Method for efficiently synthesizing ergothioneine by biological enzyme method
By screening and optimizing ergothioneine synthase, constructing a truncated enzyme and optimizing reaction conditions, the problems of feedback inhibition and low transport efficiency in the ergothioneine synthesis process of bio-fermentation were solved, and efficient ergothioneine synthesis was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-04
- Publication Date
- 2026-03-13
AI Technical Summary
In the existing bio-fermentation method for synthesizing ergothioneine, the ergothioneine accumulated in cells exerts feedback inhibition on the synthase, resulting in low product transport efficiency, and the host's endogenous metabolism competes for carbon sources and energy.
Ergothionein synthases from different species were screened, their gene codons were optimized and recombinant expression was performed, and truncated ergothionein synthases were constructed. In vitro enzymatic catalysis was carried out with hexamethasone as a substrate, and the reaction conditions were optimized to improve the synthesis efficiency.
It significantly increased the yield of ergothioneine, reaching 12 g/L within 8 hours and a maximum of 51.5 g/L in a 5L fermenter within 10 hours, solving the problems of feedback inhibition and low translocation efficiency.
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Figure CN121653091A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for the efficient synthesis of ergothioneine using a bioenzymatic approach, belonging to the field of synthetic biology. Background Technology
[0002] Ergothioneine (EGT) is a naturally occurring amino acid, first isolated from ergot fungi in 1909. Its structure was determined in 1911, revealing it to be a histidine betaine derivative. Ergothioneine is a natural sulfur-containing amino acid and a powerful natural antioxidant. Compared to other thiols such as glutathione, ergothioneine exhibits higher thermal stability, better pH stability, and is less prone to spontaneous oxidation. Due to its potent antioxidant properties and various biological functions, including anti-inflammatory, anti-aging, and oxidative stress prevention, it is widely used in food, cosmetics, and pharmaceuticals. In nature, it is primarily synthesized by specific bacteria and fungi and cannot be synthesized by plants, animals, or humans.
[0003] Currently, ergothioneine can be prepared through chemical synthesis and bio-fermentation. Chemical synthesis requires sophisticated processes for synthesis and purification. Compared to chemical synthesis and natural biological extraction, bio-fermentation offers advantages such as lower cost and readily available raw materials. However, it has a longer fermentation cycle, and microbial fermentation demands significant modification of the strain's metabolic pathways. This requires knocking out genes affecting metabolites to enhance the expression of genes involved in the synthetic pathway. Furthermore, some genes are difficult to knock out and may even severely impact strain growth, necessitating a substantial time investment in the initial stages of strain construction.
[0004] The biosynthesis of ergothioneine has been extensively elucidated in various microorganisms, with the two most widely studied biosynthetic pathways being the bacterial pathway, particularly that of Mycobacterium smegmatis (…). Mycobacterium smegmatis The synthesis of ergothionein is catalyzed by five ergothionein synthases: EgtA, EgtB, EgtC, EgtD, and EgtE. In this synthetic pathway, EgtD, acting as a histidine methyltransferase, transfers three methyl groups from SAM to L-histidine (L-His) to form hescisine (HER); simultaneously, L-cysteine (L-Cys) and L-glutamate (L-Glu) provide γ-glutamylcysteine (γ-GC) under the catalysis of EgtA; subsequently, iron(II)-dependent EgtB catalyzes the formation of hescisyl-γ-glutamylcysteine sulfoxide (γ-GC-HER) from γ-GC and HER; then, the amide hydrolase EgtC removes L-Glu, producing hescisylcysteine sulfoxide (Cys-HER); finally, the CS lyase EgtE, dependent on pyridoxal 5-phosphate (PLP), catalyzes the formation of ergothionein.
[0005] The fungal route is mainly carried out by Neurospora crassa (… Neurospora crassaEgt1 is a bifunctional enzyme that catalyzes two consecutive reactions: L-histidine (L-His) reacts with S-adenosylmethionine (SAM) to form hydatidine (HER), which then reacts with L-cysteine (L-Cys) and oxygen under the action of Egt1 to synthesize hydatid cysteine sulfoxide (Cys-HER). Egt2 is a pyridoxal phosphate-dependent CS lyase that catalyzes the conversion of Cys-HER to EGT. Current literature reports the preparation of ergothioneine via biofermentation, where fed-batch fermentation in a 5-liter fermenter resulted in a final EGT titer of 7.2 g / L after 96 hours of fermentation.
