Lipomyces starkeyi strain for synthesis of ergothioneine and use thereof

Abstract

This invention discloses a *Trichoderma parareesei* strain for synthesizing ergothioneine and its applications, belonging to the fields of genetic engineering and bioengineering technology. This invention achieves heterologous ergothioneine synthesis by expressing *TrEGT1* and *TrEGT2* derived from *Trichoderma parareesei* in *Trichoderma parareesei*. By screening different promoters driving *TrEGT1* and *TrEGT2*, the optimal ergothioneine synthesis gene combination was obtained. Further, multiple copies of the gene combination were integrated, and the synthesis of precursor histidine was enhanced to improve the ergothioneine synthesis capacity of *Trichoderma parareesei*, achieving a shake-flask fermentation yield of 1200±12.3 mg / L and a 5L fermentation tank yield of 8.6 g / L. This invention integrates a heterologous metabolic pathway into *Trichoderma parareesei*, achieving high-efficiency ergothioneine production and facilitating low-cost, large-scale production of ergothioneine and its products.

Description

Technical Field

[0001] This invention relates to the *Yamylostella lipolytica* strain for synthesizing ergothioneine and its applications, belonging to the fields of genetic engineering and bioengineering technology. Background Technology

[0002] Ergothioneine (ERG) is a thiol-containing histidine derivative that protects cells from oxidative damage caused by excessive reactive oxygen species (ROS). ERG is primarily found in its thione tautomer under physiological pH conditions and possesses a relatively high reduction potential (-60 mV), making it more stable and resistant to auto-oxidation compared to other thiol-containing antioxidants such as glutathione. As a powerful antioxidant and cell protectant, ERG is believed to have beneficial effects on human health, such as anti-inflammatory, anti-aging, antidepressant properties, and the ability to protect against UV damage. Recently, ERG has been evaluated as an additive ingredient in food and cosmetics, which will increase market demand and the exploration of ERG production methods. ERG is ubiquitous in higher organisms, such as plants and mammals; however, it is only biosynthesized by bacteria and fungi, such as cyanobacteria, actinomycetes, and basidiomycetes (mushrooms). For humans, mushrooms are the primary dietary source of ERG. Studies have shown that consuming mushrooms can reduce the risk of cognitive dysfunction and cancer. Due to its antioxidant properties, ERG is presumably the compound responsible for this effect. In a transgenic Caenorhabditis elegans model of Alzheimer's disease with accumulation of human β-amyloid protein, ERG has been shown to improve health and lifespan.

[0003] Therefore, ERG is a leading candidate for new dietary supplements for healthy aging. Currently, only chemically synthesized ERG is available on the market. However, toxic byproducts from the chemical synthesis of vitamins or nutritional supplements raise consumer safety concerns, and the stringent purification process increases production costs. In contrast, the microbial fermentation method for synthesizing ergothioneine offers greater advantages in terms of environmental friendliness.

[0004] Ergothioneine is biosynthesized from its precursors histidine, cysteine, and S-adenosylmethionine. In the fungal pathway, histidine is methylated three times by Egt1 to form histidine trimethyl inner salt. Subsequently, the Egt1 enzyme ligates cysteine ​​to generate cyclohexylcysteine ​​sulfoxide, which is then dissociated from the intermediate by the cyclohexylcysteine ​​sulfoxide lyase Egt2. Finally, sulfur reduction produces ERG. The bacterial synthesis of ergothioneine is more complex. A disadvantage of the bacterial pathway is the use of five enzymes instead of two, and ATP is used to form intermediates from other pathways, including glutamate, which must be cleaved in subsequent steps. Furthermore, *Escherichia coli*, as a pathogenic microorganism, cannot be used in the production of food, pharmaceuticals, and related fields. De novo synthesis of ergothioneine has been achieved by expressing the ergothioneine biosynthetic pathway in a microbial host, but the yield is low, making industrial-scale production difficult. As a typical unconventional oil-producing yeast, Yarrowia lipolytica is an internationally recognized safe and reliable microbial production host. It has complete genetic manipulation tools and a powerful reduction system, making it an ideal host for ergothioneine production. Summary of the Invention

[0005] This invention provides a lipophilic yeast engineered strain for synthesizing ergothionein, which expresses ergothionein synthesis protein 1 TrEGT1 derived from Trichoderma parareesei and cyclohexylcysteine ​​sulfoxide lyase TrEGT2 derived from Trichoderma parareesei in the starting strain.

