Ergothioneine synthetase from chaetomium globosum, coding gene, recombinant yeast strain containing gene and application

Abstract

The invention discloses a chaetomium globosum sourced ergothioneine synthetase CgEgt2 and a coding gene cgegt2 of the chaetomium globosum sourced ergothioneine synthetase CgEgt2. The amino acid sequence and the nucleotide sequence of the chaetomium globosum sourced ergothioneine synthetase CgEgt2 are shown as SEQ ID NO.2 and SEQ ID NO.1. The invention further discloses a preparation method of the The invention provides a Kluyveromyces marxianus recombinant strain KMNC-WZ02 containing a cgegt2 gene and an ncegt1 gene, the preservation number of the Kluyveromyces marxianus recombinant strain KMNC-WZ02 is CGMCC (China General Microbiological Culture Collection Center) NO.29599, a two-copy strain of the KMNC-WZ02 is constructed, and the ergothioneine yields of the KMNC-WZ02 and the two-copy strain of the KMNC-WZ02 respectively reach 466 mg / L and 590.3 mg / L after the KMNC-WZ02 and the two-copy strain of the KMNC-WZ02 are fermented for 7 days. The method provided by the invention is of great significance to excavation of EGT synthetases from different sources and construction of efficient EGT production strains.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering, and particularly relates to ergothioneine synthase from Chaetomium globosum, a coding gene, a recombinant yeast strain containing the gene and application. BACKGROUND

[0002] Ergothioneine (mercaptohistidine trimethyl inner salt, EGT) is a sulfur-containing histidine derivative, which was first discovered by French researchers when studying Claviceps purpurea that destroys grains. It is a natural antioxidant and is found in high levels in higher mushroom fungi and fermented foods (Tian et al., British Journal of Nutrition. 2023, 129(1): 104-114). Its antioxidant capacity is much higher than that of vitamin C (Beelman et al., Journal of Nutritional Science, 2020, 9: e52), and it can scavenge free radicals and protect DNA from oxidative damage. EGT not only has strong antioxidant capacity, but also has anti-inflammatory, anti-aging and inhibiting melanin accumulation effects (Liu et al., Molecules, 2023, 28(4): 1648). In addition, compared with vitamin C and glutathione, two commonly used antioxidants, EGT has more stable chemical properties. Therefore, EGT has broad application prospects in the food, cosmetics and biological medicine industries. However, due to the high production cost of EGT at present, its large-scale market application is limited, and the use of synthetic biology to produce EGT has great application potential.

[0003] Currently, microorganisms that can naturally synthesize EGT include Schizosaccharomyces pombe, Rhodotorula mucilaginosa, Claviceps purpurea and Neurospora crassa. Fungal synthesis of EGT requires two key enzymes, Egt1 and Egt2, of which Egt1 catalyzes the synthesis of the intermediate product Cys-HER from histidine trimethyl inner salt (hercynine, HER) and cysteine, and continues to be converted to ergothioneine by Egt2. With the functional verification of the coding genes of Egt1 and Egt2 enzymes of N. crassa and C. purpurea, researchers found that the activity of enzyme genes from different sources and the effect of different enzyme gene combinations were different (van et al., Metabolic Engineering, 2022, 70: 129-142). However, there are few known enzyme gene sequences from fungi, which limits the demand for heterologous expression of ergothioneine synthase to produce EGT.

[0004] Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, which is more resistant to high temperature than Saccharomyces cerevisiae, and can still have good growth performance at 37℃. This advantage can reduce the cost of cooling bioreactors and reduce the risk of contamination in industrial production. At the same time, Kluyveromyces marxianus is a strain that naturally has the ability to metabolize more types of sugars, such as lactose and xylose (Gombert et al., Applied Microbiology and Biotechnology. 2016, 100(14): 6193-6208). In addition, it is reported that it is the fastest growing eukaryote described to date, with the ability to synthesize and secrete proteins (Lane et al., Antonie Van Leeuwenhoek, 2011, 100(4): 507-519). In recent years, Kluyveromyces marxianus has been used as a cell factory for the production of bioethanol and various enzymes (Qiu et al., Biotechnol Adv. 2023, 64: 108125). Using Kluyveromyces marxianus as a new yeast chassis cell to produce ergothioneine will have great biotechnological application potential. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies in the prior art, and provide a new source of ergothioneine synthase, a coding gene, a recombinant yeast strain containing the gene, and an application. The technical solutions are specifically as follows.