[0006] The current challenges in microbial fermentation synthesis of ergothioneine are as follows: First, ergothioneine synthesis requires three amino acids—histidine, methionine, and cysteine—as precursors, and the synthesis process requires a large amount of S-adenosylmethionine (SAM) as a methyl donor; second, intracellular accumulation of ergothioneine may exert feedback inhibition on ergothioneine synthase; and finally, the efficiency of transporting the generated ergothioneine product out of the cell is low, and the host's endogenous metabolism competes for carbon and energy during the synthesis process. Summary of the Invention
[0007] To address the shortcomings of the prior art, this invention provides a method for the efficient synthesis of ergothioneine using a bioenzymatic approach. The aim is to solve the technical problems in the prior art, such as the feedback inhibition of ergothioneine synthase by intracellular accumulation, the low efficiency of transporting the generated ergothioneine product out of the cell, and the competition for carbon and energy by endogenous metabolism in the host during the synthesis process.
[0008] The first technical solution provided by the present invention is a truncated ergothionein synthase, the amino acid sequence of which is shown in SEQ ID NO.6.
[0009] The second technical solution provided by the present invention is to encode the gene for the truncated ergothionein synthase described in the first technical solution.
[0010] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.
[0011] The fourth technical solution provided by the present invention is to express the truncated ergothionein synthase described in the first technical solution, or to contain the gene described in the second technical solution, or to transform recombinant cells into the recombinant vector described in the third technical solution.
[0012] In some embodiments, the recombinant cells use Escherichia coli as a host.
[0013] The fifth technical solution provided by the present invention is a method for improving the catalytic activity of ergothioneine synthase, wherein the method involves truncating the ergothioneine synthase parent with the amino acid sequence shown in SEQ ID NO.3 to obtain a truncated ergothioneine synthase with the amino acid sequence shown in SEQ ID NO.6.
[0014] The sixth technical solution provided by the present invention is a method for synthesizing ergothionein, wherein the method uses ergothionein synthase Cfegt1 with an amino acid sequence as shown in SEQ ID NO. 3 or the truncated ergothionein synthase described in the first technical solution, and ergothionein synthase Ncegt2 with an amino acid sequence as shown in SEQ ID NO. 2 as catalysts to convert hexamethasone into ergothionein.
[0015] In some embodiments, 1 mM ascorbic acid, 2 mM DTT, 1 mM PLP, 1 mM TCEP, 0.2 mM ferrous sulfate heptahydrate, 20 g / L HER and an initial concentration of 2 g / L cysteine are added to the crude enzyme solution containing the catalyst, and 2 g / L cysteine is added every half hour to carry out the catalytic reaction at a temperature of 30°C.
[0016] In some embodiments, 1 mM ascorbic acid, 10 mM DTT, 1 mM PLP, 1 mM TCEP, 0.2 mM ferrous sulfate heptahydrate, and 0.1 g BSA are added to the crude enzyme solution of the catalyst. The substrate HER is added at a flow rate of 12 g / L / h, and the cysteine solution is added at a flow rate of 15 g / L / h. The pH is maintained at 7 and the reaction temperature is 30°C during the reaction.
[0017] The seventh technical solution provided by the present invention is the application of ergothioneine synthase, the truncated ergothioneine synthase described in the first technical solution, the gene described in the second technical solution, the recombinant vector described in the third technical solution, and the recombinant cells described in the fourth technical solution in the preparation of ergothioneine or ergothioneine-containing products. The amino acid sequence of the ergothioneine synthase is shown in SEQ ID NO.3.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention screens ergothionein synthases from different species and selects... Cfegt1 , Fgegt1 and Vaegt1 After codon optimization, recombinant expression was performed to obtain a crude enzyme solution, which catalyzes the synthesis of ergothioneine. This ergothioneine synthase... Cf The reaction of egt1 and ergothioneine synthase Egt2 for 8 hours resulted in an ergothioneine yield of 12 g / L. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) CfBy truncating egt1, a higher yield of ergothioneine is achieved, reaching 15 g / L after 8 hours of reaction. In a 5 L fermenter, a maximum yield of 51.5 g / L of ergothioneine was obtained after 10 hours of catalytic reaction. Attached Figure Description
[0019] Figure 1 This is a schematic diagram of the synthesis pathway of ergothionein in this invention.