[0006] In one embodiment, the amino acid sequence of the ergothionein synthetic protein 1 is shown in SEQ ID NO.1; and the amino acid sequence of the cyclohexylcysteine ​​sulfoxide lyase is shown in SEQ ID NO.2.

[0007] In one embodiment, the starting strain is Yarrowia lipolytica Po1f, as disclosed in the paper "Combining 26srDNA and the Cre-loxP System for Iterative Gene Integration and EfficientMarker Curation in Yarrowia lipolytica".

[0008] In one embodiment, the *Yamyces lipolytica* strain is *Yamyces lipolytica* Po1f, *Yamyces lipolytica* E129, *Yamyces lipolytica* E150, *Yamyces lipolytica* Po1d, *Yamyces lipolytica* Po1g, or *Yamyces lipolytica* Po1h.

[0009] In one implementation, TrEGT1 and TrEGT2 are expressed either by free expression via plasmids or by integration into the genome.

[0010] In one embodiment, the TrEGT1 and TrEGT2 genes are expressed using pYlxp as an expression vector. In another embodiment, the TrEGT1 and TrEGT2 genes are integrated into the genome.

[0011] In one implementation, the TrEGT1 and TrEGT2 are integrated into the genome in multiple copies, with a copy number ≥ 3.

[0012] In one implementation, TrEGT1 and TrEGT2 are integrated into the 26S rDNA site of the genome.

[0013] In one embodiment, the promoters regulating TrEGT1 and TrEGT2 include, but are not limited to, the strong promoter TEFin or the strong promoter Hp4d; the nucleotide sequence of the strong promoter TEFin is shown in SEQ ID NO.5; the nucleotide sequence of the strong promoter Hp4d is shown in SEQ ID NO.6.

[0014] In one implementation, TrEGT1 is started with the strong starter TEFin, and TrEGT2 is started with the strong starter Hp4d.

[0015] In one embodiment, the nucleotide sequence encoding TrEGT1 is shown in SEQ ID NO.3, and the nucleotide sequence encoding TrEGT2 is shown in SEQ ID NO.4.

[0016] In one embodiment, the *Yarrowia lipolytica* also integrates the histidine synthase gene YALI0_A15950g, the nucleotide sequence of which is shown in SEQ ID NO.7.

[0017] The present invention also provides the application of the engineered *Yamylostella lipolytica* strain in the production of ergothioneine.

[0018] In one embodiment, the bacterial culture that has reached the logarithmic growth phase is inoculated into YPD medium, and the OD reaches 0.6-0.8 after inoculation. The culture is then fermented at 28-30°C and 200-220 rpm.

[0019] In one embodiment, the culture medium used for seed culture is YPD medium, containing: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and natural pH.

[0020] In one embodiment, the bacterial culture that has reached the logarithmic growth phase is inoculated into YPD medium, and the OD reaches 0.6-0.8 after inoculation. The culture is then fermented at 28-30°C and 200-220 rpm.

[0021] In one embodiment, the fermentation is carried out in a fermentation tank with a capacity of 5L or more, and the fermentation time is ≥148h.

[0022] In one embodiment, the fermentation process also includes feeding; the feeding is the addition of glucose to bring the glucose concentration in the fermentation system to 0.5–1 g / L.

[0023] This invention also claims protection for the recombinant strain, or the method of producing ergothioneine, or the method of synthesizing ergothioneine using Yeastia lipolytica, in the production of food, medicine, and chemical products.

[0024] Beneficial Effects: This invention utilizes *Trichoderma parareesei* PO1f as the host, expressing the ergothioneine synthesis protein 1 TrEGT1 from *Trichoderma parareesei* and the cyclohexylcysteine ​​sulfoxide lyase TrEGT2 from *Trichoderma parareesei*, thus constructing a sufficiently efficient *Trichoderma parareesei* strain capable of ergothioneine synthesis. Further, by screening different promoters to drive TrEGT1 and TrEGT2, integrating multiple copies of the combined genes TrEGT1 and TrEGT2, and enhancing the precursor histidine, the ergothioneine synthesis capacity of *Trichoderma parareesei* was improved, achieving a shake-flask yield of 1200±12.3 mg / L and a 5L fermenter yield of 8.6±0.13 g / L. This invention integrates a heterologous metabolic pathway into *Trichoderma parareesei*, achieving high ergothioneine production and laying the foundation for subsequent low-cost ergothioneine synthesis. Attached Figure Description

[0025] Figure 1 A metabolic diagram illustrating the heterologous synthesis of ergothionein in Yeast Extract.

