A thermostable fatty acid photodecarboxylase mutant based on continuous site combinatorial mutation and its application

Abstract

The present invention discloses a thermostable fatty acid photodecarboxylase mutant based on continuous site combinatorial mutation and its application, belonging to the technical field of enzyme engineering. In the present invention, a continuous site combinatorial mutant library was constructed for the fatty acid photodecarboxylase CvFAP derived from Chlorella vulgaris, and an 8-site combinatorial mutant M8 was screened. The optimal reaction temperature of the combinatorial mutant M8 is 45 °C, which is 15 °C higher than that of CvFAP. Moreover, M8 retains 50% activity at 55 °C, while CvFAP has almost no activity at this temperature. The present invention breaks through the limitation that the optimal catalytic temperature of conventional CvFAP is 30 °C and the substrate fatty acid depends on organic solvents such as dimethyl sulfoxide for solubilization. At 55 °C above the melting point of myristic acid and without the assistance of organic solvents for solubilization, the whole cell containing the mutant M8 was used to efficiently catalyze myristic acid to generate tridecane. The present invention provides a prospect for the application of fatty acid photodecarboxylase under high temperature and without organic co-solvent conditions.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to a thermostable fatty acid photodecarboxylase mutant based on continuous site combinatorial mutation and its application. Background Art

[0002] Fatty acid photodecarboxylase (FAP) was discovered in microalgae and can catalyze the conversion of fatty acids into biofuels such as alkanes or alkenes, thereby reducing the dependence on non-renewable resources such as petroleum. Therefore, it has a broad application background. However, there are multiple problems in the application of fatty acid photodecarboxylase. For example, its thermal stability is low, the half-life is less than 60 min, and the activity decreases significantly above 50 °C. It has also been reported that it is sensitive to photo-stability, and the protein tertiary structure of FAP is damaged under long-term blue light irradiation, resulting in a decrease in enzyme activity.

[0003] The most typical fatty acid photodecarboxylase is derived from Chlorella variabilis NC64A. The optimum temperature of this enzyme is 30 °C, and its activity at 50 °C is about 35% of the optimum temperature. Above 55 °C, this enzyme has almost no activity. However, fatty acid substrates are usually poorly soluble in water. Currently, in the process of catalyzing fatty acid decarboxylation reaction based on FAP, fatty acids are usually dissolved in organic co-solvents such as dimethyl sulfoxide. Limited by the influence of organic co-solvents on enzyme activity, the concentration of fatty acid substrates usually cannot be increased to a high level. Common reports are in the range of 10 - 65 mM, which limits its industrial-scale application.

[0004] Among the common fatty acid substrates of fatty acid photodecarboxylase, the melting point of lauric acid is 44.2 °C, the melting point of myristic acid is 54.4 °C, and the melting point of palmitic acid is 62.9 °C. If a fatty acid decarboxylase mutant with heat resistance and high stability can be developed, especially one that still retains activity above 55 - 60 °C, it will effectively solve the problems of its low thermal stability and difficult solubility of substrates, and has great potential in industrial applications.

[0005] Common thermal stability improvement techniques include amino acid mutation in the distal loop region, introduction of interactions such as hydrogen bonds, etc., mainly including site-directed mutation and iterative combinatorial mutation. The advantage of site-directed mutation is that it can improve the stability of the enzyme by precisely modifying specific amino acid residues, while iterative combinatorial mutation allows multiple mutations to be introduced simultaneously to enhance the overall thermal stability of the enzyme. However, during the transformation process of FAP, due to the synergistic effect between adjacent amino acids and their mutual influence, it is difficult to accumulate the effects of beneficial mutations. However, so far, there has been no report on the related research of directly optimizing and strengthening the interaction between adjacent amino acids through continuous mutation, reducing the non-additive influence during the subsequent mutation site combination process, and thus obtaining a thermostable fatty acid photodecarboxylase mutant. Summary of the Invention