[0020] Figure 2 This is a diagram of the PBAD-Ncegt1-Ncegt2 carrier of the present invention.
[0021] Figure 3 This is a diagram of the PBAD-Cfegt1-Ncegt2 carrier of the present invention.
[0022] Figure 4 This is a diagram of the PBAD-Fgegt1-Ncegt2 carrier of the present invention.
[0023] Figure 5 This is a diagram of the PBAD-Vaegt1-Ncegt2 carrier of the present invention.
[0024] Figure 6 A comparison of different ergothionein synthases synthesizing ergothionein.
[0025] Figure 7 A comparison of ergothionein synthesis between ergothionein synthases Cfegt1 and Tcfegt1.
[0026] Figure 8 To detect protein expression using polyacrylamide gel electrophoresis.
[0027] Figure 9 It is a standard of 350 mg / L ergothioneine.
[0028] Figure 10 The content of ergothionein synthesized by enzymatic reaction. Detailed Implementation
[0029] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0030] Test method: Ergothioneine detection method: Liquid chromatography samples were prepared by diluting with 70% acetonitrile solution, and ergothioneine standards were dissolved in 70% acetonitrile solution. An Agilent HPLC system (Agilent 1200 Infinity Series 1260, Agilent Technologies) and an Agilent ZORBAX NH2 column (4.6 × 250 mm, 5 μm) were used. The mobile phase was acetonitrile / deionized water (70:30, v / v) at a flow rate of 1.0 mL / min. The ergothioneine absorption peak was observed at 254 nm.
[0031] Raw materials used in the examples: 1. pBAD / HisA plasmid is from Thermo Fisher Scientific.
[0032] 2. The source of Escherichia coli BW25113 is Thermo Fisher Scientific.
[0033] 3. LB medium: 5g yeast extract, 10g peptone, 10g sodium chloride.
[0034] Example 1: Construction of Escherichia coli expression vector refer to Figure 1 The synthetic pathway of the fungus *Neurospora crassa* was determined, and a literature review was conducted. Neurospora crassa (Source) Ncegt1 , Ncegt2 Ergothioneine synthase has a wide range of applications; therefore, two genes from this species were used as controls. Enzyme activity comparisons were performed in a database using intelligent computing software to screen ergothioneine synthases from different species and select [the appropriate synthases]. Cfegt1 , Fgegt1 and Vaegt1 Gene synthesis was performed after codon optimization. The amino acid sequences of Ncegt1 protein are shown in SEQ ID NO. 1, Ncegt2 protein in SEQ ID NO. 2, Cfegt1 protein in SEQ ID NO. 3, Fgegt1 protein in SEQ ID NO. 4, and Vaegt1 protein in SEQ ID NO. 5. The nucleotide sequences of the corresponding encoding genes are SEQ ID NO. 7-11, respectively.
[0035] Using pBAD / HisA plasmid (purchased from Thermo Fisher Scientific) as a template, and pBAD-F and pBAD-R as primers, the vector fragment was amplified. Ncegt1 Using the gene as a template, the fragment was amplified using primers F1 and R1. Ncegt1 To synthesize Ncegt2Using the gene as a template, and F2 and R2 as primers, fragments were amplified. Ncegt2 Synthetic Cfegt1 Using the gene as a template, primers F3 and R3 were used for amplification to obtain the fragment. Cfegt1 .synthesis Fgegt1 Using the gene as a template, and F4 and R4 as primers, fragments were amplified. Fgegt1 .synthesis Vaegt1 Using the gene as a template, and F5 and R5 as primers, fragments were amplified. Vaegt1 The plasmid pBAD- for synthesizing ergothioneine was constructed by ligating three fragments using the Novizan Recombinant Cloning Kit (CloneMultiS One Step Cloning Kit, (C113-02)). Ncegt1 - Ncegt2 pBAD- Cfegt1 - Ncegt2 pBAD- Fgegt1 - Ncegt2 pBAD- Vaegt1 - Ncegt2 (See Figures 2-5 The cells were transformed into competent Trans1-T1 (Phage Chemically Comptent Cell) cells using chemical transformation, and positive clones were selected to extract plasmids for sequencing.