[0026] Figure 2 Chromatogram (A) and mass spectrum (B) of ergothionein produced by the engineered strain under YPD culture.

[0027] Figure 3 To produce ergothioneine in a 5L fermenter. Detailed Implementation

[0028] (I) Culture medium

[0029] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride. Add 20 g / L agar powder to prepare LB solid medium.

[0030] YNB medium: Yeast Nutrition Base 67.4 g / L, glucose 20 g / L, amino acids (appropriate amino acids may be omitted as needed; specifically: uracil is added to strains carrying the leucine selection tag, leucine is added to strains carrying the uracil selection tag, and no amino acids are needed for strains carrying both tags; the concentration is 5 g / L uracil and 10 g / L leucine).

[0031] YPD medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L.

[0032] (II) Preparation of competent cells of *Yamylostella lipolytica*: Competent cells of *Yamylostella lipolytica* were prepared using the Frozen-EZ Yeast Transformation II kit. The cells were cultured at 30°C in 10 mL of YPD medium to a medium order of 100 mg / L (OD). 600 =0.8-1.0). The following steps are performed at room temperature.

[0033] 1. Centrifuge the cells at 3500 rpm for 5 minutes and aspirate the supernatant;

[0034] 2. Add 10 mL of EZ1 solution to wash the precipitate, centrifuge the precipitate cells again, and remove the supernatant;

[0035] 3. Add 1 mL of EZ2 solution to resuspend the precipitated cells.

[0036] (III) Transformation of Yeast Extract:

[0037] 1. Mix 50 μL of competent cells with 0.2–1 μg of DNA (less than 5 μL); add 500 μL of EZ3 solution and mix thoroughly.

[0038] 2. Incubate at 30℃ for 45 minutes, vortexing gently 2-3 times during incubation to mix thoroughly;

[0039] 3. Take 50-150 μL from the conversion mixture and transfer it to an appropriate auxotrophic plate;

[0040] 4. Transformants were grown after incubation on a plate at 30℃ for 3 days.

[0041] (iv) Extraction of ergothionein: Take 1 ml of fermentation broth and mix it thoroughly with an equal volume of methanol solution, centrifuge to collect the supernatant, and pass it through a membrane for liquid phase detection.

[0042] (V) Ergothionein HPLC determination: The determination was performed using Shimadzu high-performance liquid chromatography. HPLC conditions: Column: InertSustain C18 250mm×4.6mm column (particle size 5μm); Mobile phase A: ultrapure water containing 1‰ trifluoroacetic acid; Mobile phase B: acetonitrile containing 1‰ trifluoroacetic acid; Mobile phase: A 10%, B 90%; Detection time: 15min; Detector wavelength: 254nm.

[0043] (vi) Yarrowia lipolytica Po1f, Yarrowia lipolytica E129, Yarrowia lipolytica E150, Yarrowia lipolytica Po1d, Yarrowia lipolytica Po1g, and Yarrowia lipolytica Po1h are all strains obtained by the inventors' team through legal means and have been published in the paper "Synthetic biology tools for engineering Yarrowia lipolytica".

[0044] Information on the remaining strains is shown in Table 1.

[0045] Table 1. Strains involved in this invention

[0046]

[0047] Example 1: Construction of a recombinant lipolytic yeast strain producing ergothioneine