[0006] A Chaetomium globosum-derived ergothioneine synthase Egt2, hereinafter referred to as CgEgt2 enzyme, has an amino acid sequence as shown in SEQ ID NO. 2.

[0007] A gene encoding the CgEgt2 enzyme, hereinafter referred to as cgegt2 gene, has a nucleotide sequence as shown in SEQ ID NO. 1.

[0008] The present application provides a Kluyveromyces marxianus recombinant strain KMNC-WZ02, which contains an ergothioneine synthesis gene egt1 and the aforementioned cgegt2 gene. The preservation number of the recombinant strain KMNC-WZ02 is CGMCC NO. 29599, the preservation date is January 13, 2024, and it is preserved in the General Microbial Center of the Chinese Microbial Strain Preservation Management Committee, located at No. 3, Yikhina, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain is in a viable state after detection.

[0009] In one embodiment of the present invention, the ergothionein synthesis gene egt1 is derived from Neurospora crassa, hereinafter referred to as the ncegt1 gene, and its nucleotide sequence is shown in SEQ ID NO.3. The nucleotide sequence of its codon optimized for Kluyveromyces martensii is shown in SEQ ID NO.4.

[0010] The application of the recombinant strain KMNC-WZ02 in the fermentation production of ergothioneine. Preferably, the culture medium for fermenting ergothioneine contains 20 g / L glycerol, 20 g / L yeast extract, and 20 g / L peptone. The preferred fermentation temperature is 30°C.

[0011] A two-copy strain constructed using recombinant strain KMNC-WZ02 as the starting strain. It can be used in the fermentation production of ergothioneine. Preferably, the culture medium for fermenting ergothioneine contains 20 g / L glycerol, 20 g / L yeast extract, and 20 g / L peptone. The preferred fermentation temperature is 30°C.

[0012] The advantages of this invention are:

[0013] This invention provides a novel ergothioneine synthase, Egt2, by determining its amino acid sequence and the nucleotide sequence encoding the enzyme, and verifying its normal expression and function in *Kluyveromyces martensii*, providing theoretical and fundamental support for related research on ergothioneine synthases. A recombinant strain, KMNC-WZ02, was obtained by combining the ncegt1 gene and the cgegt2 gene provided in this invention into *Kluyveromyces martensii*. Its EGT production yield during fermentation is superior to the best reported enzyme gene combination to date, namely the combination of the ncegt1 gene from *N. crassa* and the cpegt2 gene from *C. purpurea*. A two-copy strain of KMNC-WZ02 was also constructed. The EGT yields of KMNC-WZ02 and its two-copy strain after 7 days of fermentation reached 466 mg / L and 590.3 mg / L, respectively. This invention is of great significance for discovering EGT synthases from different sources, constructing efficient EGT-producing strains, and promoting the application of EGT in various fields such as cosmetics and pharmaceuticals. Attached Figure Description

[0014] Appendix Figure 1 This is the technical process for big data mining of ergothionein synthase genes in Example 1 of the present invention.

[0015] Appendix Figure 2 Example 2 of the present invention describes the yield of ergothionein heterologously expressed in the chassis of *Kluyveromyces martensii* using the endogenous cgegt2 gene obtained through big data mining.

[0016] Appendix Figure 3Comparison of the ergot alkaloid production of the recombinant Kluyveromyces marxianus strain heterologously expressing the best reported combination of enzyme genes (ncegt1 gene from N. crassa and cpegt2 gene from V. purpurea) of Example 3 of the present application with the recombinant strain KMNC-WZ02 of the present application.

