Yarrowia lipolytica strain for synthesizing ergothioneine and application of Yarrowia lipolytica strain

Abstract

The invention discloses a Yarrowia lipolytica strain for synthesizing ergothioneine and application of the Yarrowia lipolytica strain, and belongs to the technical field of genetic engineering and bioengineering. According to the invention, a key enzyme TrEGT1 is subjected to semi-rational transformation, so that the ergothioneine yield of a strain expressing a mutant is increased by 240.9%, precursor metabolic network screening is further modularized, precursor flux is enhanced, a competitive approach of a precursor is knocked out and weakened, and strategies such as fermentation process optimization and the like are further optimized; and when the constructed strain is fermented in a 5L bioreactor, the yield of ergothioneine can reach 10.3 g / L. The highest-level ergothioneine synthesis reported at present is realized, the feasibility of synthesizing the ergothioneine by using the Yarrowia lipolytica as the chassis cell is verified, and a reference value is provided for efficiently synthesizing the ergothioneine by using other chassis cells.

Description

Technical Field

[0001] The invention relates to a Yarrowia lipolytica strain for synthesizing ergothioneine and application thereof, belonging to the technical field of genetic engineering and bioengineering. Background Art

[0002] Ergothioneine (EGT) is a natural rare sulfur-containing histidine derivative with powerful antioxidant, anti-aging, anti-inflammatory and cell protection from ultraviolet damage and other biological functions. Therefore, it is widely used in food, cosmetics and medicine, such as antioxidants, anti-aging ingredients, nutritional supplements, etc.

[0003] At present, the main market source of ergothioneine is extracted from plants such as mushrooms, but this method has many disadvantages: long production cycle, low yield, high cost, and environmental pollution. In contrast, the synthesis of ergothioneine using cell factories is a green and sustainable production method that can effectively replace traditional plant extraction methods. As an unconventional yeast, Yarrowia lipolytica has a wide substrate spectrum and can metabolize a variety of substrates such as glucose, glycerol, lipids and alkanes. The metabolic flexibility of this yeast makes it an ideal host for the synthesis of ergothioneine. In recent years, researchers have made significant progress in constructing a de novo synthesis pathway of ergothioneine in Yarrowia lipolytica through metabolic engineering and gene editing technology. The goal of this study is to construct a de novo synthesis pathway of ergothioneine in Yarrowia lipolytica, and to further increase the yield of ergothioneine through strategies such as systematic optimization of metabolic pathways, strengthening the expression of key enzymes, and improving substrate utilization efficiency, laying a solid foundation for its industrial production. This will not only help reduce production costs, but also meet the market's growing demand for ergothioneine and promote its widespread application in more fields. Summary of the invention

[0004] The invention provides an ergothioneine synthase TrEgt1 mutant, which has mutations of one or more amino acids at positions 492 and 786 on the basis of a parent.

[0005] In one embodiment, the mutant is based on the parent TrEgt1, and the histidine at position 492 is mutated to valine to obtain the mutant H492V.

[0006] In one embodiment, the mutant is obtained by mutating the histidine at position 492 to valine and the tyrosine at position 786 to alanine based on the parent, thereby obtaining the mutant H492V / Y786A.

[0007] In one embodiment, the parent TrEGT1 has an amino acid sequence as shown in Genbank Accession No.: XP_006968620.1.

[0008] In one embodiment, the nucleotide sequence encoding the parent is shown as SEQ ID NO.1.

[0009] The present invention also provides a gene encoding the mutant.

[0010] The invention also provides an expression cassette containing the gene.

[0011] In one embodiment, the expression cassette has a "P TEF -TrEGT1 Y786A / H492V -T XPR2 -P HP4D -TrEGT2-T XPR2 "Shown structure.

[0012] In one embodiment, the promoter P TEF The nucleotide sequence of the promoter is shown in SEQ ID NO.14; HP4D The nucleotide sequence is shown in SEQ ID NO.16.

[0013] The present invention also provides a recombinant microorganism expressing the mutant.

