A sulfur transferase egt-st4, related biological materials, and methods of use and preparation of ergothioneine

Abstract

The present application relates to the technical field of microbial engineering, and specifically discloses a sulfur transferase EGT-ST4, related biological materials, and a method for preparing ergothioneine. The amino acid sequence of the sulfur transferase EGT-ST4 is shown in SEQ ID NO:1. The present application also provides biological materials containing the nucleic acid molecules. The recombinant E. coli can achieve high-efficiency soluble expression of EGT-ST4, and the enzyme activity can reach 8342 U / L in a 5 L fermenter. Using the recombinant bacteria or the catalyst prepared therefrom, ergothioneine can be generated from histidine betaine in the presence of inorganic polysulfides, and when the substrate concentration is 45 g / L, the product titer can reach 48.1 g / L, and the molar conversion rate can reach 92.0%. The present application provides an efficient and stable enzyme source and catalytic process for the industrial production of ergothioneine, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, and in particular to a thiotransferase EGT-ST4, related biomaterials, their applications, and a method for preparing ergothioneine. Background Technology

[0002] Ergothioneine (EGT) is a natural sulfur-containing histidine derivative with antioxidant and cell-protective biological functions, and it has high application value in food, cosmetics, pharmaceuticals, and health products. The human body cannot synthesize ergothioneine and relies mainly on exogenous intake. Therefore, developing efficient, economical, and scalable ergothioneine preparation technologies is of great significance.

[0003] Currently, ergothioneine can be prepared through chemical synthesis, bio-fermentation, and in vitro enzymatic conversion. Among these methods, in vitro enzymatic conversion offers advantages such as mild reaction conditions, high catalytic selectivity, relatively well-defined reaction system composition, and ease of process control, thus demonstrating promising potential for industrial application.

[0004] In the reported enzymatic synthesis pathways of ergothionein, hercynine (HER) can be used as a substrate to catalyze the production of ergothionein through an enzyme with sulfur transfer catalysis in the presence of a sulfur-containing donor. This type of reaction pathway has fewer steps and a relatively simple substrate system, providing a feasible scheme for the in vitro biocatalytic preparation of ergothionein.

[0005] Existing research indicates that EanB proteins derived from *Chlorobium limicola* are enzymes capable of catalyzing the sulfur transoxidation of histidine betaine to produce ergothioneine. However, the expression of wild-type EanB in heterologous hosts and its industrial catalytic applications still face certain limitations.

[0006] For example, when wild-type EanB is expressed in commonly used heterologous expression hosts such as Escherichia coli, it is prone to low soluble expression levels and the formation of insoluble aggregates of some recombinant proteins, thus limiting the amount of soluble enzyme protein with catalytic activity obtained. At the same time, there is still room for improvement in the unit enzyme protein catalytic efficiency of wild-type EanB, making it difficult to obtain a high overall catalytic capacity under the same fermentation volume or the same amount of enzyme.

[0007] Furthermore, increasing substrate concentration, extending reaction time, or subjecting to high-load catalysis may further reduce the catalytic activity and functional stability of wild-type EanB, thereby limiting substrate conversion efficiency and the increase in ergothioneine final product concentration. These issues increase the cost of enzyme preparation and the amount of enzyme used in the actual reaction process, hindering its further application in the large-scale production of ergothioneine.

[0008] Therefore, there is still a need in this field to develop a thiotransferase with high heterologous soluble expression level, high catalytic activity, good substrate loading adaptability and functional stability, and to establish a corresponding recombinant expression system and ergothioneine preparation method, so as to improve the production efficiency of ergothioneine biocatalytic process and reduce production cost. Summary of the Invention

[0009] In view of this, the purpose of the present invention is to provide a thiotransferase EGT-ST4, related biomaterials and their applications, and a method for preparing ergothionein, so as to solve the problems existing in the prior art.

[0010] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, this invention provides a novel thiotransferase, named EGT-ST4. This novel thiotransferase is capable of catalyzing the conversion of histidine betaine to ergothioneine in the presence of inorganic polysulfides.

[0011] In one embodiment, the novel thiotransferase EGT-ST4 has the amino acid sequence shown in SEQ ID NO:1, or is a protein having at least 90%, 95%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1 and having the activity of catalyzing the conversion of histidine betaine to ergothionein.

[0012] In a second aspect, the present invention provides a biomaterial related to the above-described thiotransferase EGT-ST4, wherein the biomaterial is at least one of the following: A1) The nucleic acid molecule encoding the aforementioned thiotransferase EGT-ST4; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant expression vector containing the nucleic acid molecule described in A1) or a recombinant expression vector containing the expression cassette described in A2); A4) A recombinant host cell containing the nucleic acid molecule described in A1), a genetically engineered host cell containing the expression cassette described in A2), or a recombinant host cell containing the recombinant expression vector described in A3).

[0013] In one embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2.

[0014] In one embodiment, the recombinant host cell is recombinant Escherichia coli.

[0015] In one embodiment, the recombinant host cell is Escherichia coli BL21(DE3).

