Sulfoxide synthase mutant and application thereof

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a sulfoxide synthase mutant and application thereof, the catalytic activity of the mutant protein is remarkably improved compared with that of parent protein, then production of ergothioneine catalyzed by sulfoxide synthase is promoted, and a new synthetic route and engineering bacteria are provided for production of ergothioneine.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a sulfoxide synthase mutant and application thereof. Background Art

[0002] Ergothioneine, also known as thiohistidine trimethylol salt (EGT), has a molecular formula of C9H 15 N3O2S was first discovered in Thiobacillus ergotii in 1909 and got its name from it. Ergothioneine is a natural antioxidant that can remove active free radicals in cells, inhibit the activity of certain oxidases and many oxidative reactions in cells. Therefore, ergothioneine is widely used in the fields of food, medicine, cosmetics, health products and biotechnology. At present, ergothioneine can be synthesized by chemical methods or extracted from natural products such as mushrooms, but these two methods have the disadvantages of low yield, many impurities and high cost. The microbial fermentation method produces ergothioneine, which has low raw material cost, environmental friendliness, simple operation, short cycle, and is suitable for industrial production, becoming a hot spot in the current research on ergothioneine production.

[0003] The aerobic and anaerobic biosynthetic pathways of EGT have been reported, mainly including the synthesis pathway of EGT in prokaryotes represented by mycobacteria, the synthesis pathway in eukaryotes represented by Neurosporacrassa, and the anaerobic biosynthetic pathway represented by Chlorobium limicola. Guided by these synthesis pathways, EGT was prepared by fermentation using natural edible mushrooms that were not genetically engineered, such as Flammulina velutipes, Pleurotus ostreatus, and Lentinusedodes. By establishing a deep fermentation control strategy for accumulating EGT, the accumulation of EGT in the shake flask fermentation broth exceeded 500 mg / L (Mei Baoliang et al., 2015); by heterologous expression of ergothioneine biosynthetic enzymes, an engineered bacterium for EGT production was constructed, and the synthesis efficiency of EGT was further improved by strategies such as modification and optimization of the synthesis pathway of precursor amino acids.

[0004] However, there are few research reports on the transformation of key enzymes in the EGT synthesis pathway. Osawa R et al. (J Agric Food Chem. 2018 Feb 7; 66(5): 1191-1196) proposed that the prokaryotic organism Mycobacterium smegmatis EgtB enzyme is the rate-limiting enzyme for the synthesis of EGT. The key enzymes in the ergothioneine synthesis pathway, including the EgtB enzyme, are modified to improve the catalytic performance of the enzyme, which is expected to improve the biosynthesis level of EGT and provide a reference for the industrial application of bioenzymatic synthesis of EGT. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a sulfoxide synthase (EgtB) mutant and its application, the catalytic activity of the mutant protein is significantly improved compared with the parent enzyme, thereby promoting the production of ergothioneine catalyzed by EgtB.

[0006] The technical solution of the present invention is as follows: The present invention provides a sulfoxide synthase EgtB mutant, wherein the substitution relative to the parent sulfoxide synthase is selected from one of the following: W410P, W410T, W410Y, W410D, W410G, W410R, W410E, W410F, R73L, R73I, R73M, R73H, R73D, W410R+R73H, W410R+R73I; each position corresponds to the amino acid sequence of the parent sulfoxide synthase shown in SEQ ID NO: 1.

[0007] The present invention also provides a gene encoding the EgtB mutant.

[0008] The present invention also provides a vector, a recombinant vector or an expression vector comprising the above encoding gene.

[0009] The present invention also provides a host cell comprising the above encoding gene or recombinant vector.

[0010] The present invention also provides the application of the EgtB mutant, using the mutant protein to catalyze histidine trimethylolate (HER) and directly convert it into histidine trimethylolate cysteine ​​sulfoxide (Cys-HER) using L-Cys as a sulfur donor.