[0017] Figure 1 shows the ergot alkaloid production of the recombinant Kluyveromyces marxianus strain heterologously expressing the best reported combination of enzyme genes (ncegt1 gene from N. crassa and cpegt2 gene from V. purpurea) of Example 3 of the present application with the recombinant strain KMNC-WZ02 of the present application. Figure 4 Comparison of the ergot alkaloid production of the two-copy recombinant strain obtained by heterologously introducing the ncegt1 gene and the cgegt2 gene in the recombinant strain KMNC-WZ02 of Example 4 of the present application with the recombinant strain KMNC-WZ02. DETAILED DESCRIPTION

[0018] The specific embodiments of the present application are described in detail below with reference to the accompanying drawings. Unless otherwise specified, the reagents and instruments used are commonly used in the art.

[0019] Example 1 Technical process of ergot alkaloid synthase gene big data mining

[0020] In this embodiment, a set of complete database is used to mine potential ergot alkaloid synthase genes. This method can effectively screen genes involved in ergot alkaloid synthesis and can be used to construct an engineered strain for efficient production of ergot alkaloid.

[0021] The specific steps of the method are as follows. First, download the existing target range species genome and transcriptome data from the NCBI database. According to the sequence similarity with the reported ergot alkaloid synthase genes and the transcriptome differential expression gene analysis, possible sequences are screened out. At the same time, the sequences of the reported ergot alkaloid synthase genes are used as a search source for Position-Specific Iterated BLAST (PSI-BLAST, https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK LOC=blasthome) search to screen out possible ergot alkaloid synthase genes. These sequences are further screened according to the methods of domain, conserved motif, intron analysis, and transcription start analysis to obtain candidate sequences. Then, phylogenetic analysis is performed to extract each branch representative sequence for three-dimensional structure simulation, which is compared with the structure of the reported active ergot alkaloid synthase. According to the TM score and root mean square deviation (RMSD), reliable ergot alkaloid synthase sequences are obtained. Then, the sequences are heterologously expressed in a host chassis to verify their functions. The overall process is shown in Figure 1

[0022] ​Through the enzyme mining process, the present application first mines the egt2 gene from Chaetomium globosum in the database, and determines the specific nucleotide sequence and amino acid sequence thereof through various technical means such as sequence alignment, structure simulation and molecular docking, as shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0023] Table 1. Structural similarity of cgegt2 homologous sequences from Chaetomium globosum and cpegt2 gene sequences from Clavieps purpurea

[0024]

[0025] The present application provides a technical process for mining ergothioneine synthase gene big data, filling the unknown blank of existing ergothioneine biosynthesis gene large-scale data mining.

[0026] Example 2 Functional characterization of cgegt2 gene

[0027] The present application takes Kluyveromyces marxianus DLY-KMSP-0209 as the starting strain, which enhances the homologous recombination efficiency of the strain (patent application number 202211350091.2, the strain DLY-KMSP-0209 is obtained by domesticating and genetically modifying the Kluyveromyces marxianus strain NBRC1777). The strain NBRC1777 contains an endogenous egt2 gene and lacks the essential gene egt1 for ergothioneine synthesis (see patent application number 202311303331.8 for details). Therefore, after knocking out the endogenous egt2 gene of DLY-KMSP-0209, the ncegt1 gene and the cgegt2 gene are introduced into the Kluyveromyces marxianus DLY-KMSP-0209, and the function of the cgegt2 gene is verified by comparing with the control strain only introducing the ncegt1 gene. The specific method is as follows.

[0028] The sequence of heterologous expression was codon-optimized for K. marxianus, and the synthetic target fragment was cloned. The ncegt1 gene fragment was connected between the K. marxianus TEF1 promoter and the INU1 terminator, and the upstream and downstream homologous arms (1000 bp each) of the insertion site ura3 were connected on the periphery of the promoter and the terminator, and then it was transformed into the E. coli DH5a cloning strain. In addition, the ncegt1 gene and the cgegt2 gene were constructed together between the endogenous TEF1 promoter and the INU1 terminator of K. marxianus and between the endogenous TEF3 promoter and the PDC1 terminator, and the upstream and downstream homologous arms (1000 bp each) of the insertion site ura3 were connected on the periphery of the fragment, and then it was transformed into the E. coli DH5a cloning strain.