[0016] In one embodiment, the nucleic acid molecule encoding the novel thiotransferase EGT-ST4 is located on a recombinant expression vector. The recombinant expression vector may be a pET series expression vector, a pBAD series expression vector, a pTrc series expression vector, a pTac series expression vector, or an equivalent expression vector thereof.

[0017] In a preferred embodiment, the recombinant expression vector is a pET-28a(+) expression vector, and the nucleic acid molecule encoding the novel thiotransferase EGT-ST4 is operatively linked to the T7 promoter.

[0018] In one embodiment, a nucleic acid molecule encoding a novel thiotransferase EGT-ST4 is fused with a fusion tag coding sequence and expressed. The fusion tag is selected from one or more of TrxA, MBP, SKP, or HaloTag.

[0019] In a preferred embodiment, the fusion tag is TrxA. The fusion tag and the novel thiotransferase EGT-ST4 can be linked via a flexible linker peptide, including but not limited to (GGGGS). n , where n is 1, 2, 3, 4 or 5, preferably (GGGGS)3.

[0020] In one embodiment, the recombinant Escherichia coli is able to express the novel thiotransferase EGT-ST4 in free form or in fusion-tagged form.

[0021] In a third aspect, the present invention provides a method for preparing the above-described thiotransferase EGT-ST4, comprising: fermenting and culturing the recombinant host cell described in any one of the above claims, and inducing expression of the thiotransferase EGT-ST4.

[0022] Specifically, a nucleic acid molecule encoding a novel thiotransferase EGT-ST4 is provided, the nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:2 or an optimized codon sequence thereof; Construct a recombinant expression vector containing the nucleic acid molecule, so that the nucleic acid molecule is operatively linked to a promoter, a ribosome binding site, and a transcription termination sequence; Optionally, a fusion tag sequence encoding TrxA, MBP, SKP, or HaloTag is ligated to the 5′ or 3′ end of the nucleic acid molecule; The recombinant expression vector was introduced into Escherichia coli host cells, and recombinant E. coli capable of expressing the novel thiotransferase EGT-ST4 were screened to obtain them. The recombinant Escherichia coli was fermented and induced to express to obtain whole-cell catalysts, resting-cell catalysts, or recombinant enzyme preparations.

[0023] The present invention also provides a method for preparing an enzyme source using the recombinant Escherichia coli, comprising the following steps: The recombinant Escherichia coli was inoculated into a seed culture medium and cultured to obtain a seed solution; The seed culture was inoculated into a fermenter and cultured using a fed-batch fermentation method. During fermentation, the pH is maintained at 6.2-7.0, preferably 6.45, by automatically adding ammonia or alkali solution; The dissolved oxygen level is controlled between 20% and 50%, preferably 35%, by adjusting the feeding rate, aeration rate, and stirring speed. When the bacterial cell density OD 600 When the temperature reaches 20-30 °C, the culture temperature is lowered to 20-30 °C, preferably 25 °C, and an inducer is added to induce expression. Induce culture for 12-30 hours, preferably 16-27 hours, collect cells or fermentation broth to obtain cells or enzyme sources containing the novel thiotransferase EGT-ST4.

[0024] In one embodiment, the inducer is IPTG with a final concentration of 0.05-1.0 mM, preferably 0.5 mM.

[0025] In one embodiment, after the recombinant E. coli is fermented in a 5 L fermenter, the cell density OD 600 The concentration of the novel thiotransferase EGT-ST4 protein reached 5.8 g / L, and the enzyme activity reached 8342 U / L, reaching 148.2.

[0026] In a fourth aspect, the present invention provides the use of the above-described thiotransferase EGT-ST4, or any of the above-described biomaterials, in the catalytic generation of ergothionein.

[0027] Specifically, it includes the following steps: The recombinant Escherichia coli was fermented and cultured to induce the expression of a novel thiotransferase, EGT-ST4. Collect the fermented cells to prepare whole-cell catalysts, resting cell catalysts, or enzyme solutions obtained by disruption; Under anaerobic or hypoxic conditions, histidine betaine, inorganic polysulfides, and the above-obtained whole-cell catalyst, resting-cell catalyst, or enzyme solution are reacted to generate ergothionein. The reaction solution was subjected to solid-liquid separation, deproteinization, chromatography, concentration and crystallization to obtain ergothioneine product.

[0028] In one embodiment, the resting cell catalyst is prepared by the following method: collecting bacterial cells after induced expression, washing them with PBS buffer at pH 7.0-7.5, and resuspending them in reaction buffer or permeabilization buffer to prepare OD.600 It is a resting cell suspension of 50-100.

[0029] In one embodiment, the permeation buffer contains 0.01%-0.5% (v / v) of Triton X-100, preferably 0.1% (v / v).

[0030] In one embodiment, the final concentration of histidine betaine in the reaction system is 13-45 g / L, preferably 15-45 g / L.

[0031] In one embodiment, the inorganic polysulfide is sodium polysulfide (Na₂S). x , where x is 2-8.

[0032] In one embodiment, the reaction temperature is 30-35 °C, the reaction pH is 7.5-8.0, and the reaction time is 24-36 hours.