[0011] The present invention also provides a method for preparing the above-mentioned EgtB mutant, which comprises carrying out gene recombination and expression by utilizing the coding gene of the EgtB mutant of the present invention or an expression vector comprising the coding gene.

[0012] The present invention also provides a method for producing ergothioneine (L-EGT), which comprises the steps of introducing the EgtB mutant into a microbial expression system containing EgtA, EgtC, EgtD and EgtE, and preparing L-EGT under suitable conditions using L-His and L-Met as substrates.

[0013] The present invention also provides a method for improving the enzymatic activity of sulfoxide synthase, which is by introducing the following substitutions into a parent sulfoxide synthase: W410P, W410T, W410Y, W410D, W410G, W410R, W410E, W410F, R73L, R73I, R73M, R73H, R73D, W410R+R73H or W410R+R73I; each position corresponds to the amino acid sequence of the parent sulfoxide synthase shown in SEQ ID NO: 1.

[0014] The beneficial effects of the present invention are as follows: 1. The EgtB protein was modified by selecting active sites through directed evolution and gene mutation, and mutants with improved activity were screened.

[0015] 2. The EgtB protein with enhanced activity is combined with other enzymes in the ergothioneine synthesis pathway (eg, EgtA, EgtC, EgtD, EgtE) for the biosynthesis of ergothioneine, thereby improving the microbial fermentation level of ergothioneine.

[0016] 3. The present invention obtains for the first time the key amino acid sites for improving the activity of EgtB enzyme, which are the 410th and 73rd active sites of the EgtB enzyme derived from Mycobacterium smegmatis. The above key amino acid sites can effectively improve the activity of EgtB enzyme through mutation and mutation combination, providing key targets for further improving the subsequent enzyme activity of the enzyme.

[0017] 4. The present invention takes Escherichia coli expression system as an example, obtains the engineering bacteria comprising EgtB mutant, and applies the engineering bacteria to thioneine production. It is found that the mutation can effectively improve thioneine production, and compared with the engineering bacteria that are not mutated, the engineering bacteria containing mutant can increase thioneine production by up to 337%. Mutant described in the present invention effectively improves the synthesis efficiency of thioneine, and provides a new synthesis route and engineering bacteria for the production of thioneine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : The amino acid sequence of the parent sulfoxide synthase of the present invention.

[0019] Figure 2 : The nucleotide sequence of the gene encoding the parent sulfoxide synthase of the present invention.

[0020] Figure 3: The plasmid map expressing sulfoxide synthase (EgtB) in the examples.

[0021] Figure 4 : The results of shake flask fermentation of alanine scanning mutant strains in the embodiment.

[0022] Figure 5 : The results of shake flask fermentation of W410 saturated mutant strain in the embodiment.

[0023] Figure 6 : The results of shake flask fermentation of R73 saturated mutant strain in the embodiment.

[0024] Figure 7 : The shake flask fermentation test results of the W410+R73 combined mutant strain in the embodiment. DETAILED DESCRIPTION

[0025] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0026] The present invention provides a sulfoxide synthase EgtB mutant, wherein the substitution relative to the parent sulfoxide synthase is selected from one of the following: W410P, W410T, W410Y, W410D, W410G, W410R, W410E, W410F, R73L, R73I, R73M, R73H, R73D, W410R+R73H, W410R+R73I; each position corresponds to the amino acid sequence of the parent sulfoxide synthase shown in SEQ ID NO: 1.

[0027] The present invention also provides a gene encoding the EgtB mutant.

[0028] The present invention also provides a vector, a recombinant vector or an expression vector comprising the above-mentioned coding gene, for example, a recombinant vector composed of the vector plasmid pTrc99a and the coding gene of the present invention.

[0029] The present invention also provides a host cell comprising the above encoding gene or recombinant vector. The host cell can be any host suitable for producing the EgtB mutant of the present invention from the gene or vector of the present invention, such as microbial expression systems such as Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae.