[0048] Using the synthesized TrEGT1 sequence (nucleotide sequence shown in SEQ ID NO.1) as a template, the TrEGT1 fragment was amplified by PCR using primer pair F1 / R1. Using the vector pYlxp (published in the paper "Combining 26s rDNA and the Cre-loxPSystem for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica") as a template, PCR amplification was performed using primer pair FG / RG, and the product was purified. The TrEGT1 fragment and the pYlxp vector backbone were recombined using the Gibson assembly method to obtain a recombinant vector. This recombinant vector was transformed into E. coli JM109, plasmids were extracted, and sequencing verification confirmed the correct recombinant vector pYlxp-PTEFin-TrEGT1-TXPR2. Using the synthesized TrEGT2 sequence (nucleotide sequence shown in SEQ ID NO.2) as a template, the TrEGT2 fragment was amplified by PCR using primer pair F2 / R2. Using the vector pYlxp as a template, PCR amplification was performed using primer pair FG2 / RG2, and the product was purified to obtain the vector backbone. The promoter Hp4d shown in SEQ ID NO.4 was synthesized. The fragments TrEGT1, Hp4d, and the vector pYlxp backbone were recombined using the Gibson assembly method to obtain a recombinant vector. The recombinant vector was transformed into *E. coli* JM109, plasmids were extracted, and sequencing was performed to verify the correct recombinant vector pYlxp-Hp4d-TrEGT2-TXPR2.

[0049] Primers F3 / R3 and F4 / R4 were designed to amplify the fragments p-PTEFin-TrEGT1-TXPR2 and pYlxp-Hp4d-TrEGT2-TXPR2 using vectors pYlxp-PTEFin-TrEGT1-TXPR2 and p-Hp4d-TrEGT2-TXPR2, respectively. Primers FG3 / RG3 were designed to amplify the backbone fragment p-pYlxp using vector pYlxp as a template. The fragments p-PTEFin-TrEGT1-TXPR2 and p-Hp4d-TrEGT2-TXPR2 were recombined with the p-pYlxp backbone using Gibson assembly to obtain a recombinant vector. This recombinant vector was transformed into *E. coli* JM109, and plasmids were extracted and sequenced for verification, confirming the correct recombinant vector pYlxp-PTEFin-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2.

[0050] The obtained recombinant vector pYlxp-PTEFin-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2 was transformed into *Yamylostella lipolytica* Po1f, *Yamylostella lipolytica* E129, *Yamylostella lipolytica* E150, *Yamylostella lipolytica* Po1d, *Yamylostella lipolytica* Po1g, and *Yamylostella lipolytica* Po1h, respectively, to obtain strains ST1603-Po1f, ST1603-E129, ST1603-E150, ST1603-Po1d, ST1603-Po1g, and ST1603-Po1h. The engineered strains were cultured in seed culture medium for 16-18 hours to obtain seed solutions, which were then inoculated into YPD medium. After inoculation, the OD reached 0.6-0.8, and fermentation was carried out at 30℃ and 220 rpm for 96 hours. Liquid chromatography confirmed that different *Yamylostella lipolyticis* strains could produce ergothioneine, and the yields are shown in Table 3. Among them, the engineered strain ST1603-Po1f produced the highest amount of ergothioneine, with an ergothioneine content of 183±6.4 mg / L in the fermentation broth of ST1603-Po1f. ST1603-Po1f will be used as the initial strain for further modification.

[0051] All primer sequences are listed in Table 2.

[0052] Table 2 Primer sequences

[0053]

[0054]

[0055] Table 3 Ergothionein yield in different *Aylocereus lipolyticus* species

[0056]

[0057] Example 2: Multi-copy integration increases ergothioneine yield

[0058] To improve ergothioneine yield and expression stability, the ergothioneine synthesis pathway was integrated into the 26S rDNA site of the genome (SEQ ID NO. 8). Primers F5 / R5 and F6 / R6 were designed to amplify the upstream and downstream homologous arms of the rDNA in the genome of *Yamaceae lipolytica* PO1f, obtaining fragment urDNA and drDNA. Primers F7 / R7 and F8 / R8 were designed to amplify the promoter pleu and tag URA fragment from the genome. Primers F9 / R9 were designed to amplify the fragment p2Ylxp-PTEFin-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2 from the vector Ylxp-PTEFin-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2. Using the Gibson assembly method, fragments urDNA, drDNA, pleu, and URA were recombined with p2Ylxp-PTEFin-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2 and the pYlxp vector backbone, respectively, to obtain the correct recombinant vector prDNAYlxp-Pleu-URA-Txpr2-Ylxp-PTEFin-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2. The recombinant vector was linearized and introduced into Po1f to obtain the integrated strain ST1606 (rDNAYlxp-Pleu-URA-Txpr2-Ylxp-PTEFin-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2). qPCR verification showed that the copy number of the integrated PTEFI-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2 was 3. Strain ST1606 was cultured in seed culture medium for 16-18 h, and then the seed culture was inoculated into YPD medium until the OD reached 0.6-0.8. Fermentation was carried out at 30℃ and 220 rpm for 96 h. The ergothioneine content in the ST1606 fermentation broth was measured to be 298±6.3 mg / L.