[0033] In one embodiment, when the concentration of histidine betaine is 45 g / L, the titer of ergothioneine product reaches 48.1 g / L after 36 hours of catalysis, and the molar conversion rate reaches 92.0%.

[0034] After the reaction is complete, bacterial cells, cell debris, and protein impurities can be removed by centrifugation, plate and frame filtration, microfiltration, or ultrafiltration. The resulting supernatant can be further subjected to cation exchange resin chromatography, desalting, concentration, and crystallization to obtain ergothioneine product.

[0035] In one embodiment, after the reaction is completed, the bacterial cells are removed by centrifugation or plate and frame filtration, and soluble protein impurities are removed by 10 kDa ultrafiltration. The resulting supernatant is separated by a strong acid cation exchange resin, impurities are washed off with water, and ergothioneine components are eluted with ammonia or other suitable eluents. The ergothioneine components are collected and concentrated under reduced pressure, and then an alcohol solvent is added for crystallization to obtain the ergothioneine product.

[0036] The beneficial effects of this invention include at least the following: (1) Provide novel thiotransferases with well-defined sequences.

[0037] This invention defines the protein shown in SEQ ID NO:1 as a novel thiotransferase EGT-ST4, and provides its encoded nucleic acid SEQ ID NO:2, which can be used as a core catalytic element for the preparation of ergothionein.

[0038] (2) Provide recombinant Escherichia coli enzyme sources suitable for fermentation scale-up.

[0039] This invention expresses a novel thiotransferase, EGT-ST4, in *E. coli*, enabling the engineered bacteria to be used as a host for recombinant enzyme preparation, a whole-cell catalyst, or a resting-cell catalyst. In a 5 L fermenter experiment, the recombinant *E. coli* cell density OD... 600 The concentration of the novel thiotransferase EGT-ST4 protein reached 5.8 g / L, and the enzyme activity reached 8342 U / L, reaching 148.2.

[0040] (3) It is beneficial to improve the soluble expression of the target enzyme.

[0041] This invention provides fusion tag expression methods such as TrxA, MBP, SKP and HaloTag, which can select appropriate tags according to the expression status of the target enzyme to improve protein folding, soluble expression ratio and catalytic activity.

[0042] (4) It can be used for the catalytic preparation of ergothionein under high substrate loading.

[0043] In a catalytic system using histidine betaine as a substrate, when the substrate concentration is 45 g / L, a molar conversion rate of 92.0% can be achieved after 36 hours of catalysis, and the ergothioneine product titer reaches 48.1 g / L.

[0044] (5) Compatible with multiple catalyst forms.

[0045] The recombinant Escherichia coli can be used directly as a whole-cell catalyst, or prepared as a resting cell catalyst. It can also be broken down and separated to obtain recombinant enzyme preparations, which makes it easy to select different process routes according to production scale, cost and purification requirements. Attached Figure Description

[0046] Figure 1 The plasmid map of the recombinant expression vector pET-28a(+)-EGT-ST4.

[0047] Figure 2 A schematic diagram of the expression cassette structure for the recombinant expression vector pET-28a(+)-EGT-ST4.

[0048] Figure 3 SDS-PAGE plot showing the effect of different fusion tags on the soluble expression of the novel thiotransferase EGT-ST4.

[0049] Figure 4 The image shows the HPLC detection results of ergothionein.

[0050] Figure 5 This is a comparison chart of ergothioneine product titers at different substrate concentrations and time points. Detailed Implementation

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0052] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0053] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Construction of recombinant Escherichia coli expressing the novel thiotransferase EGT-ST4 Based on the nucleotide sequence shown in SEQ ID NO:2, a gene fragment encoding a novel thiotransferase EGT-ST4 was synthesized. This gene fragment was ligated into the pET-28a(+) expression vector, allowing it to be operatively linked to the T7 promoter, resulting in the recombinant expression vector pET-28a(+)-EGT-ST4 (SEQ ID NO:3), the map of which is shown below. Figure 1 , Figure 2 As shown.

[0054] The recombinant expression vector was transformed into Escherichia coli BL21(DE3) competent cells, and the recombinant Escherichia coli BL21(DE3) / pET-28a(+)-EGT-ST4 expressing the novel thiotransferase EGT-ST4 was obtained by resistance plate screening and colony PCR verification.

[0055] To improve the soluble expression of the novel thiotransferase EGT-ST4, expression vectors containing fusion tags of TrxA (SEQ ID NO:5), MBP (SEQ ID NO:4), SKP (SEQ ID NO:6), or HaloTag (SEQ ID NO:7) were further constructed. The fusion tags were linked to the novel thiotransferase EGT-ST4 via a flexible linker peptide (GGGGS)3, resulting in TrxA-EGT-ST4, MBP-EGT-ST4, SKP-EGT-ST4, and HaloTag-EGT-ST4 expression constructs, respectively.