[0029] The E. coli transformation conditions were as follows: 5 μL of the ligation product was added to 50 μL of competent cells, mixed gently, and incubated on ice for 25 min, then heat shocked at 42°C for 90 s in water, quickly ice-bathed, and incubated for 2 min, then 500 μL of LB medium without antibiotics was added, mixed, and incubated at 37°C, 200 rpm for 1 h, then the bacterial solution was centrifuged at 3000 rpm for 1 min, 400 μL of supernatant was discarded, the bacterial solution was suspended, and plated on solid LB plates containing antibiotics (Amp) and incubated at 37°C for 12-16 h.

[0030] Colony PCR was used for positive clone screening, and the screening method was as follows: single colonies were randomly picked from the transformation plate and cultured in 1.5 mL centrifuge tubes containing liquid medium. Each tube was numbered, and 1 μL of each tube was used as a template for PCR detection, and the remaining culture was stored at 4°C. The colonies that tested positive were stored on plates or in glycerol tubes for future use. Sequencing showed that the yeast transformation fragment of the target gene was successfully cloned.

[0031] Max Kluver transformation method: take 1 mL of overnight DLY-KMSP-0209 yeast, centrifuge at 8000 rpm, take the bacterial body, wash once with sterile water, then wash twice with 1x LiAc / TE, centrifuge at 8000 rpm, and wash the supernatant thoroughly. Add 10 μL carrier DNA, 2-3 μg of DNA fragment to be transformed, and 1-1.5 μg of gRNA plasmid containing Cas9 protein and hygromycin gene expression frame to the bacterial body. Add 600 μL PEG solution (40% PEG 4000, 0.1 M LiAc, 10 mM Tris-HCl pH 7.5, 1 mM EDTA) and a certain volume of DTT (final concentration of DTT is 10 mM). After mixing thoroughly, 30°C water bath for 15 min, 47°C water bath for 15 min. Then discard the supernatant, add 800 μL YPD and incubate for 2 h, then centrifuge at 8000 rpm, discard the supernatant, suspend the bacterial body with 100 μL sterile water, and spread on an antibiotic (hygromycin) plate and cultivate at 30°C for two days.

[0032] Genome amplification was used for positive clone screening. The screening method was as follows: randomly pick single colonies from the transformation plate and place them in 1.5 mL centrifuge tubes containing liquid YPD medium and cultivate for 12 h. Take 500 μL from each tube for genome extraction, and the remaining glycerol was preserved at -20°C. Number each tube, add 100 μL of 200 mM LiAc, 1% SDS, and incubate at 70°C for 15 min, resuspend with 300 μL of 100% ethanol, centrifuge at 12000 rpm for 5 min, remove the supernatant, add 200 μL of 70% ethanol, wash twice at 1200 rpm for 2 min, and naturally blow dry for 15-30 min. Add 50 μL of sterile water to dissolve, which is the crude solution containing the genome. Take 1 μL from each tube as a template for PCR detection. The transformants that are positive in the detection are correct recombinant Max Kluver strains verified by sequencing. Ferment in YPD medium, and use ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (AB5500, USA) to detect the ergothioneine synthesis ability for 7 days. The chromatographic column is ACQUITY UPLC BEH Hilic (50x2.1mm, 1.7 μm, Waters), the temperature is 40°C, the mobile phase A is water plus 0.1% formic acid and 5 mM ammonium formate, the mobile phase B is a mixture of acetonitrile plus 0.1% formic acid and 5 mM ammonium formate, the flow rate is 0.4 mL / min, and the injection volume is 1 μL. The results are shown in Figure 2

[0033] Figure 2 ​​It can be seen that the ergothioneine synthesis ability of Cgegt2 enzyme is successfully verified in the K. marxianus chassis. In the chassis with the endogenous egt2 gene knocked out, the strain constructed by combining the cgegt2 gene with the ncegt1 gene can synthesize 303.3 mg / L of EGT in 7 days in a 2% (g / L) yeast powder, 2% (g / L) peptone, and 2% (g / L) glycerol medium.