[0006] To solve the above problems, the object of the present invention is to provide a thermostable fatty acid photodecarboxylase mutant based on continuous site combinatorial mutation and its application, which solves the technical problem that the natural fatty acid photodecarboxylase CvFAP has almost no catalytic activity above 50°C, and can be used to catalyze the decarboxylation of fatty acids and the synthesis of alkanes under high temperature conditions exceeding the melting point of fatty acids and without the assistance of organic solvents for dissolution.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] In the first aspect, the present invention provides a thermostable fatty acid photodecarboxylase mutant, and the amino acid sequence of the thermostable fatty acid photodecarboxylase mutant is as shown in SEQ ID NO.3.

[0009] Based on the above technical solution, further, the thermostable fatty acid photodecarboxylase mutant reduces the free energy of the overall protein structure through molecular dynamics stability calculation and site-directed mutagenesis to increase protein stability. The mutant is obtained by performing continuous site combinatorial mutation on the fatty acid photodecarboxylase of amino acid residues at positions 77-645 in SEQ ID NO.1. The mutation sites include: mutating Q at position 410 to N, mutating K at position 413 to T, mutating G at position 422 to F, mutating G at position 423 to R, mutating R at position 424 to S, mutating G at position 426 to P, mutating T at position 428 to A, and mutating S at position 429 to T, a total of 8-site mutation combination (the amino acid residue numbers are numbered according to the full length of SEQ ID NO.1).

[0010] In the second aspect, the present invention provides a gene encoding the above thermostable fatty acid photodecarboxylase mutant.

[0011] Based on the above technical solution, further, the nucleotide sequence of the gene is as shown in SEQ ID NO.5.

[0012] In the third aspect, the present invention provides a recombinant plasmid into which the gene encoding the above thermostable fatty acid photodecarboxylase mutant is inserted.

[0013] In the fourth aspect, the present invention provides a recombinant engineering strain carrying and capable of expressing the above recombinant plasmid.

[0014] Based on the above technical solution, further, the recombinant engineering strain is Escherichia coli.

[0015] Fifth aspect, the present invention provides the use of the above-mentioned thermostable fatty acid photodecarboxylase mutant, a recombinant plasmid inserted with a gene encoding the above-mentioned thermostable fatty acid photodecarboxylase mutant, and a recombinant engineering bacterium carrying and capable of expressing the above-mentioned recombinant plasmid in catalyzing the decarboxylation of fatty acids to synthesize alkanes.

[0016] Based on the above technical solution, further, the fatty acids include palmitic acid, lauric acid, and myristic acid.

[0017] Based on the above technical solution, further, the reaction of catalyzing the decarboxylation of fatty acids to synthesize alkanes is carried out at a temperature higher than the melting point of the fatty acid and without adding an organic solvent for solubilization.

[0018] Based on the above technical solution, further, the reaction temperature for catalyzing the decarboxylation of fatty acids to synthesize alkanes is 45 - 60 °C, preferably 55 - 56 °C, and is carried out in a conventional buffer solution under blue light irradiation.

[0019] Based on the above technical solution, further, the buffer solution includes Tris-HCl buffer solution and PBS buffer solution.

[0020] Based on the above technical solution, further, the buffer solution is 100 mM Tris-HCl, pH 8.5.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention constructs a continuous 8-site combinatorial mutant M8 containing Q410N, K413T, G422F, G423R, R424S, G426P, T428A, and S429T (the amino acid residue numbers are numbered according to the full length of SEQ ID NO.1) for the CvFAP derived from wild-type Chlorella variabilis, and uses it to catalyze the production of pentadecane or tridecane from palmitic acid or myristic acid. The present invention confirms that the amino acid residues at positions 410, 413, 422, 423, 424, 426, 428, and 429 have a great influence on the thermostable performance of the fatty acid photodecarboxylase. The relative activity of the combinatorial mutant M8 is increased by 3 times compared with the wild-type CvFAP at 50 °C. Particularly, at 55 °C, the combinatorial mutant M8 can achieve the efficient conversion of myristic acid into tridecane without the need for organic solvents such as dimethyl sulfoxide and methanol to solubilize the fatty acid substrate, while the wild-type CvFAP has almost no catalytic activity under the same conditions. The present invention provides a certain basis for improving the thermostable performance of this enzyme, and its combinatorial mutant M8 improves the application potential of this enzyme in the field of catalyzing fatty acids to produce biofuels in the absence of a cosolvent. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Results of molecular dynamics analysis of CvFAP from Chlorella at different temperatures (300K and 330K) and determination of the unstable region 410 - 429

