CN117327597A - Kluyveromyces marxianus for producing ergothioneine and construction method and application thereof - Google Patents

Abstract

The invention discloses a Kluyveromyces marxianus strain KME1-WZ01 for producing ergothioneine, the preservation number is CGMCC No.27310, the Kluyveromyces marxianus strain contains exogenous ergothioneine synthetic gene Egt1 and endogenous gene Egt2, and the nucleotide sequence of the Egt2 gene is shown as SEQ ID No. 1. Amplification primer pairs of the Egt2 gene and Egt2 enzyme expressed by the amplification primer pairs are also provided. The KME1-WZ01 can increase the yield of ergothioneine under the condition that precursors histidine and methionine are added into a fermentation medium. The genetically modified bacterium of the invention only needs to be transferred into an exogenous Egt1 gene, has simple operation and provides a simpler genetic operation module for synthesizing ergothioneine from the head by utilizing a yeast chassis. The invention firstly utilizes the Kluyveromyces marxianus as the chassis strain to produce the ergothioneine, has simple fermentation method and provides a new path for synthesizing the ergothioneine by a biological method.

Description

Kluyveromyces marxianus for producing ergothioneine and construction method and application thereof

Technical Field

The invention relates to the field of bioengineering, in particular to Kluyveromyces marxianus for producing ergothioneine, and a construction method and application thereof.

Background

Ergothioneine (EGT) is a natural, sulfur-containing histidine derivative that is found mainly in part of fungi and bacteria. Ergothioneine was at the earliest a sulfur-containing compound found in the study of ergot fungi that destroy rye grains. Because of its excellent antioxidant activity, it is a natural antioxidant with anti-photoaging and antioxidant effects, scavenging free radicals, protecting DNA from oxidative damage. EGT has far higher antioxidant capacity than vitamin C (Beelman et al Journal of Nutritional Science,2020,9, e 52). The ergothioneine is expensive, so that the technology capable of efficiently producing the EGT has great market value.

The EGT in fungi is synthesized by two enzymes Egt1 and Egt2, and microorganisms which can naturally synthesize the EGT are reported to be schizosaccharomyces pombe, rhodotorula mucilaginosa, ergot, neurospora crassa and the like, wherein the rhodotorula mucilaginosa reports that the highest yield is 38mg/L of ergothioneine production through hydrogen peroxide stress stimulation (Xiong et al, food Bioscience,2023, 53, 102745). Analysis of natural synthetases from natural ergothioneine-producing strains is a necessary path for understanding the synthetic pathways of ergothioneine. With functional verification of the Egt1 and Egt2 genes of Neurospora crassa and Clavipita, researchers have found that the activities of enzyme genes from different sources and the combined effects of different enzyme genes are different (van et al Metabolic Engineering,2022, 70:129-142). However, the currently published enzyme genes from fungi are limited, and more enzyme genes need to be found and modified, so that the efficient production of ergothioneine is promoted to meet the industrial requirement.

Kluyveromyces marxianus (Kluyveromyces marxianus) is an emerging host for heterologous protein synthesis, is also a craftree negative yeast, does not perform aerobic ethanol fermentation, and exceeds the traditional Saccharomyces cerevisiae in some important applications. Kluyveromyces marxianus is more tolerant to high temperature than Saccharomyces cerevisiae, can still have better growth performance at 37 ℃, and has the capabilities of protein synthesis and high secretion (Lane et al Antonie Van Leeuwenhoek,2011, 100, 507-519). The yeast is a biosafety fungus, and can utilize various carbon sources including glucose, xylose, glycerol, lactose and the like. In recent years, kluyveromyces marxianus has been applied as a cell factory for the production of bioethanol and various enzymes (YIbin Qia et al Kluyveromyces as promising yeast cell factories for industrial bioproduction: from bio-functional design to applications. Biotechnol Adv,2023, 64, 108125), but the production of ergothioneine using this yeast has not been reported. Therefore, the Kluyveromyces marxianus is used as chassis cells to produce ergothioneine through a proper transformation strategy, and the method has huge biotechnology application potential.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a Kluyveromyces marxianus (Kluyveromyces marxianus) strain KME1-WZ01 for producing ergothioneine, which contains ergothioneine synthetic genes Egt1 and Egt2, wherein the Egt2 gene is an endogenous gene of the Kluyveromyces marxianus, the nucleotide sequence of the endogenous gene is shown as SEQ ID NO.1, the amino acid sequence of Egt2 gene expression is shown as SEQ ID NO.2, and the Egtl gene is an exogenous gene. No report on Kluyveromyces marxianus containing endogenous Egt2 gene is found in the prior art. The enzymes expressed by the Egt1 and Egt2 genes are hereinafter abbreviated as Egt1 and Egt2 enzymes, and the Egt2 enzyme is also referred to as selenocysteine lyase.