[0036] Table 1 Primers and Sequences
[0037] Example 2: Transformation and Induced Expression of Ergothionein Synthesis Plasmid To prepare electrocompetent cells, 50 µL of BW25113 glycerol bacteria were inoculated into 5 mL of antibiotic-free LB medium and cultured overnight. The cells were then transferred to 100 mL of antibiotic-free LB medium at a 1% inoculation rate and cultured at 37°C until OD500. 600=0.6, pre-cool on ice for 20 min, centrifuge the bacterial culture at 4000 rpm for 10 min, discard the supernatant, resuspend the cells in pre-cooled 10% glycerol, centrifuge at 4000 rpm for 10 min, and wash the cells twice with 10% glycerol. Resuspend the cells in 1 mL of pre-cooled 10% glycerol and aliquot into sterile EP tubes (100 µL per tube). Take five tubes of competent cells, add 1 µL of the plasmid constructed in Example 1, add the above system to a 2 mm electroporation cuvette, electroporate at 2.5 kV, quickly add 900 µL of antibiotic-free LB medium, resuspend, transfer to sterile EP tubes, incubate at 37°C, 200 rpm for 60 min, centrifuge at 4000 rpm for 5 min, discard part of the supernatant, resuspend, plate on LB agar containing 100 mg / L ampicillin, incubate upside down at 37°C overnight, pick single clones for PCR identification, and use the correct strains for subsequent ergothioneine synthesis.
[0038] Single colonies were inoculated into 5 mL of LB broth containing Amp (100 mg / L) resistance and cultured overnight at 37°C and 220 rpm. The bacterial culture obtained in the previous step was then transferred at a 1% inoculation rate to 200 mL of LB broth containing Amp (100 mg / L) resistance and cultured at 37°C and 220 rpm on a shaker. 600 When the OD value reaches 0.6-0.8, add arabinose to a final concentration of 2 g / L and induce at 30℃ for 16 h to reach OD. 600 4. Centrifuge the fermentation broth at 7000 rpm for 15 min at 4°C, discard the supernatant, and resuspend the bacterial cells in 40 mL of 50 mM HEPES buffer (pH 7.5) until the OD of the bacterial culture is reached. 600 The value is 20. After resuspending the bacterial solution, the solution is broken up by low-temperature ultrasonication. When the bacterial solution is clear, the supernatant is collected by low-temperature centrifugation. The supernatant is the crude enzyme solution.
[0039] Example 3: Synthesis of Ergothioneine via Crude Enzyme Reaction The limiting factors in ergothioneine synthesis are the requirement of histidine, methionine, and cysteine as precursors. Excessive cysteine concentration can be toxic to cells. While cysteine is highly soluble in acidic conditions, its sulfhydryl group is easily oxidized by atmospheric oxygen under neutral or weakly alkaline conditions, gradually converting into disulfide-bonded cysteine. Furthermore, the synthesis process requires a large amount of S-adenosylmethionine (SAM) as a methyl donor, consuming significant amounts of ATP. Therefore, the enzymatic synthesis of ergothioneine in this invention directly uses herbicides (HER) as a substrate in an in vitro enzymatic reaction. Since cysteine is easily oxidized under neutral conditions, it is added intermittently using a freshly prepared substrate.