[0056] Recombinant expression vectors of EGT-ST4 carrying four different fusion tags (MBP, Skp, HaloTag, and TrxA) were transformed into Escherichia coli BL21 (DE3) strain. After induction culture, protein samples were prepared, and the expression levels of recombinant proteins were analyzed by SDS-PAGE electrophoresis. Figure 3 In the figure, lane M represents the protein molecular weight standard (marker), with molecular weight markers of approximately 33 kDa, 45 kDa, 66 kDa, and 98 kDa from bottom to top. From left to right, four fusion tag groups are set: MBP, Skp, halotag, and TrxA, each with three sample lanes. The samples loaded in each lane are the total cell lysate, supernatant, and precipitate after induction by the corresponding strain, respectively. The subscripts 1, 2, and 3 in each group represent the total lysate, supernatant, and precipitate, respectively: in the MBP group, m1, m2, and m3 correspond to the total lysate, supernatant, and precipitate, respectively; in the Skp group, s1, s2, and s3 correspond to the total lysate, supernatant, and precipitate, respectively; in the halotag group, h1, h2, and h3 correspond to the total lysate, supernatant, and precipitate, respectively; and in the TrxA group, t1, t2, and t3 correspond to the total lysate, supernatant, and precipitate, respectively. All four fusion tag constructs successfully induced the production of the corresponding molecular weight fusion EGT-ST4 target protein in the host bacteria, achieving efficient expression of the novel thiotransferase EGT-ST4. Comparing protein band characteristics, soluble expression levels, and subsequent catalytic activity assays, the TrxA fusion expression system demonstrated superior soluble expression of the target protein and significantly better recombinant enzyme catalytic activity. Therefore, the TrxA fusion expression construct was selected as the preferred vector for subsequent fermentation optimization and resting cell catalytic experiments.

[0057] Example 2: High-density fermentation of recombinant Escherichia coli and preparation of enzyme source The recombinant Escherichia coli obtained in Example 1 was inoculated into a 5 L fermenter with an initial liquid volume of 3 L, and cultured using a fed-batch fermentation strategy. The pH was maintained at 6.45 by automatically adding 25% (w / w) ammonia, and the dissolved oxygen level was controlled at 35% by adjusting the feed rate, aeration rate, and stirring speed.

[0058] 1. Preparation of seed liquid (1) Preparation of primary seed liquid Take 50 μL of bacterial culture from recombinant Escherichia coli BL21(DE3) / pET-28a(+)-TrxA-EGT-ST4 glycerol strain stored at -80 °C and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL of LB liquid medium. The LB liquid medium contains kanamycin at a final concentration of 50 μg / mL.

[0059] The inoculated culture was incubated at 37 ℃ and 220 rpm with shaking for 10–12 h until the bacterial OD of the culture was reached. 600 Achieving a pH of 2.5–3.0 yields a first-grade seed solution.

[0060] (2) Preparation of secondary seed liquid The primary seed culture was transferred at an inoculation rate of 5% (v / v) to a 1 L Erlenmeyer flask containing 300 mL of modified TB medium. The modified TB medium contained kanamycin at a final concentration of 50 μg / mL.

[0061] Incubate at 37 ℃ and 220 rpm with shaking for 6–8 h until the bacterial culture OD reaches its maximum. 600 When the pH reaches 3.0–4.0, a secondary seed culture is obtained. Samples are taken for microscopic examination to confirm that the bacterial cell morphology is normal and free from contamination by other microorganisms.

[0062] 2. Fermentation tank inoculation and culture conditions 300 mL of secondary seed culture was aseptically inoculated into a 5 L fermenter containing 3.0 L of modified TB medium. The ratio of the secondary seed culture volume to the initial culture medium volume in the fermenter was 1:10, and the initial culture volume after inoculation was approximately 3.3 L.

[0063] The fermentation temperature was set according to the cell growth stage and the induction expression stage. During fermentation, 25% (w / w) ammonia was used as a pH adjuster to maintain the pH of the culture system at 6.45. The 25% ammonia not only adjusted the pH of the culture system but also served as an inorganic nitrogen source to supplement the nitrogen supply.

[0064] 3. Fed-feeding culture strategy The fermentation process includes a batch culture stage, a progressive feeding stage, and a steady feeding stage.

[0065] (1) Batch cultivation stage The batch culture phase lasts from 0 to 6 hours after inoculation. During this phase, no feed solution is added, and the feed rate is 0. The bacteria utilize the carbon and nitrogen sources in the basal medium for growth.

[0066] After culturing to the predetermined induction time point, an inducer (final concentration of 0.5 mM) was added to the fermentation system, and the induction temperature was set to 25°C to initiate the expression of EGT-ST4. The type of inducer, final concentration, and induction temperature can be set according to the expression characteristics of the expression construct described in Example 1.

[0067] (2) Incremental feeding stage The feeding phase lasts from 0 to 15 hours after induction, which is approximately 6 to 21 hours after inoculation.

[0068] The initial feeding rate was set at 5 mL / h, and then gradually increased based on changes in dissolved oxygen and pH. The feeding rate was increased by 1–2 mL / h per hour, gradually increasing from 5 mL / h to 25–30 mL / h within 15 hours. During this phase, the feeding rate was controlled at 5–30 mL / h.