The preservation number of the strain KME1-WZ01 is CGMCC No.27310, and the strain is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with the preservation date of 2023, 5 and 10 days and the preservation address: beijing, chaoyang area, north Chen Xili No.1, 3, china academy of sciences, microbiological institute. The strain tested was in a viable state.

Preferably, the ergothioneine synthesis gene Egt1 is derived from Neurospora crassa (Neurospora crassa), and the nucleotide sequence of the ergothioneine synthesis gene Egt1 is shown as SEQ ID NO. 5.

The construction method of the strain KME1-WZ01 comprises the following steps: transforming the plasmid of the Neurospora crassa ergothioneine synthetic gene Egt1 into an escherichia coli DH5 alpha clone strain, adopting colony PCR to carry out positive clone screening, transforming a target DNA fragment into a Kluyveromyces marxianus original strain, and obtaining a Kluyveromyces marxianus recombinant strain after sequencing verification of a transformant with positive PCR detection, wherein the laboratory is named as KME1-WZ01.

The invention also discloses an ergothioneine synthetic gene Egt2, wherein the Egt2 gene is derived from Kluyveromyces marxianus KME1-WZ01, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

The invention also discloses an amplification primer pair of the ergothioneine synthetic gene Egt2, wherein the sequence of a forward primer of the amplification primer pair is shown as SEQ ID NO.3, and the sequence of a reverse primer is shown as SEQ ID NO. 4. The amplification primer pair can rapidly and accurately obtain the encoding gene of Egt 2.

The invention also discloses an enzyme expressed by the ergothioneine synthetic gene Egt2, and the amino acid sequence of the Egt2 enzyme is shown as SEQ ID NO. 2.

The application of Kluyveromyces marxianus KME1-WZ01 in producing ergothioneine, including but not limited to producing ergothioneine by a fermentation method, can also be used as chassis cells for further modification.

The invention also provides a method for producing ergothioneine by Kluyveromyces marxianus KME1-WZ01, wherein the fermentation culture medium comprises 20g/L of carbon source, 10-20g/L of yeast powder and 20g/L of peptone, and the carbon source is selected from glucose, xylose or glycerol, but is not limited to the listed carbon source types.

Preferably, the carbon source is glycerol.

In fermentation production, precursors histidine and methionine may also be added to the fermentation medium to increase the yield of ergothioneine. The addition amount of histidine and methionine is preferably 1g/L.

The invention has the advantages that:

provides a novel Kluyveromyces marxianus recombinant strain KME1-WZ01 capable of producing ergothioneine. The invention also provides an endogenous ergothioneine synthesis gene Egt2 derived from Kluyveromyces marxianus and an enzyme expressed by the same, and the ergothioneine can be produced without simultaneously introducing exogenous Egt1 genes and Egt2 genes as reported at present. The genetically modified bacterium of the invention only needs to be transferred into an exogenous Egt1 gene, has simple operation and provides a simpler genetic operation module for synthesizing ergothioneine from the head by utilizing a yeast chassis. During the fermentative production of ergothioneine by KME1-WZ01, the production of ergothioneine is increased by the addition of the precursors histidine and methionine. In a 3L fermentation tank, the ergothioneine with the concentration of 630mg/L can be synthesized in 7 days without adding a precursor, so that the method has good development and application potential and provides a new path for synthesizing the ergothioneine by a biological method.

SEQ ID NO.1 (nucleotide sequence of Egt2 gene of Kluyveromyces marxianus KME1-WZ 01):

SEQ ID NO.2 (amino acid sequence of the Egt2 enzyme of Kluyveromyces marxianus KME1-WZ 01):

SEQ ID NO.3 (forward primer):

5’-ATGAGCTCGATTCCATTTGGACATC-3’。

SEQ ID NO.4 (reverse primer):

5’-TCATGAGCTTGAAGCTTCTTTCCTC-3’。

SEQ ID NO.5 (Neurospora crassa Egt1 gene nucleotide sequence):

SEQ ID NO.6 (nucleotide sequence optimized for Neurospora crassa Egt 1):

SEQ ID NO.7 (amino acid sequence expressed by Neurospora crassa Egt1 gene):

drawings

FIG. 1 shows the yield of ergothioneine in YPD medium from Kluyveromyces marxianus strain KME1-WZ01 of the present invention.