[0059] All primer sequences are listed in Table 3.

[0060] Table 4 Primer sequences

[0061]

[0062]

[0063] Example 3: Enhancing the supply of precursor histidine to increase ergothionein production

[0064] To further increase ergothionein production, it is necessary to enhance the supply of histidine precursors. Primers F10 / R10 were designed to amplify the histidine synthase YlHis (Gene ID: 2905745) from the genome of *Agropyron lipolytica* PO1f. Using the vector pYlxp as a template, PCR amplification was performed with primers FG / RG, and the product was purified. The YlHis fragment and the pYlxp vector backbone were recombined using Gibson assembly to obtain a recombinant vector. This recombinant vector was transformed into *E. coli* JM109, and plasmids were extracted and sequenced for verification, yielding the correct recombinant vector pYlxp-PTEFin-YlHis-TXPR2. Primers F11 / R11 and F12 / R12 were designed, and the fragments PTEFI-YlHis-TXPR2 and rDNA Ylxp-Pleu-URA-Txpr2-Ylxp-PTEFin-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2 were amplified using pYlxp-PTEFin-YlHis-TXPR2 and rDNA Ylxp-Pleu-URA-Txpr2-Ylxp-PTEFin-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2, respectively. The fragment vector T1-TXPR2-Hp4d-TrEGT2-TXPR2 was recombined with the pYlxp backbone using the Gibson assembly method to obtain a recombinant vector. This recombinant vector was transformed into *E. coli* JM109, and plasmids were extracted and sequenced for verification, yielding the correct recombinant vector prDNAYlxp-Pleu-URA-Txpr2-Ylxp-PTEFin-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2-PTEFin-YlHis-TXPR2. The recombinant vector was linearized and introduced into ST1606 to obtain the integrated strain ST1608 (rDNAYlxp-Pleu-URA-Txpr2-Ylxp-PTEFin-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2-PTEFin-YlHis-TXPR2). Strain ST1608 was cultured in seed culture medium for 16-18 hours, and then the seed culture was inoculated into YPD medium to achieve an OD of 0.6-0.8. Fermentation was then carried out at 30℃ and 220 rpm for 96 hours. The ergothioneine content in the ST1606 fermentation broth was measured to be 1200±12.3 mg / L.

[0065] All primer sequences are listed in Table 4.

[0066] Table 5 Primer sequences

[0067]

[0068] Example 4: Horizontal fermentation of ergothionein in a 5L fermenter

[0069] The engineered *Yacinthia lipolytica* strain ST1608 constructed in Example 3 was streaked onto YPD solid medium plates using an inoculation loop and cultured at 30°C for 2-3 days to obtain an activated engineered strain. The activated strain was then inoculated into 50mL / 250mL YPD medium for primary seed culture, and cultured at 220rpm, 30°C, and shake-flask at natural pH for 16-24 hours to obtain the OD value. 600 The primary seed culture was prepared at a concentration of 5-8. Then, the primary seed culture was inoculated at a rate of 5% into 1L shake flasks containing 200mL of YPD medium for secondary seed culture at 30℃ and 220rpm for 16-24 hours to obtain OD. 600 The secondary seed culture was prepared at pH 5-8 with a natural pH. Then, 8% of the secondary seed culture was inoculated into a 5L fermenter containing 2.5L of fermentation medium. The initial aeration rate was 3.5L / min, the initial stirring speed was 300rpm, and the culture temperature was 30℃. The initial pH was controlled at 6 using 8mol / L KOH. When the initial dissolved oxygen level dropped to 20%, it was maintained at 20% by adjusting the stirring speed (300-1200rpm) and air flow rate (3.5-10L / min). When the glucose level dropped to 0.5g / L-1g / L, 800g / L glucose was added to maintain the residual glucose concentration at 0.5-1g / L. Ergothionein was measured periodically.