[0069] During feeding, dissolved oxygen should be controlled at 30%–40% of air saturation. When other culture parameters remain stable and dissolved oxygen continues to rise above 50%, it indicates that the available carbon source in the culture medium may be insufficient. In this case, the feeding rate should be increased by 2–3 mL / h. When dissolved oxygen drops significantly, the feeding rate should be reduced accordingly to avoid carbon source accumulation and the generation of metabolic byproducts.

[0070] (3) Stabilized feeding stage The stable feeding phase lasts 15–27 hours after induction, which is approximately 21–33 hours after inoculation.

[0071] During this stage, the feeding rate should be maintained at 25–35 mL / h, and fine-tuned based on dissolved oxygen feedback. The allowable adjustment range for the feeding rate is 20–40 mL / h. When dissolved oxygen increases and other control parameters do not change significantly, the feeding rate should be increased appropriately; when dissolved oxygen decreases, the feeding rate should be decreased appropriately.

[0072] 4. Dissolved oxygen control During fermentation, the dissolved oxygen setting is set to 35%, and the dead zone is controlled at ±5%, that is, the dissolved oxygen is preferably maintained at 30% to 40% of the air saturation.

[0073] The initial aeration rate for fermentation was set to 1 vvm, and then converted based on the actual volume of the fermentation broth. The aeration rate can be adjusted from 0.5 to 2.0 vvm.

[0074] The initial stirring speed for fermentation was set to 400 rpm, with an adjustment range of 300–800 rpm. The stirring speed and dissolved oxygen were cascaded and controlled, as follows: When dissolved oxygen is above 40%, gradually reduce the stirring speed by about 50 rpm each time; when dissolved oxygen is below 30%, gradually increase the stirring speed by about 50 rpm each time; when dissolved oxygen is in the range of 30% to 40%, maintain the current stirring speed.

[0075] The stirring speed should not be varied at a rate not exceeding 100 rpm / min to reduce the impact of sudden changes in stirring conditions on cell growth and protein expression.

[0076] When dissolved oxygen is below 25%, prioritize increasing the stirring speed; if the stirring speed reaches 800 rpm and the dissolved oxygen is still below 25%, further increase the aeration rate, with a maximum aeration rate of 2.0 vvm. If necessary, the feed rate can be appropriately reduced based on the above adjustments to prevent insufficient oxygen supply.

[0077] 5. Modified TB culture medium The basal medium used for fermentation is a modified TB medium, which contains the following components per liter: Glycerin 8 g, tryptone 12 g, yeast extract 24 g, K2HPO4 12.54 g and KH2PO4 2.31 g.

[0078] The amount of glycerol was reduced from 12 g / L in conventional TB medium to 8 g / L in order to reduce the carbon source concentration in the early stage of fermentation, control the initial growth rate of the cells, and reduce the accumulation of metabolic byproducts such as acetic acid.

[0079] K2HPO4 and KH2PO4 constitute a phosphate buffer system, which is used to buffer pH changes caused by metabolic acid production during bacterial growth.

[0080] One mL of 1000× micronutrient stock solution is also added to each liter of basal culture medium. The concentrations of each component in the 1000× micronutrient stock solution are as follows: FeSO4·7H2O 0.5 g / L, CaCl2·2H2O 0.2 g / L, MnSO4·H2O 0.1 g / L, ZnSO4·7H2O 0.1 g / L, CoCl2·6H2O 0.02 g / L, Na2MoO4·2H2O 0.02 g / L, CuCl2·2H2O 0.01 g / L, and H3BO3 0.01 g / L.

[0081] The trace element storage solution was sterilized by filtration through a 0.22 μm filter membrane and then stored at 4 ℃ in the dark.

[0082] 0.3 mL of Antifoam 204 defoamer is also added to each liter of culture medium. If foam appears during fermentation, an appropriate amount of defoamer can be added as needed.

[0083] All components of the basal culture medium, except for the micronutrient stock solution, antifoaming agent, and kanamycin, were autoclaved at 121 °C for 20 min. After sterilization, the medium was cooled to room temperature, and the filtered and sterilized micronutrient stock solution and antifoaming agent were added under aseptic conditions. Kanamycin was added before inoculation to a final concentration of 50 μg / mL.

[0084] The feed solution was formulated as follows: 500 g / L glycerol, 100 g / L tryptone, 50 g / L yeast extract, and 10 g / L MgSO4·7H2O. Glycerol was sterilized separately at 115 °C for 15 min; the remaining components, including tryptone, yeast extract, and MgSO4·7H2O, were sterilized at 121 °C for 20 min. After cooling, all components were aseptically mixed to obtain the feed solution.

[0085] 6. Fermentation Results Fermentation results showed that enzyme activity gradually increased after induction, reaching a peak of 8342 U / L after 27 hours of fermentation. At this time, the concentration of the novel thiotransferase EGT-ST4 protein reached 5.8 g / L, and the cell density OD... 600 It reached 148.2.

[0086] The results showed that the recombinant Escherichia coli could achieve high-level expression of the novel thiotransferase EGT-ST4 (with TrxA) under high-density fermentation conditions, and could be used as an enzyme source or cell catalyst for the preparation of ergothionein.