[0014] The present invention also provides an engineered Yarrowia lipolytica strain with high ergothioneine production, which expresses TrEGT1 and TrEGT2, or a mutant of TrEGT1 and TrEGT2, from Trichoderma reesei on the basis of a starting strain.

[0015] In one embodiment, the overexpression includes but is not limited to integrating the gene encoding TrEgt1 or a mutant thereof into the AXP site and / or the D17 site.

[0016] In one embodiment, the promoter P TEF Expression and P HP4D The ergothioneine pathway genes are expressed and synthesized; the ergothioneine pathway genes include TrEGT1 and TrEGT2, or a TrEGT1 mutant and TrEGT2.

[0017] In one embodiment, the engineered strain further enhances the expression of a sulfur assimilation module; the genes of the sulfur assimilation module include but are not limited to one or more of genes MET2, MET3, MET5, MET10, MET14, and MET16.

[0018] In one embodiment, the nucleotide sequence of gene MET2 is shown as SEQ ID NO.3; the nucleotide sequence of gene MET3 is shown as SEQ ID NO.4; the nucleotide sequence of gene MET5 is shown as SEQ ID NO.5; the nucleotide sequence of gene MET10 is shown as SEQ ID NO.6; the nucleotide sequence of gene MET14 is shown as SEQ ID NO.7; and the nucleotide sequence of gene MET16 is shown as SEQ ID NO.8.

[0019] In one embodiment, the engineered strain also has enhanced supply of the precursor amino acids histidine, cysteine ​​and / or methionine.

[0020] In one embodiment, the strengthening of the supply of precursor amino acids comprises expressing one or more of the following genes: SAM2, MET6, CYS3, CYS4, and HIS1, a gene having feedback inhibition on the regulation of histidine metabolism.

[0021] In one embodiment, the genes SAM2, MET6, CYS3, CYS4, and HIS1 are respectively integrated at the E3 site.

[0022] In one embodiment, the genes MET6 and HIS1 are integrated together at the E3 site.

[0023] In one embodiment, the genes MET6 and CYS3 are also integrated together at the E3 site.

[0024] In one embodiment, the genes MET6 and HIS1 are integrated together at the E3 site, and the gene CYS3 is integrated at the E4 site.

[0025] In one embodiment, the engineered Yarrowia lipolytica strain also has a gene SPE2 for degrading the precursor amino acid SAM or a gene STR2 for degrading Cys knocked out.

[0026] In one embodiment, the starting strain is Yarrowia lipolytica ΔKu70; the Yarrowia lipolytica ΔKu70 is disclosed in the paper "Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica.".

[0027] The present invention also provides the application of the Yarrowia lipolytica engineered bacteria in producing ergothioneine.

[0028] In one embodiment, the engineered Yarrowia lipolytica is fermented in YPD medium at 30° C. for 120 to 144 hours.

[0029] In one embodiment, the engineered bacteria of Yarrowia lipolytica are inoculated into a fermentation medium, fermented at 25-30° C. for 168 hours, and the dissolved oxygen level is controlled at 15-20%; the pH is controlled at 5.0±0.2 during the fermentation process; and glucose is fed when the glucose is lower than 1 g / L to keep the glucose concentration at 0.1-1 g / L.

[0030] In one embodiment, an inorganic salt medium is also fed; the inorganic salt medium contains: 7.5 g / L (NH 4 ) 2 SO 4 ,14.4g / L KH 2 PO 4 ,0.5g / L MgSO 4 7H 2 O.

[0031] In one embodiment, the fermentation is for at least 144 hours.

[0032] The present invention also claims to protect the application of the engineered Yarrowia lipolytica in producing ergothioneine-containing products in the fields of food, medicine and chemical industry.

[0033] Beneficial effects:

[0034] 1. The present invention screens exogenous ergothioneine synthesis genes in lipolytic yeast and screens promoter genes, constructs a P TEF Promoter driven expression of TrEGT1, and in P HP4D The strain expressing TrEGT2 driven by the promoter had an EGT production of 81.2 mg / L.