[0030] The present invention also provides the application of the EgtB mutant, using the mutant protein to catalyze histidine trimethylolate (HER) and directly convert it into histidine trimethylolate cysteine ​​sulfoxide (Cys-HER) using L-Cys as a sulfur donor.

[0031] The EgtB mutants of the present invention have improved enzyme activity compared to their parent EgtB, for example, in some embodiments, the activity is increased by more than 50%, in other embodiments, the activity is increased by more than 100%, in other embodiments, the activity is increased by more than 200%, and in other embodiments, the activity is increased by more than 300%.

[0032] The present invention also provides a method for preparing the above-mentioned EgtB mutant, which method is carried out by genetic recombination and expression using the coding gene of the EgtB mutant of the present invention or an expression vector containing the coding gene. Genetic recombination methods and expression hosts known to those skilled in the art can be used, and a culture medium and culture conditions suitable for host expression can be selected. The method can also include a step of recovering the EgtB mutant, which may involve a step of separating or purifying the EgtB mutant from the host culture or expression product, and can be carried out using any method known to those skilled in the art.

[0033] The present invention also provides a method for producing ergothioneine (L-EGT), which comprises the steps of introducing the EgtB mutant into a microbial expression system (such as an Escherichia coli expression system) containing EgtA, EgtC, EgtD and EgtE, and preparing L-EGT under suitable conditions using L-His and L-Met as substrates.

[0034] In the above-mentioned method for producing ergothioneine, the preferred culture medium composition is: glucose 20-40 g / L, yeast extract 1-4 g / L, peptone 2-5 g / L, sodium citrate 1-2 g / L, KH2PO41-2 g / L, MgSO4·7H2O 1-2 g / L, FeSO410-20 mg / L, V B·mix (1,3,5,12) 1-2 mg / L, V H 1-2 mg / L, V B6 1-10 mg / L, MnSO41-10 mg / L, the rest is water, pH 7.0-7.2.

[0035] The present invention also provides a method for improving the enzymatic activity of sulfoxide synthase, which is by introducing the following substitutions into a parent sulfoxide synthase: W410P, W410T, W410Y, W410D, W410G, W410R, W410E, W410F, R73L, R73I, R73M, R73H, R73D, W410R+R73H or W410R+R73I; each position corresponds to the amino acid sequence of the parent sulfoxide synthase shown in SEQ ID NO: 1.

[0036] The following definitions are used in the present invention: 1. Nomenclature of amino acid and DNA sequences Amino acid residues are referred to using the generally accepted IUPAC nomenclature, either by three-letter abbreviations or by single-letter symbols. DNA nucleic acid sequences are referred to using the generally accepted IUPAC nomenclature.

[0037] 2. Identification of EgtB mutants The mutated amino acid in the EgtB mutant is represented by "amino acid replaced by the original amino acid position". For example, W410F means that the amino acid residue at position 410 in the amino acid sequence from the N-terminus to the C-terminus is replaced by phenylalanine (F) from the tryptophan (W) of the parent EgtB. The numbering method of the amino acids of EgtB in the present invention is based on the sequence shown in SEQ ID NO: 1.

[0038] 3. The amino acid sequence of the parent sulfoxide synthase (SEQ ID NO: 1) is as follows Figure 1 As shown; the coding gene sequence (SEQ ID NO: 2) is as shown Figure 2 shown.

[0039] The present invention will be described in more detail below through specific examples. Unless otherwise defined, the technical and scientific terms used in the following examples have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0040] Example 1. Preparation method of mutants 1.1 Primer design Using the primer design software primer5, with the EgtB enzyme sequence as a template, primers were designed at the beginning and end of the target gene; with the EgtB enzyme sequence as a template, amplification primers containing the mutation site bases were designed at the mutation site. The upstream and downstream were amplified separately by PCR, and then the recombinant fragments were prepared by overlapping PCR. The PCR amplification system is as follows:

[0041] The overlapping PCR system is as follows:

[0042] PCR reaction conditions (Bao Bio PrimeSTAR HS enzyme): pre-denaturation (95°C) for 5 min; then 30 cycles: denaturation (98°C) for 10 s, annealing ((Tm-3 / 5)°C) for 15 s, extension at 72°C (this enzyme activity extends about 1 kb in 1 min); continue extension at 72°C for 10 min; maintain (4°C).