[0024] Figure 2 Partial amino acid site distribution map of the 410 - 429 amino acid residue region with low structural stability obtained from the structural analysis of CvFAP from Chlorella

[0025] Figure 3 Results of affinity chromatography purification of the consecutive site combinatorial mutant M8 of CvFAP

[0026] Figure 4 Results of determination of the melting temperature T m of CvFAP (a) and the combinatorial mutant M8 (b)

[0027] Figure 5 Results of determination of t 1 / 2 of CvFAP and the combinatorial mutant M8

[0028] Figure 6 Results of the optimal reaction temperature of CvFAP and the combinatorial mutant M8

[0029] Figure 7 Results of the activity of CvFAP and the combinatorial mutant M8 in catalyzing palmitic acid substrate at 50°C

[0030] Figure 8 Results of the activity of CvFAP and the combinatorial mutant M8 in catalyzing myristic acid substrate without cosolvent at 55°C

[0031] Figure 9 Standard curve for quantitative analysis of pentadecane (a) and tridecane (b) by gas chromatography

[0032] Figure 10 Gas chromatography analysis of each alkane and its retention time Detailed implementation mode

[0033] To better understand the above technical solution, the exemplary embodiments of the present invention will be described in more detail below. It should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0034] Example 1 Structural stability analysis of fatty acid photodecarboxylase and screening of consecutive site combinatorial mutation sites

[0035] Using the resolved Chlorella - derived CvFAP structure (Protein Data Bank ID: 5NCC) as the initial model, the missing residue atoms were added using spdbv. At a temperature of 300K, systematic molecular dynamics simulations were carried out using the AMBER14SB biomolecular force field. The optimal substrate palmitic acid ligand was optimized by Gaussian and then docked with the protein using Autodock. The topology file was generated through the sobtop program, and the small - molecule RESP charges were generated using Multiwfn. The TIP3P water model was used, and the steepest - descent method was used to minimize the system energy to eliminate unfavorable initial contacts and stresses. Using as the cutoff distance to handle real - space interactions, and the PME method was used to handle long - range electrostatic interactions. After energy minimization (maximum energy < 1000 kJ / mol / nm), NVT equilibration was carried out at 300K and 330K with a time step of 2 fs, and NPT equilibration was carried out at 1 bar pressure for 500 ps, and finally 100 ns of molecular dynamics simulations were performed. Finally, structural analysis was carried out using RMSF and Pymol software. The results are as Figure 1 shown, and the amino - acid site distribution of the amino - acid residue region of positions 410 - 429 with relatively low RMSF structural stability was determined as Figure 2 shown.

[0036] Example 2 Construction of consecutive site combinatorial mutants of fatty acid photodecarboxylase

[0037] After codon optimization for Escherichia coli respectively, the full - length coding gene CvFAP (SEQ ID NO.4) of fatty acid photodecarboxylase derived from Chlorella, with the transit peptide deleted (amino - acid residues 1 - 76), was synthesized (corresponding to amino - acid residues 77 - 645 in SEQ ID NO.1), and cloned into the BamⅠ / HindⅢ restriction sites of the expression vector pET28a to obtain the recombinant plasmid pET28a - CvFAP. The CvFAP mutant provided by the present invention was obtained by mutating the nucleotide sequence shown in SEQ ID NO.4, and the recombinant expression amino - acid sequence of wild - type CvFAP is SEQ ID NO.2.