[0035] 2. The present invention increases the ergothioneine production of the strain expressing the mutant H492V / Y786A by 240.9% by semi-rational transformation of the key enzyme TrEgt1.

[0036] 3. The present invention further integrates the transformed TrEGT1 into the yeast genome, and by strengthening the sulphur assimilation module and enhancing the supply of precursor amino acids, and knocking out the SPE2 consuming the precursor amino acid SAM, promotes the conversion of the precursor amino acid to thioneine, further improves the yield of thioneine, and makes the strain constructed reach 516.6mg / L of thioneine after 24-well plate fermentation 144h.

[0037] 4. The bacterial strain constructed by the present invention is fermented in a 5L fermentor tank, and the thioneine output of 10.6g / L can be obtained, which is the highest thioneine level reported so far to be synthesized de novo by microorganisms, and has industrial application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the metabolism of heterologous ergothioneine synthesis in Yarrowia lipolytica.

[0039] Figure 2 To screen exogenous ergothioneine synthase and optimize promoter production.

[0040] Figure 3 This is a map of ergothioneine production after semirational modification of ergothioneine synthase and site-directed integration.

[0041] Figure 4 Figure 2. Ergothioneine production of engineered strains after strengthening the sulfur assimilation module.

[0042] Figure 5 Figure 1. Ergothioneine production of engineered strains after enhancing precursor amino acid flux.

[0043] Figure 6 The graph shows the ergothioneine production in a 5 L fermenter using fed-batch culture. DETAILED DESCRIPTION

[0044] (I) Culture medium

[0045] LB medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L. Add 20g / L agar powder to prepare LB solid medium.

[0046] YNB medium: Yeast Nutrition Base 67.4 g / L, glucose 20 g / L, amino acids (5 g / L uracil, 10 g / L tryptophan, 10 g / L leucine, 10 g / L histidine, and appropriate deletion of corresponding amino acids as needed).

[0047] YPD medium: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L.

[0048] Inorganic salt medium: 7.5 g / L (NH 4 ) 2 SO 4 ,14.4g / L KH 2 PO 4 ,0.5g / L MgSO 4 7H 2 O.

[0049] (II) Preparation of competent cells of Y. lipolytica: The competent cells of Y. lipolytica were prepared using the Frozen-EZYeast Transformation II kit and cultured with 5 mL YPD medium at 30°C until the OD 600 =0.8-1.0). The following steps were carried out at room temperature.

[0050] 1. Centrifuge the cells at 3500 rpm for 5 min and remove the supernatant;

[0051] 2. Add 10 mL of EZ1 solution to wash the precipitate, centrifuge the precipitated cells again, and aspirate the supernatant;

[0052] 3. Add 1 mL of EZ2 solution to resuspend the pelleted cells.

[0053] (III) Transformation of Yarrowia lipolytica:

[0054] 1. Scrape the yeast colony from the YPD plate and inoculate it into YPD liquid medium, then place it in a constant temperature incubator at 30°C and grow for 16-22 hours;

[0055] 2. Prepare yeast transformation buffer solution (if multiple transformations are required, magnification × n): 50% PEG3350, added in a volume of 90 μL; 2M lithium acetate (M CHCOOLi 65.99), volume 5 μL; ssDNA, volume 5 μL, ssDNA needs to be boiled in water for 3 min before adding to the transformation buffer, and then put on ice for cooling;

[0056] 3. Use a pipette to draw 500 μL of bacterial solution into a centrifuge tube, centrifuge the tube at an appropriate speed, and discard the supernatant to obtain yeast cells. Transfer the yeast to a centrifuge tube containing 100 μL of transformation buffer, slowly blow and mix, add 0.25-0.5 μg of plasmid DNA or linear DNA, mix thoroughly and oscillate for 2 minutes (add at least 0.25-0.5 μg of DNA to each plasmid);

[0057] 4. Incubate the centrifuge tube containing the transformation mixture in a metal bath at 30°C for 30-45 min, oscillate the mixture for 15 s every 10 min, and then heat shock at 39°C for an additional 10 min to improve transformation efficiency;

[0058] 5. Transfer the centrifuge tube to a centrifuge and centrifuge at 8000 rpm for 2 minutes. Pour out the supernatant in a sterile clean bench, add an appropriate amount of sterile water to resuspend the bacteria, and add it to the YPD solid medium. Use a sterile coating rod to evenly spread on the YPD solid medium, and then place the medium in a constant temperature incubator at 30°C for cultivation.