[0043] 1.2 Preparation of linear vector: The vector was linearized by inverse PCR amplification.

[0044] 1.3 Plasmid construction method: The target fragment is obtained by PCR, and the plasmid is amplified by reverse PCR. DNA gel recovery is performed to obtain the target fragment and the vector fragment. After homologous recombination, they are transformed into Escherichia coli DH5α competent cells and coated with resistance plates. Single colonies identified as positive are selected for culture and plasmid extraction.

[0045] 1.4 Recombination reaction: All recombinases used are enzymes of the ClonExpress ® II One Step Cloning Kit series. Recombination conditions: 37°C, 30 min. The recombinant system is as follows:

[0046] 1.5 Transformation of plasmid: Take 10 μL of reaction solution and add it to 100 mL DH5α competent cells. Mix gently and place on ice for 20 min. Heat shock at 42℃ for 45-90 s. Place on ice for 2-3 min. Add 900 μL SOC and resuscitate at 37℃ for 1 h. Centrifuge at 8000 rpm for 2 min. Discard part of the supernatant and keep about 200 μL to resuspend the bacteria and apply it to a plate containing 100 mg / L ampicillin. Invert the plate and culture it at 37℃ overnight. After a single colony grows on the plate, identify it by colony PCR and select positive recombinants.

[0047] 1.6 Clone identification: Inoculate PCR-positive colonies into LB medium containing 100 mg / L ampicillin and culture overnight to incubate the bacteria. Extract the plasmid and identify it by enzyme digestion.

[0048] Table 1 Primers for PCR amplification (saturation mutation of W410 and R73 sites)

[0049]

[0050] Example 2. Construction of mutant strains and fermentation test 2.1 Construction of alanine mutagenesis strain The EgtB enzyme was molecularly docked with the substrates γ-glutamylcysteine ​​and histidine betaine using Discovery studio software to simulate their potential interactions. According to the results of molecular docking, the amino acid residue sites (Asp72, Tyr75, Arg73, Gln49, Asp76, Tyr388, His412, Gln50, His46, Phe78, Asp411, Arg415, Phe379, Trp410, Trp42, Pro378) were obtained, and the amino acids at the corresponding sites were replaced with alanine by designing primers to obtain mutant genes. egtB D72A 、egtB Y75A 、egtB R73A 、egtB Q49A 、egtB D76A 、egtB Y388A 、egtB H412A 、egtB Q50A 、 egtB H46A 、egtB F78A 、egtB D411A 、egtB R415A 、egtB F379A 、egtB W410A 、egtB W42A 、egtB P378A , then the mutant fragment was combined with pTrc99a ( Figure 3 )Linearized vector was connected to obtain recombinant plasmid, and the recombinant plasmid was transferred to DH5α competent cells. After coating and culture, several clones on the recombinant reaction transformation plate were picked for colony PCR identification. The colonies identified as positive by colony PCR were cultured and the plasmid was extracted. Then the plasmid was transformed into ergothioneine production strain SX180110 (MG1655 ∆ lacIZ ::P xylF -T7RNAP,∆mlc :: mlc*,∆purR,∆tdcD ::P trc -hisG,∆yghX ::P trc - hisDCBHAFI,∆ilvG::P trc -egtBCDE,∆mbhA ::P trc -gshA,∆tehB ::Ptrc -egtE ncr ) to obtain the alanine scanning mutagenesis strain.