[0038] Primers were designed to construct a mutant library. Using the recombinant plasmid pET28a - CvFAP as the template and a pair of oligonucleotides with mutation sites as the amplification primers, PrimeSTAR high - fidelity enzyme was used to amplify the fragment and the vector, and the OK ClonDNA Ligation Kit II cloning enzyme was used to ligate the fragment and the vector to obtain a recombinant plasmid library with specific consecutive mutation sites.

[0039] The target gene was amplified using the amplification primers shown in Table 1, where the underlined part indicates the mutation site. Primers with F in their names represent upstream primers, and those with R represent downstream primers:

[0040] Table 1 Nucleotide Sequences of Amplification Primers

[0041]

[0042]

[0043] The CvFAP gene containing specific mutation sites was amplified using PrimeSTAR Max high-fidelity enzyme and OK Clon DNA Ligation Kit II cloning enzyme. It should be noted that the template plasmid, i.e., the wild-type plasmid, was mixed in the cloning system. Therefore, the template plasmid was removed by digesting the PCR product with DpnⅠ enzyme. Then, 20 μL of the digested product was added to Escherichia coli DH5α competent cells and spread on an LB solid plate containing 50 μg / mL kanamycin resistance. After culturing inverted at 37 °C for 12 hours, a plate colony library of mutants could be obtained. The relevant mutant sequences were confirmed to be correct by gene sequencing.

[0044] Example 3 Recombinant Expression of Fatty Acid Photodecarboxylase Continuous Site Combinatorial Mutants

[0045] The plasmids constructed in Example 2 were separately transformed into Escherichia coli BL21(DE3) competent cells. After screening on plates containing kanamycin, strains carrying the above recombinant plasmids were obtained respectively. Single clone strains carrying the above recombinant plasmids were picked from the plates and inoculated into 100 mL of TB liquid medium for overnight culture. They were cultured at 37 °C and 220 rpm for 2 - 3 h until OD 600 reached 0.6 - 0.8. IPTG with a final concentration of 0.3 mM was added to induce the expression of the target protein, and the culture was continued at 16 °C for 20 h. The bacterial cells were collected by centrifugation at 4500 rpm for 15 min at 4 °C, and then freeze-dried for subsequent experiments.

[0046] Example 4 Purification of Fatty Acid Photodecarboxylase Continuous Site Combinatorial Mutants

[0047] All target proteins were purified by Ni-NTA affinity chromatography. Recombinant Escherichia coli expressing CvFAP or mutant M8 was collected by centrifugation and resuspended in buffer A (50 mM PBS buffer, pH 8.5). After sonication, the cell lysate was centrifuged at 12,000 g (4 °C, 45 min), and the supernatant was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with buffer A. Then, the imidazole concentration was increased from 10 mM to 100 mM to elute the target protein, which was collected in different tubes. After SDS-PAGE analysis, the target protein was collected and buffer-exchanged into 50 mM PBS buffer, pH 8.5, to remove the high concentration of imidazole. The results were as Figure 3 shown. The purified target protein was snap-frozen in liquid nitrogen and stored at -80 °C or used for subsequent tests.

[0048] Example 5 Enzyme Activity Detection Method and Product Quantitative Analysis of Fatty Acid Photodecarboxylase

[0049] A standard curve for quantifying the product alkane was established by gas chromatography. Using ethyl acetate as the solvent, a 1 mg / mL pentadecane standard solution was prepared, and a pentadecane standard curve with a concentration range of 0.1 mg / mL - 1 mg / mL was established. Pentadecane standard solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1 mg / mL were prepared for gas chromatography analysis to obtain the pentadecane standard curve. The relationship between the peak area and concentration of pentadecane was y = 0.0019x + 0.0017, R 2 = 0.9999, where y represents the gas chromatography response peak area (mAU) of the target product, and x represents the concentration (mM) of the product pentadecane. Similarly, the standard curve of tridecane was obtained. The relationship between the peak area and concentration of tridecane was y = 0.0022x + 0.0048, R 2 = 0.9999. The standard curves of pentadecane and tridecane were as Figure 9 shown.