[0059] (four) HPLC determination of thioneine: with variable wavelength detector and Agilent ZORBAX Nikpase XDB-C18 chromatographic column, adopt SHIMADZU LC-20AT high performance liquid chromatography system to analyze thioneine containing sample. During HPLC analysis, mobile phase A is 0.1% trifluoroacetic acid, and mobile phase B is methanol containing 0.1% trifluoroacetic acid. Flow velocity is 1mL / min, and detection wavelength is 254nm (tryptophan detection wavelength=220nm), injection volume is 10 μL, and column temperature is 30 ℃. Elution gradient is 0-15min, 1% B; 15-18 minutes, 1-60% B; 18-23 minutes, 60-60% B; 23-26 minutes, 60-1% B; 26-30 minutes, 1% B.

[0060] (V) The strain information is shown in Table 1.

[0061] Table 1 Strains involved in the present invention

[0062]

[0063]

[0064]

[0065] (VI) Integration sites involved in specific implementations:

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] Example 1: Construction of the synthesis pathway of ergothioneine in Yarrowia lipolytica

[0074] To produce EGT in Y. lipolytica, known EGT synthases from fungi were screened, including NcEgt1 from Neurospora crassa (N. crassa) (Genbank accession number: XP_956324.3), NcEgt1 (Genbank accession number: XP_001728131.1), CpEgt1 from C. purpurea (Genbank accession number: CCE33591.1), CpE gt2 (Genbank accession number: CCE33140.1) and TrEgt1 (Genbank accession number: XP_006968620) and TrEgt2 (Genbank accession number: XP_006968735) from Trichoderma reesei (T. reesei) were used to explore the effects of promoters of different strengths on the expression of EGT synthase, and some promoters commonly used to express genes in Y. lipolytica were screened, such as pTEF (nucleotide sequence as shown in SEQ ID NO.14), pTDH (nucleotide sequence as shown in SEQ ID NO.15) and pHP4D (nucleotide sequence as shown in SEQ ID NO.16). All heterologous genes were synthesized by the company to obtain plasmid templates containing the target genes, among which the nucleotide sequence of gene TrEGT1 is shown in SEQ ID NO.1; the nucleotide sequence of gene TrEGT2 is shown in SEQ ID NO.2.

[0075] Taking strain ST1601 as an example, the construction method is as follows:

[0076] First, the primers for heterologous genes were designed using Snapgene software, and the target genes were amplified using primers TrEGT1-F / TrEGT1-R and TrEGT2-F / TrEGT2-R to obtain TrEgt1 and TrEgt2 gene fragments; the target genes were integrated into pHR-AXP-hrGFP (Addgene Plasmid #84613), and the target gene fragments with homology arms of the AXP plasmid were amplified using primers AXP-BF / AXP-BR to obtain the integrated fragments. During the yeast transformation process, the transformed strains were obtained by adding the PCR amplified fragments and the Cas9 plasmids of the corresponding sites. The constructed engineered bacteria were named ST1601 to ST1613.

[0077] The engineered bacteria ST1601 to ST1613 were fermented in YPD medium at 30°C and 220 rpm for 144 h, and the fermentation broth was extracted and identified by liquid phase. Figure 2 As shown, the engineered strains constructed in this embodiment all achieved the synthesis of ergothioneine, and the strain ST1612 expressed by TrEGT1 driven by promoter pTEF and TrEGT2 driven by promoter pHP4D had the highest EGT titer of 81.2 mg / L, which was 44.6% higher than that of ST1601 strain.