[0034] Example 3 Ergothioneine synthesis level of KMNC-WZ02 strain constructed by heterologous expression of ncegt1 gene and cgegt2 gene without knocking out the endogenous egt2 gene of the chassis

[0035] The specific construction steps of the KMNC-WZ02 recombinant strain are as follows: the ncegt1 gene and the cgegt2 gene are respectively constructed between the endogenous TEF1 promoter and the INU1 terminator and between the endogenous TEF3 promoter and the PDC1 terminator of K. marxianus, and then the upstream and downstream homologous arms (1000 bp each) of the insertion site ura3 are connected to the periphery of the fragments, and then the fragments are transformed into the E. coli DH5a cloning strain. Further, the target fragment is introduced into the DLY-KMSP-0209 strain to obtain the correct yeast transformant. The specific E. coli transformation method and the yeast transformation method are shown in Example 2, which will not be repeated here.

[0036] The specific construction steps of the control strain (the optimal enzyme combination reported) are as follows: the ncegt1 gene and the cpegt2 gene are respectively constructed between the endogenous TEF1 promoter and the INU1 terminator and between the endogenous TEF3 promoter and the PDC1 terminator of K. marxianus, and then the upstream and downstream homologous arms (1000 bp each) of the insertion site ura3 are connected to the periphery of the fragments, and then the fragments are transformed into the E. coli DH5a cloning strain. Further, the target fragment is introduced into the DLY-KMSP-0209 strain to obtain the correct yeast transformant. The specific E. coli transformation method and the yeast transformation method are shown in Example 2, which will not be repeated here.

[0037] The specific fermentation process is as follows: 20 μL of three parallel yeast transformants stored in a -80°C refrigerator is inoculated into 1 mL of YPD liquid medium, and then cultured at 30°C, 200 rpm on a shaker for 24 h, and then transferred into 100 mL of YPD seed medium (i.e. YPD liquid medium), and then cultured at 37°C on a shaker for 18 h. The initial OD 6000.1 μL of the culture medium was inoculated into YPG medium (glycerol 20 g / L, yeast extract 20 g / L, peptone 20 g / L), sealed with a breathable sealing film, and fermented at 200 rpm and 30°C for 7 days. A 1 mL sample was then placed in a 2 mL centrifuge tube. Next, 500 μL of 0.5 mm diameter glass beads were added to the centrifuge tube, which was then placed in a pre-cooled metal module at -20°C, and the cells were disrupted using a cell disruptor. The disrupted mixture was centrifuged at 12000 rpm, and the supernatant was used for ergothioneine yield detection. 20 μL of the fermentation broth was diluted 30-fold with 580 μL of a 1:1 volume ratio of methanol and acetonitrile. After passing through a 0.22 μm filter membrane, the ergothioneine content was detected using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (AB5500, USA). The detection conditions were the same as in Example 2, and the results are as follows. Figure 3 As shown.

[0038] Depend on Figure 3 It can be seen that the KMNC-WZ02 recombinant strain has the ability to synthesize ergothionein, and can synthesize 466 mg / L of EGT, which is about 58% higher than the reported optimal enzyme combination (296.1 mg / L).

[0039] Example 4: Ergothioneine production of a two-copy strain of the KMNC-WZ02 recombinant strain.

[0040] This example measures the yield of ergothionein in a recombinant strain that heterologously expresses the second copy of the ncegt1 and cgegt2 genes in the KMNC-WZ02 recombinant strain.

[0041] The specific steps for constructing the two-copy strain are as follows: Using the recombinant strain KMNC-WZ02 as the starting strain, the heterologous ncegt1 and cgegt2 genes were constructed between the endogenous TEF1 promoter and INU1 terminator of *Kluyveromyces martensii*, and between the endogenous TEF3 promoter and PDC1 terminator. Then, homologous arms (1000 bp each) upstream and downstream of the insertion site Leu2 were ligated around the fragments. This was then transformed into an *E. coli* DH5α clone strain. The target fragment was further introduced into the KMNC-WZ02 strain to obtain the correct yeast transformant. Specific *E. coli* transformation methods and yeast transformation methods are described in Example 2 and will not be repeated here.