[0051] 2.2 Selection of key sites for site-directed mutagenesis Take 10 μL of glycerol bacteria (mutant strain) and inoculate it into 5 mL of LB, and culture it at 37°C, 220 rpm for 12 h. Then transfer the seed liquid to a shake flask containing 30 mL of LB medium at a 3% inoculation rate, and culture it at 37°C, 220 rpm for 12 h. Then transfer the seed liquid to a fermentation bottle containing 30 mL of fermentation medium at a 10% inoculation rate, and culture it at 37°C, 220 rpm for 24 h. After the fermentation is completed, treat the fermentation broth according to the fermentation broth treatment method and perform high-performance liquid chromatography detection. The fermentation results are shown in Figure 4 As shown in Table 2, the W410A and R73A mutant strains showed a significant difference compared with the control strain SX220460 (SX180110 / pTrc99a -egtB msm ) The EGT production increased most significantly, by 243% and 224% respectively, so W410 and R73 were judged to be the key sites for EgtB to bind to the substrate.

[0052] Table 2 Alanine mutant strain shake flask fermentation results

[0053] 2.3 Construction of saturated mutant strains By designing primers (Table 1), tryptophan at position 410 and arginine at position 73 were mutated to the remaining 18 amino acids to obtain mutant genes. egtB W410F 、egtB W410L 、egtB W410I 、egtB W410M 、egtB W410V 、egtB W410S 、egtB W410P 、 egtB W410T 、egtB W410Y 、egtB W410H 、egtB W410Q 、egtB W410N 、egtB W410K 、egtB W410C 、egtB W410R、egtB W410G 、 egtB W410D 、egtB W410E and egtB R73F 、egtB R73L 、egtB R73I 、egtB R73M 、egtB R73V 、egtB R73S 、egtB R73P 、 egtB R73T 、egtB R73Y 、egtB R73H 、egtB R73Q 、egtB R73N 、egtB R73K 、egtB R73C 、egtB R73W 、egtB R73G 、egtB R73D 、 egtB R73E After that, the mutant gene fragment was connected to the pTrc99a linearized vector to obtain a recombinant plasmid, and the recombinant plasmid was transferred to DH5α competent cells. After coating and culturing, several clones on the recombination reaction transformation plate were picked for colony PCR identification, and the colony identified as positive by colony PCR was cultured and the plasmid was extracted. After that, the plasmid was transformed into the ergothioneine production strain SX180110 to obtain a saturated mutant strain.

[0054] 2.4 Obtaining mutants with improved catalytic activity and thioneine-producing strains thereof The saturated mutant strain and the control strain SX220460 were subjected to shake flask fermentation experiments and tested. The fermentation results are shown in Figure 5 , Figure 6As shown in Tables 3 and 4, the ergothioneine production capacity of mutant strains such as W410P, W410T, W410Y, W410D, W410R, W410G, W410E, W410F, R73L, R73I, R73M, R73H or R73D was improved, and the EGT yield was increased by 80%, 92%, 110%, 112%, 219%, 160%, 265%, 49%, 110%, 285%, 118%, 264% or 115% respectively compared with the control strain, indicating that these mutant enzymes have higher catalytic activity than the parent enzyme.

[0055] Table 3 Shake flask fermentation results of W410 site saturated mutant strain

[0056] Table 4 Results of shake flask fermentation of R73 site saturated mutant strain

[0057] Shake flask fermentation: After activating the strain, prepare the seed solution, inoculate 10% of the inoculum into a 500 mL Erlenmeyer flask (final volume 30 mL), seal with nine layers of gauze, and culture at 37°C, 200 r / min with shaking. During the fermentation process, the pH was maintained at 7.0 by adding ammonia water; 60% (m / v) glucose solution was added to maintain the fermentation process.