[0040] The crude enzyme solution obtained in Example 2 was used for in vitro catalytic production of ergothioneine. The 5 mL crude enzyme reaction system (based on final concentration) contained: 1 mM ascorbic acid, 2 mM DTT, 1 mM PLP, 1 mM TCEP, 0.2 mM ferrous sulfate heptahydrate, 20 g / L HER, and an initial concentration of 2 g / L cysteine, with 2 g / L cysteine added every half hour. The reaction was carried out at 30°C. Ergothioneine content was determined at different time points. Ergothioneine was efficiently synthesized under the catalysis of ergothioneine synthase Egt1 and ergothioneine synthase Egt2. Figure 6 The results showed that the ergothionein production plasmid pBAD- constructed in this application... Cfegt1 - Ncegt2 The highest concentration of ergothionein was observed during catalytic synthesis, reaching 12 g / L after 8 hours of reaction.
[0041] Example 4: Truncated ergothionein synthase Cfegt1 Egt1 is a bifunctional enzyme with two catalytic domains: an S-adenosylmethionine (SAM)-dependent methyltransferase domain and a sulfoxide synthase domain, catalyzing a two-step reaction. In this invention, the enzyme-catalyzed reaction uses HER as a substrate; therefore, Egt1 is truncated, retaining only the domain with sulfoxide synthase activity, to obtain the truncated fragment Tcfegt1. Using the pBAD / HisA plasmid as a template and pBAD-F and pBAD-R as primers, the vector fragment is amplified. The synthesized... Ncegt2 Using the gene as a template, and F2 and R2 as primers, fragments were amplified. Ncegt2 Synthetic Cfegt1 Using the gene as a template, fragment T was amplified using primers F6 and R6. cfegt1 The plasmid for synthesizing ergothioneine was constructed by ligating three fragments using the Novozymes Recombinant Cloning Kit (CloneMultiS One Step Cloning Kit, (C113-02)). The plasmid was then transformed into Trans1-T1 (Phage Chemically Comptent Cell) competent cells using chemical transformation, and positive clones were selected for plasmid extraction and sequencing.
[0042] Table 2 Primers and Sequences
[0043] The constructed plasmid was transformed into BW25113 competent cells, and crude enzyme solution was prepared according to the method in Example 2. The prepared crude enzyme solution was then tested according to the system in Example 3, reacting at 30°C. The ergothionein content was determined at different time points. Figure 7 The results show that the truncated Tc fegt1 The production of ergothionein is higher, reaching 15 g / L after 8 hours of reaction.
[0044] Example 5: Expression of ergothioneine synthase protein Algothioneine synthase single-gene plasmid was constructed using pBAD / HisA plasmid as a template and pBAD-F and pBAD-R primers for amplification of the vector fragment. Ncegt2 Using the gene as a template, and F7 and R7 as primers, fragments were amplified. Ncegt2 Synthetic Cfegt1 Using the gene as a template, primers F8 and R8 were used for amplification to obtain fragment T. cfegt1 The two fragments were ligated using the Novizan Recombinant Cloning Kit (CloneMultiS One Step Cloning Kit, (C112-02)) to construct the plasmid pBAD-T. cfegt1 and pBAD- Ncegt2 The truncated amino acid sequence of Tcfegt1 is shown in SEQ ID NO. 6. The cells were transformed into competent Trans1-T1 (Phage Chemically Comptent Cell) cells using chemical transformation, and positive clones were selected for plasmid extraction and sequencing.
[0045] Table 3 Primers and Sequences
[0046] The constructed plasmid pBAD-T cfegt1 and pBAD- Ncegt2 Transformed into BW25113 competent cells, single colonies were picked from the plate and inoculated into 5 ml of LB broth containing Amp (100 mg / L) resistance, and cultured overnight at 37°C with shaking. The bacterial culture from the previous step was then transferred at a 1% inoculation rate to 1 L of LB broth containing Amp (100 mg / L) resistance, and cultured at 37°C with shaking until OD (outcome limit) was reached. 600 When the pH reaches 0.6-0.8, add arabinose to a final concentration of 2 g / L and induce at 30°C for 16 h. Centrifuge the fermentation broth at 4°C and 5000 rpm for 15 min, discard the supernatant, and resuspend the cells in 20 mL of 50 mM PBS buffer (pH 7.5) until the OD value of the bacterial culture is reached. 600 The concentration was 200. The resuspended bacterial culture was then disrupted by low-temperature ultrasonication, and the supernatant was collected by low-temperature centrifugation.