[0070] like Figure 3 As shown, from hour 1 to 10, the cell growth is in the lag phase, with slow cell growth. From hour 11 to 48, the cell growth is in the logarithmic growth phase, during which the cell growth is rapid. From hour 48 to 108, the cell growth slows down. Finally, after 168 hours of fermentation, the OD... 600 The concentration of ergothionein reached 180±2.35, and the yield of ergothionein reached 8.6±0.13 g / L.

[0071] Comparative Example 1

[0072] The specific implementation method is the same as in Example 1, except that pathways were constructed using NcEGT1 (SEQ ID NO. 9) from Neurosporacrassa and CpEGT2 (SEQ ID NO. 10) from Claviceps purpurea, respectively. Vectors Ylxp-PTEFin-NcEGT1-TXPR2-PTEFin-CpEGT2-TXPR2, Ylxp-PTEFin-TrEGT1-TXPR2-PTEFin-CpEGT2-TXPR2, and Ylxp-PTEFin-NcEGT1-TXPR2-PTEFin-CpEGT2-TXPR2 were constructed. Sufficient vectors were introduced into *Yoloxacum mongolicum* Po1f according to the method in Example 1 to obtain engineered strains. The engineered strains were cultured in seed culture medium for 16-18 h to obtain seed liquid, which was then inoculated into YPD medium. After inoculation, the OD reached 0.6-0.8, and fermentation was carried out at 30°C and 220 rpm for 96 h. Liquid chromatography confirmed that *Aylocereus lipolyticus* can produce ergothioneine, as shown in Table 6.

[0073] Table 6. Ergothionein yield of recombinant bacteria expressing different genes.

[0074]

[0075] Comparative Example 2

[0076] The specific implementation method is the same as in Example 1, except that different combined vectors are constructed using the promoters TEFin (SEQ ID NO. 5) and Hp4d (SEQ ID NO. 6) with TrEGT1 and TrEGT2: Ylxp-PTEFin-TrEGT1-TXPR2-PTEFin-TrEGT2-TXPR2, Ylxp-Hp4d-TrEGT1-TXPR2-PTEFin-TrEGT2-TXPR2, and Ylxp-Hp4d-TrEGT1-TXPR2-Hp4d-TrEGT2-TXPR2. The recombinant plasmids are then introduced into *Yolopsis lipolyticus* Po1f to obtain engineered bacteria. The engineered strain is cultured in seed culture medium for 16-18 hours to obtain seed liquid, which is then inoculated into YPD medium. After inoculation, the OD reaches 0.6-0.8, and fermentation is carried out at 30°C and 220 rpm for 96 hours. Liquid chromatography confirmed that *Yamylostella lipolytica* can produce ergothioneine, as shown in Table 7.

[0077] Table 7. Effects of different promoters on ergothionein yield

[0078]

[0079] 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 *Yamylostella lipolytica* strain for synthesizing ergothioneine, characterized in that, Using *Agropyron lipolytica* Po1f as the starting strain, expression was derived from... Trichoderma parareesei Ergothionein 1 TrEGT1 And expresses the origin of Trichoderma parareesei Cyclohexylcysteine ​​sulfoxide lyase TrEGT2 The amino acid sequence of the ergothionein synthesis protein 1 is shown in SEQ ID NO.1; the amino acid sequence of the cyclohexylcysteine ​​sulfoxide lyase is shown in SEQ ID NO.2; TrEGT1 and TrEGT2 Multiple copies are integrated into the genome; regulation TrEGT1 and TrEGT2 The promoters include the strong promoter TEFin or the strong promoter Hp4d; the nucleotide sequence of the strong promoter TEFin is shown in SEQ ID NO.5; the nucleotide sequence of the strong promoter Hp4d is shown in SEQ ID NO.6; the engineered *Yamylostella lipolytica* strain also integrates and expresses the histidine synthase gene. YlHis The gene YlHis The nucleotide sequence is shown in Gene ID: 2905745.

2. A method for producing ergothioneine, characterized in that, The engineered *Yamylostella lipolytica* strain described in claim 1 was cultured in a fermentation medium at 28-30°C for a period of time.

3. The method according to claim 2, characterized in that, Feed is also added during the fermentation process.

4. The method according to claim 3, characterized in that, The feed is glucose, which is added to bring the glucose concentration in the fermentation system to 0.5~1 g / L.

5. The use of the engineered *Yamylostella lipolytica* strain according to claim 1, or the method according to any one of claims 2 to 4, in the production of products containing ergothioneine.

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