[0058] The preferred medium composition is: glucose 20 g / L, yeast extract 4 g / L, peptone 5 g / L, sodium citrate 2 g / L, KH2PO4 2 g / L, MgSO4·7H2O 2 g / L, FeSO4 20 mg / L, V B·mix (1,3,5,12) 2 mg / L, V H 2 mg / L, V B6 10 mg / L, MnSO4 10 mg / L, the rest is water, pH 7.0-7.2.

[0059] Fermentation broth treatment method: Take 1 mL of fermentation broth, centrifuge at 13000 rpm for 3 min, take the supernatant and filter it with a 0.22 μm filter membrane for HPLC analysis.

[0060] HPLC conditions: the mobile phase was 2% (v / v) acetonitrile, the flow rate was 0.7 mL / min, the chromatographic column was a Titank C18 column (250 mm×4.60 mm, 5 μm), the column temperature was 30°C, the UV detection wavelength was 257 nm, and the injection volume was 10 μL.

[0061] 2.5 Mutation site combinations Construction of W410, R73 double-site mutant geneegtB W410R / R73I 、egtB W410E / R73I 、egtB W410R / R73H 、 egtB W410E / R73H After that, the mutant gene fragment was connected to the pTrc99a linearized vector to obtain a recombinant plasmid, and the recombinant plasmid was transferred to DH5α competent cells. After coating and culturing, several clones on the recombination reaction transformation plate were picked for colony PCR identification, and the colony identified as positive by colony PCR was cultured and the plasmid was extracted. After that, the plasmid was transformed into the ergothioneine production strain SX180110 to obtain a double-site mutant strain.

[0062] 2.6 Shake flask fermentation of double-site mutant strains The four double-site mutant strains were subjected to shake flask fermentation experiments. The fermentation results were as follows: Figure 7 As shown, the EGT production of the W410R+R73H and W410R+R73I mutant strains after 24 h of shake flask fermentation was increased by 337% and 203% respectively compared with the control strain, indicating that the combined mutation of the mutation sites can further improve the catalytic activity of sulfoxide synthase and thus promote the production of ergothioneine.

[0063] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A sulfoxide synthase EgtB mutant, characterized in that: The substitution of the EgtB mutant relative to the parent sulfoxide synthase is selected from one of the following: W410P, W410T, W410Y, W410D, W410G, W410R, W410E, W410F, R73L, R73I, R73M, R73H, R73D, W410R+R73H, W410R+R73I; each position corresponds to the amino acid sequence of the parent sulfoxide synthase shown in SEQ ID NO:

1.

2. The coding gene of the EgtB mutant according to claim 1.

3. A recombinant vector, characterized in that: Contains the coding gene according to claim 2.

4. A host cell, characterized in that Comprising the coding gene according to claim 2 or the recombinant vector according to claim 3.

5. The host cell according to claim 4, characterized in that The host cell is Escherichia coli.

6. Use of the EgtB mutant according to claim 1, wherein the mutant protein is used to catalyze the conversion of histidine trimethylolate (HER) into histidine trimethylolate cysteine ​​sulfoxide (Cys-HER) using L-Cys as a sulfur donor.

7. A method for preparing the EgtB mutant of claim 1, comprising performing gene recombination and expression using a gene encoding the EgtB mutant or an expression vector comprising the gene encoding the EgtB mutant.

8. A method for producing ergothioneine (L-EGT), comprising the steps of introducing the EgtB mutant of claim 1 into a microbial expression system containing EgtA, EgtC, EgtD and EgtE, and preparing L-EGT under suitable conditions using L-His and L-Met as substrates.

9. The method according to claim 8, characterized in that The microbial expression system is Escherichia coli.

10. A method for improving the enzymatic activity of a sulfoxide synthase, the method comprising introducing the following substitutions into a parent sulfoxide synthase: W410P, W410T, W410Y, W410D, W410G, W410R, W410E, W410F, R73L, R73I, R73M, R73H, R73D, W410R+R73H or W410R+R73I; each position corresponds to the amino acid sequence of the parent sulfoxide synthase shown in SEQ ID NO: 1.

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