[0078] Table 2 Primers and sequences

[0079]

[0080] Example 2: Semi-rational transformation of the key enzyme TrEgt1

[0081] (1) In order to improve the activity of the key enzyme TrEgt1 and promote the effective conversion of histidine to ergothioneine, the key enzyme TrEGT1 was semi-rationally modified. In order to infer the interaction between the enzyme and the substrate, the enzyme and the substrate were docked and analyzed. And further through molecular docking, it was clarified that the catalytic pocket is composed of residues such as TYR, HIS, LEU, ASN, GLN, etc. Alanine scanning was performed on 18 residues within 5 angstroms of the substrate pocket. In order to obtain the TrEgt1 mutant gene, the H492A mutant was taken as an example: the original sequence of TrEgt1 was used as the starting sequence, and H492-F / H492-R primers were designed respectively to introduce alanine mutation at the H492 site.

[0082] According to the method of Example 1, on the basis of the ST1612 strain, a fragment containing the TrEgt1 mutant gene was integrated at the D17 site and the strain expressing the mutant was fermented according to the method of Example 1, and the fermentation conditions were the same as in Example 1. The results showed that the first round of mutation results showed that the production of thioneine of the integrated expression of H492A and Y786A mutants was 146 mg / L and 135 mg / L, respectively, which was 80.6% and 66.6% higher than the production of wild-type thioneine synthase TrEgt1 (81 mg / L). Figure 3 ).

[0083] (2) The second round of mutation was based on the results of the first round of mutation. A492 and A786 were selected for site-directed mutagenesis. A492 and A786 were further mutated to other hydrophobic residues with smaller side chains (glycine G, valine V or isoleucine I) to facilitate the substrate to enter the catalytic pocket. Taking the A492V mutant as an example, the specific steps were as follows: using the H492A-TrEgt1 sequence constructed above as the starting sequence, the A492-F / A492-R primers were designed respectively, and the valine mutation was introduced at position 492 of H492A to construct the mutant A492V.

[0084] The constructed bacterial strain was fermented according to the method of Example 1, wherein, the accumulation of bacterial strain thioneine expressing A492V mutant reached 158mg / L, which was improved by 95.1% compared to wild-type TrEGT1; Subsequently, starting from A492V-TrEGT1, Y786 was mutated to the hydrophobic residue (alanine A, glycine G, valine V, isoleucine I) with smaller side chain, and it was found that the TrEGT1 mutant expressing with Y786A / A492V mutations could increase the bacterial strain thioneine output to 195mg / L, which was improved by 140.7% and 23.4% compared to wild-type TrEGT1 and A492V-TrEGT1, respectively. The Y786A-A492V-TrEGT1 mutant screened was site-integrated at D17 site on ST1612 strain genome with reference to the method of step (1), and the strain was fermented according to the method of Example 1. It was found that after one round of site-integration, output was significantly improved, and the accumulation of ergothioneine reached 220mg / L ( Figure 3 ).

[0085] The mutant gene was integrated again at the A3 site, but the accumulation of ergothioneine did not continue to increase significantly, indicating that ergothioneine synthase may have met the capacity for intracellular synthesis.

[0086] Table 3 Primers and sequences

[0087]

[0088] Example 3: Enhancement of sulfur assimilation module

[0089] In order to promote the supply of precursor sulfur-containing amino acids, the endogenous genes MET2 (nucleotide sequence as shown in SEQ ID NO.3), MET3 (nucleotide sequence as shown in SEQ ID NO.4), MET5 (nucleotide sequence as shown in SEQ ID NO.5), MET10 (nucleotide sequence as shown in SEQ ID NO.6), MET14 (nucleotide sequence as shown in SEQ ID NO.7), and MET16 (nucleotide sequence as shown in SEQ ID NO.8) of Yarrowia lipolytica are overexpressed to further increase the production of ergothioneine.