FIG. 2 shows the yield of ergothioneine after knockout of the endogenous Egt2 gene of the strain KME1-WZ01.

FIG. 3 shows ergothioneine production by fermentation of strain KME1-WZ01 at 30℃and 37℃using glucose and xylose as carbon sources.

FIG. 4 shows ergothioneine production by strain KME1-WZ01 fermented for 7 days with glucose, xylose and glycerol as carbon sources.

FIG. 5 shows the results of increasing ergothioneine production by adding histidine and methionine precursors to strain KME1-WZ01 using glucose as a carbon source.

FIG. 6 shows the yield of ergothioneine from strain KME1-WZ01 in a 3L fermenter.

Detailed Description

The following detailed description of the invention provides specific embodiments with reference to the accompanying drawings. The examples are not intended to identify specific experimental procedures or conditions, which may be performed according to conventional procedures or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Kluyveromyces marxianus strain KME1-WZ01 (KME 1-WZ01 is a name given to recombinant strains in the laboratory) is prepared by taking Kluyveromyces marxianus NBRC1777 as an initial strain, and NBRC1777 can be purchased from Japanese biological resource center (Biological Resource Center, NITE (NBRC), JAPAN).

KME1-WZ01 retains the function of the endogenous gene Egt2 of NBRC1777 and its enzyme expression of Egt2, so that the endogenous Egt2 gene of KME1-WZ01 and the endogenous Egt2 gene of NBRC1777 are the same gene and the enzyme expressed by them is the same enzyme.

The ergothioneine synthetic gene Egt1 of Neurospora crassa is synthesized by Beijing qing department biotechnology Co., ltd, and the nucleotide sequence of the ergothioneine synthetic gene is shown as SEQ ID NO. 5. Optimizing according to the codon of Kluyveromyces marxianus, and the optimized nucleotide sequence is shown as SEQ ID NO. 6. The amino acid sequence expressed by the ergothioneine synthetic gene Egt1 of Neurospora crassa is shown as SEQ ID NO. 7.

YPD medium (glucose 20g/L, yeast powder 10g/L, peptone 20 g/L). YPX medium (xylose 20g/L, yeast powder 10g/L, peptone 20 g/L). YPG medium (glycerol 20g/L, yeast powder 20g/L, peptone 20 g/L).

The ergothioneine detection method comprises the following steps: 1mL of fermentation broth is sampled in a 2mL centrifuge tube, then 500 mu L of glass beads with the diameter of 0.5mm are added into the centrifuge tube, and the mixture is placed in a pre-cooled metal module at the temperature of minus 20 ℃ and cells are broken by a cell breaker. The crushed mixture was centrifuged at 12000rpm and the supernatant was used for the yield detection of ergothioneine. 20. Mu.L of the supernatant was taken and 1:1 was diluted 30-fold with 580. Mu.L of methanol and acetonitrile, and after passing through a 0.22 μm filter, the content of ergothioneine was measured by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (AB 5500, USA). The column was ACQUITYUPLC BEH Hilic (50X 2.1mM,1.7 μm, waters) at 40℃with mobile phase A being water plus 0.1% formic acid and 5mM ammonium formate and mobile phase B being acetonitrile plus 0.1% formic acid and 5mM ammonium formate at a flow rate of 0.4mL/min with a sample injection of 1. Mu.L.

EXAMPLE 1 Kluyveromyces marxianus NBRC1777 endogenous ergothioneine Egt2 Gene mining and validation

Genome information of Kluyveromyces marxianus NBRC1777 is obtained for analysis, and the genome is found to have an Egt2 homologous sequence through BLAST comparison, and the nucleotide sequence of the Egt2 gene is shown as SEQ ID NO. 1.

The specific steps are as follows, the published Egt2 amino acid sequence from Neurospora crassa (NCBI accession number: XP_ 956324.3) is input in the BLAST window, homologous alignment is carried out in Kluyveromyces marxianus genome, homologous amino acid sequences with similarity of 32% are found, and the result is shown as SEQ ID No. 2. The genome level analysis shows that the gene is located on chromosome 6, the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the gene is the Kluyveromyces marxianus-derived Egt2 gene with the function of excavation and verification. Furthermore, the structure of these homologous amino acid sequences was simulated by Alphafold 2, and their structural similarity to Neurospora crassa was compared by TM-score, and the results are shown in table 1.