[0050] The fatty acid photodecarboxylase reacts under 450 nm blue light at 50 °C and 55 °C respectively. Among them, the catalytic reaction of palmitic acid is carried out at 50 °C in 1.0 mL Tris-HCl buffer (pH 8.5, 100 mM) containing 20% DMSO as a co-solvent, which is mainly used for mutant screening and activity evaluation. In addition, the catalytic reaction of myristic acid is carried out at 55 °C in 5.0 mL Tris-HCl buffer (pH 8.5, 100 mM) without DMSO as a co-solvent. The reaction solution is extracted with twice the volume of ethyl acetate for substrates and products, and detected by gas chromatography. The specific detection conditions are as follows: chromatographic column: HP-5 (250 * 4.6 mm * 5 μm), using nitrogen as the carrier gas, split ratio 50:1, injection volume 1 μL, injector temperature 250 °C, detector temperature 300 °C, nitrogen flow rate of 1 mL / min, and the column oven temperature changes as shown in Table 2. The retention times of various alkanes are as Figure 10 shown. The enzyme activity is evaluated by the production amounts of the products pentadecane and tridecane.

[0051] Table 2 Column oven temperature gradient of gas chromatography

[0052]

[0053] Example 6 Thermal stability index melting temperature T of CvFAP and its mutant M8 m Determination

[0054] Melting temperature T m The measurement was carried out by a Bio-Logic MOS-500 spectrometer. The protein concentration was 0.05 mg / mL and dissolved in 50 mM phosphate buffer at pH 8.0. At 25 °C, using a cuvette with a path length of 1 mm, the CD spectrum of CvFAP was collected at intervals of 0.1 nm in the range of 190 to 260 nm. The average and smoothing of the average of three scans were performed, processed using CDToolX-Windows 10 version software, and plotted as the mean residue ellipticity (θ) in units of deg cm 2 . For the thermal denaturation measurement, the ellipticity was measured at 222 nm, from 20 °C to 86 °C, and tested in increments of 3 °C at a rate of 90 °C / h. The thermal melting point (T m ) was calculated by Boltzmann sigmoidal fitting using OriginLab OriginPro. The T m of CvFAP obtained by fitting was 49.24 °C, and the T m of the combined mutant M8 was 58.48 °C, which was 9.24 °C higher than that of CvFAP. The results are as Figure 4 shown.

[0055] Stability index t of Example 7 CvFAP and its mutant M8 1 / 2 Determination

[0056] Under the controlled temperature condition of 50 °C, the recombinant bacterial powder and the substrate were mixed and incubated together for CvFAP and its mutant M8. Samples were taken at regular intervals, and the remaining enzyme activity was measured at 30 °C. The time required for the initial activity of the enzyme to decrease by 50% was defined as the half-life (t 1 / 2 ). Using 10 mM palmitic acid as the substrate and 1 mg of Escherichia coli whole cell catalyst containing an equal amount of CvFAP or the combined mutant M8, the reaction was carried out in 1.0 mL Tris-HCl buffer (pH 8.5, 100 mM) containing 20% DMSO as a co-solvent under the condition of 450 nm blue light. The t of CvFAP was measured 1 / 2 to be 55 min, and the t of the combined mutant M8 1 / 2 was 385 min, which was 7 times higher than that of CvFAP. The results are as Figure 5 shown

[0057] Determination of the optimal activity temperature of the fatty acid photodecarboxylase mutant