[0042] The specific fermentation process is as follows: 20 μL of three parallel yeast transformants stored at -80℃ were inoculated into 1 mL of YPD liquid medium and cultured at 30℃ and 200 rpm for 24 h. Then, the culture was transferred to 100 mL of YPD seed medium (i.e., YPD liquid medium) and cultured at 37℃ for 18 h. The fermentation was then carried out according to the initial OD... 600For 0.1 inoculation into YPG medium (glycerol 20 g / L, yeast powder 20 g / L, peptone 20 g / L), and sealed with a breathable sealing film, 1 mL of sample was taken from the device in a 2 mL centrifuge tube after fermentation at 200 rpm, 30°C for 7 days. Then, 500 μL of glass beads with a diameter of 0.5 mm were added to the centrifuge tube, and the cell was broken by a cell disrupter in a pre-cooled metal module at -20°C. The mixture after breaking was centrifuged at a speed of 12000 rpm, and the supernatant was used for ergothioneine yield detection. 20 μL of the above fermentation liquid was added with 580 μL of methanol and acetonitrile solvents in a volume ratio of 1:1 to dilute 30 times, and after membrane filtration through a 0.22 μm filter membrane, the content of ergothioneine was detected by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (AB5500, USA). The detection conditions were the same as in Example 2, and the detection results are shown in Table 2. Figure 4

[0043] As can be seen from Figure 4 , the ergothioneine synthesis ability of the two-copy strain constructed in the embodiment is further improved on the basis of the recombinant strain KMNC-WZ02, and 590.3 mg / L of ergothioneine can be synthesized within 7 days.

[0044] In summary, the Cgegt2 gene obtained by big data mining has the ability to synthesize ergothioneine, and the recombinant strain KMNC-WZ02 provided has excellent ergothioneine synthesis ability, and can synthesize 466 mg / L of EGT within 7 days, which is about 58% higher than the reported optimal enzyme combination constructed recombinant strain. The two-copy strain constructed by taking the recombinant strain KMNC-WZ02 as a starting strain can synthesize 590.3 mg / L of ergothioneine within 7 days, and its ergothioneine synthesis ability is further improved.

[0045] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the method of the present application, a number of improvements and supplements can also be made, which should also be considered as the protection scope of the present application.​

Claims

1. A ergothioneine synthase Egt2 of Chaetomium globosum origin, characterized in that, The amino acid sequence of which is shown as SEQ ID NO.

2.

2. A gene egt2 encoding the ergothioneine synthase Egt2 according to claim 1, characterized in that, The nucleotide sequence of which is shown as SEQ ID NO.

1.

3. A recombinant strain of Kluyveromyces marxianus, KMNC-WZ02, characterized in that, The ergothioneine synthesis gene egt1 and the coding gene egt2 of claim 2; The preservation number of the recombinant strain KMNC-WZ02 is CGMCC NO. 29599, and the preservation date is January 13, 2024, and the preservation is in the China General Microbiological Culture Collection Center.

4. The recombinant bacterial strain KMNC-WZ02 according to claim 3, characterized in that, The ergothioneine synthesis gene egt1 is derived from Neurospora crassa, and the nucleotide sequence thereof is shown as SEQ ID NO. 3, and the codon-optimized nucleotide sequence suitable for the Kluyveromyces marxianus is shown as SEQ ID NO.

4.

5. The application of the recombinant strain KMNC-WZ02 of claim 3 in the fermentation production of ergothioneine.

6. Use according to claim 5, characterized in that, The fermentation medium for producing ergothioneine is glycerol 20 g / L, yeast powder 20 g / L and peptone 20 g / L.

7. A two-copy strain constructed by taking the recombinant strain KMNC-WZ02 of claim 3 as a starting strain.

8. The application of the two-copy strain of claim 7 in the fermentation production of ergothioneine.

9. Use according to claim 8, characterized in that, The fermentation medium for producing ergothioneine is glycerol 20 g / L, yeast powder 20 g / L and peptone 20 g / L.

10. Use according to claim 5 or claim 8, characterised in that, The fermentation temperature for producing ergothioneine is 30℃.

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