[0087] Table 1. Variation of fermentation process parameters of recombinant Escherichia coli in a 5 L fermenter (n=3 batches, mean ± SD)

[0088] illustrate: (1) Induction time point: As shown in the table above, the actual OD during induction 600 The value is 22.1, not the exact 20.0. This is because the OD increases rapidly during fermentation; in practice, it is measured in "OD". 600 The window for triggering the response is set to "reach the range of 20-25".

[0089] (2) Changes in specific activity: Before induction (0-6 hours), the enzyme activity of the total protein measured was very low due to the near absence of EGT-ST4 expression, so the specific activity data was not meaningful. After induction, EGT-ST4 gradually accumulated, and the specific activity continued to rise, reaching a peak of 1490 U / g total protein at 27 hours.

[0090] (3) OD 600 Empirical formula for conversion between wet and wet weight: Wet bacterial volume (g / L) ≈ 0.36 × OD 600 (R) 2 =0.998), which was obtained by fitting multiple measurements under the experimental conditions.

[0091] (4) The concentration of soluble protein was determined by Bradford method of the supernatant after centrifugation of crude enzyme solution at 12,000 g. The proportion of EGT-ST4 in soluble protein was estimated by SDS-PAGE grayscale scanning.

[0092] Example 3: Preparation of Ergothioneine by Resting Cell Catalysis The following experimental and control groups were set up: Experimental group (EGT-ST4 resting cell group): Recombinant E. coli BL21(DE3) resting cells (OD) expressing EGT-ST4 (pET-28a(+) vector, with TrxA fusion tag, N-terminal His6) were used. 600 =80, 2.5 mL / 10 mL reaction solution), resuspended in permeation buffer containing 0.1% Triton X-100.

[0093] Control group (wild-type EanB resting cells): using cells expressing wild-type EanB... Chlorobium limicola Recombinant E. coli BL21(DE3) resting cells containing EanB enzyme (WT enzyme, NCBI accession number: WP_012467399.1) were constructed using the same method as EGT-ST4 (pET-28a(+) vector, without fusion tag, N-terminal His6), and the same culture and induction conditions (OD). 600 =80, 2.5 mL / 10 mL reaction solution, containing 0.1% Triton X-100).

[0094] The bacterial cells induced in Example 2 were collected by centrifugation, washed twice with HEPES buffer at pH 7.5, and then resuspended in permeabilization buffer containing 0.1% (v / v) Triton X-100 to prepare OD. 600 The resting cell suspension was 80%, and the resting cell suspension (OD) 600 In the 80), the wet weight concentration of the bacterial cells is approximately 28.8 g wet bacterial cells / L suspension.

[0095] Reaction buffer: 50 mM HEPES (pH 7.5). Substrate molar ratio: 10 mL reaction solution, histidine betaine (final concentration 15 g / L, ~76.1 mM), sodium polysulfide (20 mM), 2.5 mL resting cell suspension. Catalyst: OD 600 =80% resting cells. The reaction was carried out under anaerobic conditions at 35 ℃ and 220 rpm for 24 hours. After the reaction, the sample was centrifuged to remove bacterial cells, and the concentration of ergothioneine in the supernatant was determined by HPLC. Figure 4 ).

[0096] HPLC detection conditions: An Agilent 1260 Infinity II liquid chromatography system equipped with a WatersXBridge C18 reversed-phase column (250 mm × 4.6 mm, 5 µm) was used. The mobile phase was 1% methanol-water solution (isocratic elution), the flow rate was 0.7 mL / min, the column temperature was 30 ℃, the detection wavelength was 260 nm, the injection volume was 5 µL, and the run time was 20 min.

[0097] When the concentration of histidine betaine was 15 g / L and the concentration of sodium polysulfide was 20 mM, ergothionein was generated after resting cells catalyzed for 24 hours with a conversion rate of about 90%, indicating that recombinant Escherichia coli expressing the novel thiotransferase EGT-ST4 can be used as a resting cell catalyst for ergothionein preparation.

[0098] Table 2. Ergothioneine (ERG) production in each experimental / control group at different reaction times in Example 3

[0099] Note: (g / L, n=3, mean ± SD). Reaction conditions: 10 mL system, histidine betaine 15 g / L (~76.1 mM), Na2S X 20 mM, 35 ℃, pH 7.5 (50 mM HEPES), anaerobic.

[0100] Example 4: Catalytic preparation of ergothioneine under high substrate concentration conditions The recombinant Escherichia coli cells obtained in Example 2 were prepared as resting cells, or an enzyme solution containing the novel thiotransferase EGT-ST4 was prepared after cell disruption and solid-liquid separation. Under anaerobic conditions, histidine betaine (HER) was used as a substrate, and sodium polysulfide was used as a sulfur donor at 10-20 mM, at 30-35 °C and pH 7.5-8.0.

[0101] Histidine betaine concentrations were set at 13 g / L, 30 g / L, and 45 g / L, respectively. Samples were taken periodically during the reaction, and after inactivation by boiling or removal of cells by centrifugation, the amount of ergothioneine (ERG) generated was detected by HPLC, and the molar conversion rate was calculated.

