSHGYLGCLGIGPGFAIGMQIAHPDRRVVQVTGDGAMGFHIQEFDTMVRHRLPIVTVILNNQVWGMSIHGQQMMYGANYNVITKLGSTQYASIAAAFGCHAERVTAFAEIAPAMARAFASGKPALVEIMTDADVVHPATVAMLGQLAEGSRDIMIPYYENIAAS

Claims

1. A formaldehyde lyase mutant, characterized in that It is a protein consisting of any of the following amino acid sequences: (1) replacing glycine at position 31 of the amino acid sequence shown in SEQ ID No. 2 with serine; (2) replacing the threonine at position 77 of the amino acid sequence shown in SEQ ID No. 2 with cysteine; (3) replacing methionine at position 287 of the amino acid sequence shown in SEQ ID No. 2 with cysteine; (4) replacing glycine at position 396 of the amino acid sequence shown in SEQ ID No. 2 with asparagine; (5) replacing glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with asparagine; (6) replacing glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with tryptophan; (7) replacing glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 with tyrosine; (8) replacing methionine at position 480 of the amino acid sequence shown in SEQ ID No. 2 with leucine; (9) replacing serine at position 481 of the amino acid sequence shown in SEQ ID No. 2 with glycine; (10) replacing tyrosine at position 571 of the amino acid sequence shown in SEQ ID No. 2 with phenylalanine; (11) The methionine at position 287 of the amino acid sequence shown in SEQ ID No. 2 is replaced by cysteine, and the glycine at position 421 is replaced by tryptophan; (12) The glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 is replaced with tryptophan, and the methionine at position 480 is replaced with leucine; (13) The glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 is replaced with tryptophan, and the serine at position 481 is replaced with glycine; (14) The glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 is replaced by tryptophan, and the tyrosine at position 571 is replaced by phenylalanine; (15) The glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 is replaced by tryptophan, and the isoleucine at position 482 is replaced by arginine; (16) The glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 is replaced by tryptophan, and the isoleucine at position 482 is replaced by phenylalanine; (17) The amino acid sequence shown in SEQ ID No. 2 is substituted with glycine at position 421 to tryptophan, and isoleucine at position 482 to glutamine; (18) The glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 is replaced by tryptophan, and the isoleucine at position 482 is replaced by alanine; (19) The glycine at position 421 of the amino acid sequence shown in SEQ ID No. 2 is replaced by tryptophan, the methionine at position 480 is replaced by leucine, and the isoleucine at position 482 is replaced by alanine.

2. A nucleic acid, characterized in that Encodes the formaldehyde lyase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: Contains the nucleic acid according to claim 2.

4. A recombinant expression transformant, characterized in that: Comprising the nucleic acid according to claim 2 or the recombinant expression vector according to claim 3.

5. A recombinant formaldehyde lyase mutant catalyst, characterized in that: Is any of the following: (1) culturing the recombinant expression transformant according to claim 4, and isolating transformant cells containing the formaldehyde lyase mutant; (2) culturing the recombinant expression transformant according to claim 4, isolating transformant cells containing the formaldehyde lyase mutant, and disrupting the transformant cells containing the formaldehyde lyase to obtain a cell disrupted liquid; (3) Cultivating the recombinant expression transformant according to claim 4, isolating transformant cells containing the formaldehyde lyase mutant, disrupting the transformant cells containing the formaldehyde lyase mutant, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the formaldehyde lyase mutant to obtain a freeze-dried enzyme powder.

6. Use of the formaldehyde lyase mutant according to claim 1 or the recombinant formaldehyde lyase mutant catalyst according to claim 5 in catalyzing the condensation of formaldehyde to synthesize 1,3-dihydroxyacetone.

7. The use according to claim 6, characterized in that The reaction system for catalyzing the condensation of formaldehyde to synthesize 1,3-dihydroxyacetone includes formaldehyde, thiamine pyrophosphate and MgSO 4。 8. The use according to claim 7, characterized in that In the application, the concentration of formaldehyde is 50-100 mM, the amount of thiamine pyrophosphate in the reaction solution is 0.1-1.5 mM, and the amount of MgSO4 is 5-15 mM.

9. The use according to claim 7, characterized in that The reaction system includes formaldehyde at a final concentration of 100 mM, 0.3 mM TPP, 10 mM MgSO4, and 2 mg / mL of the formaldehyde lyase mutant according to claim 1 or the recombinant formaldehyde lyase mutant catalyst according to claim 5.

10. The use according to claim 7, characterized in that The enzymatic reaction was carried out in potassium phosphate buffer (pH 8.0) at 30°C and 800 rpm.

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