[0047] Pack 5 mL of Ni-NTA medium into the chromatography column and allow it to settle naturally. Rinse the column twice with 5-10 column volumes of deionized water. Equilibrate the column three times with 5-10 column volumes of PBS buffer. Add 10 mL of supernatant to the equilibrated nickel column and pass it through the column twice at a slow flow rate. Elute the target protein using elution buffer containing 250 mM imidazole. Collect the eluent in a clean test tube and perform SDS-PAGE protein electrophoresis on the collected eluent. Figure 8 ).
[0048] Example 6 Enzyme-catalyzed synthesis of ergothionein in a 5 L fermenter Following the crude enzyme solution preparation method in Example 2, 1.5 L of crude enzyme solutions of Tcfegt1 and Ncegt2 were prepared respectively. The following were added to the crude enzyme solutions (based on final concentrations): 1 mM ascorbic acid, 10 mM DTT, 1 mM PLP, 1 mM TCEP, 0.2 mM ferrous sulfate heptahydrate, and 0.1 g BSA. The substrate HER was added at a flow rate of 12 g / L / h, and cysteine solution was added at a flow rate of 15 g / L / h. The pH was maintained at 7 and the reaction temperature at 30°C during the reaction. Ergothionein was detected in the reaction solution at different time points. The detection results are shown below. Figures 9-10 , Figure 9 This is a standard sample of ergothioneine at 350 mg / L. Figure 10 The enzyme-catalyzed reaction produced a maximum of 51.5 g / L ergothioneine in 10 hours.
[0049] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A truncated ergothionein synthase, characterized in that, The amino acid sequence of the truncated ergothionein synthase is shown in SEQ ID NO.
6.
2. The gene encoding the truncated ergothionein synthase as described in claim 1.
3. A recombinant vector carrying the gene of claim 2.
4. Recombinant cells expressing the truncated ergothionein synthase of claim 1, or containing the gene of claim 2, or transformed with the recombinant vector of claim 3.
5. The recombinant cell according to claim 4, characterized in that, The recombinant cells used Escherichia coli as the host.
6. A method for improving the catalytic activity of ergothionein synthase, characterized in that, The method involves truncating the ergothioneine synthase parent with the amino acid sequence shown in SEQ ID NO.3 to obtain the truncated ergothioneine synthase with the amino acid sequence shown in SEQ ID NO.
6.
7. A method for synthesizing ergothioneine, characterized in that, The method involves using ergothionein synthase Cfegt1 (amino acid sequence as shown in SEQ ID NO. 3) or the truncated ergothionein synthase as described in claim 1, and ergothionein synthase Ncegt2 (amino acid sequence as shown in SEQ ID NO. 2) as catalysts to convert hexamethasone into ergothionein.
8. The method according to claim 7, characterized in that, 1 mM ascorbic acid, 2 mM DTT, 1 mM PLP, 1 mM MTCEP, 0.2 mM ferrous sulfate heptahydrate, 20 g / L HER, and an initial concentration of 2 g / L cysteine were added to the crude enzyme solution containing the catalyst. 2 g / L cysteine was added every half hour to catalyze the reaction.
9. The method according to claim 7, characterized in that, Add 1 mM ascorbic acid, 10 mM DTT, 1 mM PLP, 1 mM TCEP, 0.2 mM ferrous sulfate heptahydrate, and 0.1 g BSA to the crude enzyme solution of the catalyst. Add the substrate HER at a flow rate of 12 g / L / h and the cysteine solution at a flow rate of 15 g / L / h. Maintain the pH at 7 and the reaction temperature at 30°C during the reaction.
10. The use of ergothioneine synthase, the truncated ergothioneine synthase of claim 1, the gene of claim 2, the recombinant vector of claim 3, and the recombinant cell of any one of claims 4-5 in the preparation of ergothioneine or ergothioneine-containing products, characterized in that, The amino acid sequence of the ergothionein synthase is shown in SEQ ID NO.3.