[0102] The results show that ( Figure 5 When the concentration of histidine betaine was 13 g / L, the molar conversion rate reached 97.8% after 24 hours of catalysis; when the concentration of histidine betaine was 45 g / L, the molar conversion rate reached 92.0% after 36 hours of catalysis, and the ergothioneine product titer reached 48.1 g / L.

[0103] The above results indicate that recombinant Escherichia coli expressing the novel thiotransferase EGT-ST4 and its enzyme source can catalyze the preparation of ergothionein under high substrate concentration conditions.

[0104] Table 3. Comparison of catalytic effects of EGT-ST4 and wild-type WT enzymes under high substrate loading conditions

[0105] Example 5: Isolation of Ergothioneine Products The isolation and purification of ergothionein includes the following steps: Step 1: Solid-liquid separation to remove bacterial cells / cell debris: Take 1 L of the reaction solution (ERG concentration 48.1 g / L) after the reaction of the 45 g / L substrate concentration group in Example 4. If crude enzyme solution is used as catalyst, centrifuge the reaction solution at 4 °C and 8,000 g for 20 minutes to remove cell debris and denatured protein precipitate; if resting cells are used as catalyst, remove bacterial cells by plate and frame filtration (filtration accuracy 0.45 μm, operating pressure 0.2 MPa). Collect approximately 0.95 L of supernatant / filtrate.

[0106] Step 2: Ultrafiltration to remove soluble protein impurities: A tangential flow ultrafiltration system (Millipore Pellicon 2, 10 kDa molecular weight cutoff, membrane area 0.1 m²) was used. 2 (Using regenerated cellulose membrane material), the supernatant obtained in step 1 was concentrated by ultrafiltration at 4 ℃ and a transmembrane pressure of 0.1-0.15 MPa. The concentration was reduced to 1 / 5 of the original volume (approximately 190 mL), and an equal volume of deionized water was added for diafiltration. This process was repeated twice to further remove small molecule impurities and residual salts. The permeate (approximately 570 mL, containing ERG) was collected. The ERG permeation rate was >95% (the 10 kDa membrane had a rejection rate of <1% for ERG with a molecular weight of 229 Da).

[0107] Step 3: Strong acid cation exchange resin chromatography: (3a) Resin and chromatography column preparation: Take a strong acid cation exchange resin, Dowex 50WX8 (H + Type, 200-400 mesh, Sigma-Aldrich Cat# 217506 or equivalent), pretreated as follows: wash with 2 BV of 2 M HCl solution to convert to H₂O. + The sample was washed with deionized water until the pH of the effluent reached 6.0-7.0; then converted to NH4 using 2 BV of 1 M NH4OH solution. + Wash with deionized water until the effluent pH reaches 6.0-7.0. The column packing size is... The column is 3.0 cm × 22 cm, with a bed volume (BV) of approximately 155 mL. The column is equilibrated with 3 BV deionized water before use.

[0108] (3b) Sample loading: Adjust the pH of the ERG permeate obtained in step 2 (approximately 570 mL) to 2.5 ± 0.2 with 6 M HCl (ERG carries a positive charge under acidic conditions and can bind to the sulfonic acid groups of the cation exchange resin), and load the sample at a flow rate of 2 BV / h (approximately 5 mL / min). The sample loading volume is approximately 3.7 BV.

[0109] (3c) Washing: After loading the sample, wash the chromatography column with 3 BV of deionized water at a flow rate of 2 BV / h to remove unadsorbed inorganic salts (from the reaction buffer and neutralization step), residual histidine betaine and other nonionic impurities. Monitor the absorbance of the wash effluent at 260 nm. After A260 drops to the baseline, proceed to the elution step.

[0110] (3d) Gradient elution: Linear gradient elution was performed using 1 M ammonia solution (NH4OH, containing 0.2 M NH4Cl as an ionic strength buffer). The gradient program was: 0–0.5 M ammonia solution (containing 0–0.1 M NH4Cl), 3 BV, for pre-elution to remove weakly adsorbed impurities; 0.5–1.0 M ammonia solution (containing 0.1–0.2 M NH4Cl), 5 BV, for elution of the target product ergothioneine. The elution flow rate was 1.5 BV / h (approximately 4 mL / min). The eluent was collected in 0.2 BV units, approximately 30 mL per tube, for a total of approximately 40 tubes.

[0111] (3e) Component pooling: The ergothioneine content in each collection tube was determined by HPLC. Components with ERG purity ≥95% (HPLC peak area normalization method, 260 nm) were pooled, usually tubes 18-28 (corresponding to the 0.55-0.85 M ammonia concentration range), with a pooling volume of approximately 330 mL.

[0112] Step 4: Reduced pressure concentration: The combined ERG components were concentrated to 1 / 5-1 / 8 of their original volume by rotary evaporation (using a Buchi R-300 rotary evaporator) under a 45 ℃ water bath and a vacuum of 0.08 MPa (the endpoint was determined by the appearance of a small amount of white crystals in the concentrate). The ERG concentration of the concentrate was approximately 220-260 g / L, and the total volume was approximately 55-65 mL.