[0058] The enzyme activities of CvFAP and its mutant M8 were measured at 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C respectively. The temperature at which the enzyme activity was the highest was defined as the optimal temperature of the enzyme. Using 10 mM palmitic acid as the substrate, the reaction was carried out in 1.0 mL Tris-HCl buffer (pH 8.5, 100 mM) containing 20% DMSO as a co-solvent, and 1 mg of Escherichia coli whole cell catalyst containing an equal amount of CvFAP or the combined mutant M8 was analyzed after reacting for 15 min under the condition of 450 nm blue light. The optimal temperature of CvFAP was measured to be 30 °C, and the optimal temperature of the combined mutant M8 was 45 °C, which was 15 °C higher than that of CvFAP. The results are as Figure 6 shown

[0059] Heat resistance activity determination of CvFAP and its mutant M8 at 50 °C

[0060] The heat-resistant enzyme activity assays of CvFAP and its mutants were respectively carried out in a total volume of 1.0 mL of Tris-HCl buffer (pH 8.5, 100 mM) containing 20% DMSO as a cosolvent at 50 °C. 0.2 mL of dimethyl sulfoxide containing palmitic acid (10 mM), 0.8 mL of Tris-HCl buffer (pH 8.5, 100 mM), and 1 mg of Escherichia coli whole-cell catalyst containing an equal amount of CvFAP or the combined mutant M8 were added to a transparent glass vial (total volume 1.5 mL). After reacting for 2 h under blue light conditions at 450 nm, the substrate and product were extracted with twice the volume of ethyl acetate and detected by gas chromatography. It was experimentally measured that the conversion rate of CvFAP was 30%, and the conversion rate of the combined mutant M8 was 90%. The results are as Figure 7 shown.

[0061] Example 10 Catalytic conversion of myristic acid to tridecane by CvFAP and its mutant M8 under cosolvent-free conditions

[0062] The heat-resistant enzyme activity assays of CvFAP and its mutants were respectively carried out in a total volume of 1.0 mL of Tris-HCl buffer (pH 8.5, 100 mM) without a cosolvent at 55 °C. 10 mM of myristic acid, 5 mL of Tris-HCl buffer (pH 8.5, 100 mM), and 10 mg of Escherichia coli whole-cell catalyst containing an equal amount of CvFAP or the combined mutant M8 were added to a transparent glass bottle (total volume 10 mL). After reacting for 12 h under blue light conditions at 450 nm, the substrate and product were extracted with twice the volume of ethyl acetate and detected by gas chromatography. It was experimentally measured that CvFAP was almost inactive, and the conversion rate of the combined mutant M8 was 66%. The results are as Figure 8 shown.

[0063] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A heat-resistant fatty acid photodecarboxylase mutant, characterized in that, The amino acid sequence of the described thermostable fatty acid photodecarboxylase mutant is as shown in SEQ ID NO.

3.

2. A gene encoding the thermostable fatty acid photodecarboxylase mutant according to claim 1.

3. The gene according to claim 2, wherein The nucleotide sequence of the described gene is as shown in SEQ ID NO.

5.

4. A recombinant plasmid into which the gene according to claim 2 or 3 is inserted.

5. A recombinant engineering strain carrying and capable of expressing the recombinant plasmid according to claim 4.

6. The recombinant engineering strain according to claim 5, characterized in that, The described recombinant engineering strain is Escherichia coli.

7. Use of the thermostable fatty acid photodecarboxylase mutant according to claim 1, the recombinant plasmid according to claim 4, or the recombinant engineering strain according to claim 5 or 6 in the catalytic decarboxylation of fatty acids to synthesize alkanes; The described fatty acid is palmitic acid, myristic acid or lauric acid.

8. The application according to claim 7, characterized in that, The reaction of catalytic decarboxylation of fatty acids to synthesize alkanes is carried out at a temperature higher than the melting point of the fatty acid and without adding an organic solvent for solubilization.

9. The application according to claim 8, wherein The reaction temperature for catalytic decarboxylation of fatty acids to synthesize alkanes is 45 - 60 °C, and it is carried out in a conventional buffer solution under blue light irradiation conditions.

10. The application according to claim 9, characterized in that, The reaction temperature for catalytic decarboxylation of fatty acids to synthesize alkanes is 55 - 56 °C.

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