[0090] The specific steps are: using primers MET2-F / MET2-R to amplify the gene MET2 from the Yarrowia lipolytica genome, using primers MET3-F / MET3-R to amplify the gene MET3 from the Yarrowia lipolytica genome, using primers MET5-F / MET5-R to amplify the gene MET5 from the Yarrowia lipolytica genome, using primers MET10-F / MET10-R to amplify the gene MET10 from the Yarrowia lipolytica genome, using primers MET14-F / MET14-R to amplify the gene MET14 from the Yarrowia lipolytica genome, using primers MET16-F / MET16-R to amplify the gene MET16 from the Yarrowia lipolytica genome, and constructing them in the A3 plasmid. Using primers A3-BF / A3-BR to amplify the target fragments containing the upstream and downstream homology arms of the A3 site. The integration fragment and about 600ng of sgRNA were transformed into the Yeast Transformation Kit Frozen-EZ Yeast Transformation II into the Yeast lipolytica engineering strain, spread on the screening solid medium, and cultured at 30°C for 3 days until colonies appeared. The obtained engineering strains ST1614 to ST1626 were fermented in YPD medium at 30°C and 220rpm for 144h, and the fermentation broth was extracted and analyzed by HPLC. The results showed that the production of ergothioneine in the recombinant engineering strain ST1625 could reach 348.7mg / L ( Figure 4 ).

[0091] Table 4 Primer sequences

[0092]

[0093] Example 4: Strengthening the supply of precursor amino acids to increase ergothioneine production

[0094] In order to promote the supply of precursor amino acids, on the basis of strain ST1625, the endogenous genes CYS3 (nucleotide sequence as shown in SEQ ID NO.9), CYS4 (nucleotide sequence as shown in SEQ ID NO.10), SAM2 (nucleotide sequence as shown in SEQ ID NO.11), HIS1 (nucleotide sequence as shown in SEQ ID NO.12), and MET6 (nucleotide sequence as shown in SEQ ID NO.13) for the synthesis of precursor amino acids of Yarrowia lipolytica were overexpressed to further increase the yield of ergothioneine ( Figure 5 ).

[0095] The specific steps are: using primers CYS3-F / CYS3-R, CYS4-F / CYS4-R, SAM2-F / SAM2-R, HIS1-F / HIS1-R, MET6-F / MET6-R, respectively, to amplify the above genes from the genome, respectively integrate the above genes at the E3 site, and construct strains named ST1627 to ST1631. Further construct strains ST1632 with MET6 and HIS1 integrated at the E3 site, strains ST1633 with MET6 and CYS3 integrated at the E3 site, and strains ST1634 with MET6 and HIS1 integrated at the E3 site and CYS3 integrated at the E4 site.

[0096] Strains ST1627 to ST1634 were fermented at 30°C for 144 h in 24-well plates containing YPD medium. The results showed that the ergothioneine content in the fermentation broth of strains ST1627 to ST1634 was significantly increased. Among them, the three key enzymes MET6, CYS3, and HIS1 were expressed in combination. After the combined expression, the accumulation of ergothioneine increased most significantly, reaching 446.6 mg / L.

[0097] After verifying that the accumulation of ergothioneine can be greatly improved by strengthening the precursor amino acid of ergothioneine, the related genes that can consume the precursor amino acid pathway are further knocked out. The specific steps are as follows: Taking the knockout of SPE2 as an example, the N20 sequence of the gene is found on the CHOPCHOP website, and the knockout cas9 plasmid is constructed with primers SPE2-dF / SPE2-dR. A 600-800bp fragment is cut off from the SPE2 sequence, and the SPE2 sequence is amplified with primers SPE2-F / SPE2-HO-R and SPE2-R / SPE2-HO-F. After the two fragments are connected to obtain the target fragment, the knockout cas9 plasmid and the target fragment are transformed into the strain according to the yeast transformation method to perform gene knockout. It was preliminarily speculated that SPE2 (S-adenosylmethionine decarboxylase) could degrade SAM, and STR2 (cystathionine synthase) could degrade Cys. SPE2 (Gene ID: 2911812) and STR2 (Gene ID: 2911236) were knocked out in strain ST1634.

[0098] The constructed strains ST1635 and ST1636 were fermented according to the method of Example 1, and the results showed that ( Figure 5 ), the accumulation of thioneine after knocking out STR2 reached 336.6mg / L; knocking out STR2 did not increase the accumulation of thioneine, and after knocking out SPE2, the accumulation of thioneine reached 516.6mg / L. This is compared to the strain without knocking out SPE2, and the yield of the strain has increased by 54.2%.