[0113] Step 5, Crystallization: Slowly add 3 times the volume of the concentrate of anhydrous ethanol (AR grade, ≥99.7%, Sigma-Aldrich Cat# 459844) to the concentrate while stirring (magnetic stirring at 100 rpm) until the final ethanol concentration is approximately 75% (v / v). The specific solvent is anhydrous ethanol (Ethanol absolute, CAS No.: 64-17-5). After stirring and mixing thoroughly, transfer the solution to a sealed, clean glass crystallizing dish. First, let it stand at room temperature (20-25 ℃) for 2 hours to allow crystal nuclei to form slowly, then transfer it to a 4 ℃ cold room and let it crystallize for 12-16 hours (overnight).

[0114] Step 6, Crystal Collection and Drying: Use a Buchner funnel (Whatman No. 1 filter paper). White crystals were collected by vacuum filtration (9 cm). The crystals were washed 2-3 times with pre-cooled anhydrous ethanol (4 ℃, 15-20 mL each time) to remove residual mother liquor from the crystal surface. The crystals were transferred to a clean petri dish and dried in a vacuum drying oven (0.08 MPa) at 40 ℃ for 6-8 hours until constant weight was obtained to obtain pure ergothioneine.

[0115] The dried ERG crystals were dissolved in deionized water (1 mg / mL) and detected using the HPLC method described in Example 3. The detection wavelength was 260 nm, and the purity was calculated using the peak area normalization method.

[0116] The results showed that the HPLC purity of ERG crystals was 99.5% ± 0.1% (n=3), and the retention time of the main peak (RT = 3.5 ± 0.02 min) was consistent with that of the ERG standard. Within the detection limit (signal-to-noise ratio S / N=3, approximately 0.01 μg), no histidine betaine, polysulfide decomposition products, or other unknown impurity peaks were detected.

[0117] The above results indicate that the ergothioneine prepared by the recombinant Escherichia coli catalysis can be obtained through conventional solid-liquid separation, ion exchange, concentration and crystallization, and is suitable for subsequent scale-up preparation.

[0118] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0119] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0120] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A thiotransferase EGT-ST4, characterized in that, Its amino acid sequence is shown in SEQ ID NO:1, or it is a protein that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:1 and has the activity of catalyzing the conversion of histidine betaine to ergothionein.

2. A biomaterial related to the thiotransferase EGT-ST4 of claim 1, characterized in that, The biomaterial is at least one of the following: A1) A nucleic acid molecule encoding the thiotransferase EGT-ST4 of claim 1; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant expression vector containing the nucleic acid molecule described in A1) or a recombinant expression vector containing the expression cassette described in A2); A4) A recombinant host cell containing the nucleic acid molecule described in A1), a genetically engineered host cell containing the expression cassette described in A2), or a recombinant host cell containing the recombinant expression vector described in A3).

3. The biomaterial according to claim 2, characterized in that, The nucleic acid molecule contains the nucleotide sequence shown in SEQ ID NO:

2.

4. The biomaterial according to claim 2 or 3, characterized in that, The recombinant host cell is recombinant Escherichia coli, preferably Escherichia coli BL21(DE3).

5. The biomaterial according to claim 2, characterized in that, The nucleic acid molecule is fused with the coding sequence of a fusion tag, wherein the fusion tag is selected from one or more of TrxA, MBP, SKP, or HaloTag; the fusion tag is coupled to the thiotransferase EGT-ST4 via inclusion (GGGGS). n Flexible linker peptides are used for linking, where n is 1, 2, 3, 4 or 5.

6. A method for preparing the thiotransferase EGT-ST4 according to claim 1, characterized in that, include: The recombinant host cell in claim 2 (A4), or the recombinant host cell containing the corresponding nucleic acid construct in claim 4 or 5, is fermented and cultured, and the expression of the thiotransferase EGT-ST4 is induced.

7. The use of the thiotransferase EGT-ST4 of claim 1, or the biomaterial of any one of claims 2-5, in the catalytic generation of ergothionein.

8. A method for preparing ergothioneine, characterized in that, include: The catalyst was mixed with histidine betaine and inorganic polysulfides to form a reaction system. After the reaction was completed, ergothionein was obtained by separation and purification. The catalyst is selected from at least one of the following: purified enzyme including the thiotransferase EGT-ST4 of claim 1, crude enzyme solution, cell lysate, whole cell catalyst, and resting cell catalyst.

9. The method according to claim 8, characterized in that, The catalyst is a whole-cell catalyst, a resting cell catalyst, or an enzyme solution obtained by cell disruption; the resting cell catalyst is prepared by the following method: collecting induced expression bacterial cells, washing with buffer, and resuspending them in a permeation buffer containing 0.01%-0.5% (v / v) Triton X-100 to prepare OD. 600 It is a resting cell suspension of 50-100.

10. The method according to claim 8 or 9, characterized in that, The final concentration of histidine betaine in the reaction system is 13-45 g / L; the inorganic polysulfide is sodium polysulfide; the reaction temperature is 30-35 ℃, the reaction pH is 7.0-9.0, preferably 7.5-8.0, and the reaction time is 24-36 hours.

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