[0099] Table 5 Primers and sequences

[0100]

[0101]

[0102] Example 5: Fermentation of strains in a 5L fermenter

[0103] The strain ST1636 constructed in Example 4 was supplemented with LEU and URA tags (the method refers to "De Novo Biosynthesis of Lutein in Yarrowia lipolytica") and amplified in a 5L bioreactor. Refer to the method in the document to supplement the LEU and URA tags. Pick a single colony of the strain and inoculate it in 10mL YPD medium, and culture it at 30°C for 18h to prepare a primary seed solution. The primary seed solution was inoculated into 200mL YNB medium at an inoculum size of 5%, and cultured at 30°C for 22h to prepare a secondary seed solution. The secondary seed solution was inoculated into an inorganic salt culture medium at an inoculum size of 8%, so that the bacterial concentration after inoculation was 0.4-0.6. The dissolved oxygen level was controlled at 10-20% by stirring and dissolved oxygen association. As the fermentation progressed, the pH value gradually decreased, so NH 3 ·H 2 O, keep the pH value of the fermentation liquid at 5.0. Start feeding when glucose is lower than 1g / L, and ferment until OD 600 Reach 6-8 times of feeding 20× inorganic salt medium (i.e., high concentration inorganic salt medium), and control the glucose concentration in the fermentation environment to range from 0.1 to 1 g / L.

[0104] Until the end of fermentation. After 168h of fermentation, OD 600 Reach 226, ergothioneine production can reach 10.3g / L ( Figure 6 ), which is the highest level of ergothioneine synthesized de novo by microorganisms reported to date.

[0105] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. An ergothioneine synthetase mutant, characterized in that Based on the parent, the histidine at position 492 is mutated to valine and / or the tyrosine at position 786 is mutated to alanine; the parent has an amino acid sequence as shown in Genbank accession number: XP_006968620.

1.

2. A polynucleotide, characterized in that For (a) or (b): (a) a gene encoding the mutant according to claim 1; (b) An expression cassette containing the gene described in (a).

3. A recombinant microorganism expressing the mutant according to claim 1.

4. An engineered strain of Yarrowia lipolytica, characterized in that TrEGT1 and TrEGT2 from Trichoderma reesei, or TrEGT1 mutant and TrEGT2 were expressed; the TrEGT1 mutant was obtained by mutating the histidine at position 492 to valine and / or the tyrosine at position 786 to alanine based on the parent; the parent had an amino acid sequence as shown in Genbank accession number: XP_006968620.

1.

5. The engineered yeast Yarrowia lipolytica according to claim 4, characterized in that The expression of the sulfur assimilation module is also enhanced; the genes of the sulfur assimilation module include but are not limited to one or more of the genes MET2, MET3, MET5, MET10, MET14, and MET16.

6. The engineered yeast Yarrowia lipolytica according to claim 4 or 5, characterized in that The supply of the precursor amino acids histidine, cysteine ​​and / or methionine is also enhanced; the enhanced supply of the precursor amino acids comprises expressing one or more of the following genes: SAM2, MET6, CYS3, CYS4, HIS1.

7. The engineered yeast Yarrowia lipolytica according to any one of claims 4 to 6, characterized in that The gene SPE2 and / or the gene STR2 are knocked out.

8. A method for preparing ergothioneine, characterized in that, The engineered Yarrowia lipolytica according to any one of claims 4 to 6 is fermented in a culture medium at 25 to 30°C.

9. The method according to claim 8, characterized in that During the fermentation process, the dissolved oxygen level is controlled at 15-20%; during the fermentation process, the pH is controlled at 5.0±0.2; when the glucose is lower than 1g / L, glucose is fed to make the glucose concentration at 0.1-1g / L.

10. Use of the engineered Yarrowia lipolytica according to any one of claims 4 to 7 in producing products containing ergothioneine in the fields of food, medicine or chemical industry.

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