Table 1 Kluyveromyces marxianus NBRC1777 Egt2 homologous Gene and sequence and structural similarity of known Neurospora crassa Egt2 Gene

The genome of NBRC1777 was extracted and cDNA was synthesized by reverse transcription with reverse transcriptase. The cDNA is used as a template, and the amplification primers are as follows:

the forward primer is: 5'-ATGAGCTCGATTCCATTTGGACATC-3' (SEQ ID NO. 3),

the reverse primer is as follows: 5'-TCATGAGCTTGAAGCTTCTTTCCTC-3' (SEQ ID NO. 4).

PCR amplification was performed using Vazyme company Phanta Max Super-Fidelity DNA polymerase. The PCR conditions were: 98 ℃ for 2min;98℃for 10sec;55 ℃,15sec;72 ℃,1min 25s;35 cycles; extending at 72℃for 5min.

The PCR product was detected by 1% agarose gel electrophoresis, and the amplified fragment size was about 1305bp, which was expected. And (3) recovering the target gene fragment by adopting a method of agarose gel electrophoresis gel recovery kit, and sequencing to obtain the nucleotide sequence shown in SEQ ID NO. 1.

The invention discovers that Kluyveromyces marxianus NBRC1777 has an endogenous Egt2 sequence for the first time, which shows that the Kluyveromyces marxianus NBRC1777 can be used as a chassis cell synthesized by ergothioneine by only introducing an exogenous Egt1 gene, and compared with the existing improved chassis, the genetic operation is simpler and more convenient.

EXAMPLE 2 construction of Kluyveromyces marxianus ergothioneine synthetic Strain KME1-WZ01 and Egt2 functional characterization

Kluyveromyces marxianus Kluyveromyces marxianus NBRC1777 is taken as an initial strain, neurospora crassa ergothioneine synthase gene Egt1 is selected and introduced into Kluyveromyces marxianus NBRC1777, the function of Egt2 is verified, and the Kluyveromyces marxianus recombinant strain KME1-WZ01 capable of synthesizing ergothioneine is obtained. The specific method comprises the following steps:

codon optimization is carried out on Neurospora crassa ergothioneine synthase gene Egt1, the method is suitable for Kluyveromyces marxianus, a synthesized target fragment is cloned, the target fragment is connected to the middle of a carrier with an endogenous TEF1 promoter and an INU1 terminator of Kluyveromyces marxianus, an upstream homologous arm and a downstream homologous arm (1000 bp respectively) of an insertion site ura3 are connected to the peripheries of the promoter and the terminator, and the target fragment is transformed into an escherichia coli DH5 alpha clone strain.

The E.coli transformation conditions were: adding 5 mu L of the connection product into 50 mu L of competent cells, lightly mixing, standing on ice for 25min, carrying out water bath heat shock for 90s at 42 ℃, rapidly carrying out ice bath, standing for 2min, adding 500 mu L of LB culture medium without antibiotics, carrying out recovery for 1h at 200rpm at 37 ℃ after mixing, centrifuging bacterial liquid at 3000rpm for 1min, discarding 400 mu L of supernatant, coating the suspension bacterial liquid on a solid LB plate with antibiotics (Amp), and carrying out inversion culture for 12-16h at 37 ℃.

Positive clone screening is carried out by colony PCR, and the screening method comprises the following steps: single colonies were randomly picked from the transformation plate and placed in liquid medium in a 1.5mL centrifuge tube for cultivation. Each tube was numbered and 1. Mu.L of each tube was used as a template for PCR detection, the remaining cultures were stored at 4℃and colonies positive for detection were stored on plates or glycerol tubes for use. Through sequencing, the yeast transformation fragment containing the Neurospora crassa ergothioneine synthase gene Egt1 gene is successfully cloned.

Kluyveromyces marxianus transformation method: 1mL of overnight NBRC1777 yeast was collected, centrifuged at 8000rpm, the cells were collected, washed once with sterile water, then washed 2 times with 1×LiAc/TE, centrifuged at 8000rpm, and the supernatant was thoroughly washed. 10 mu L of carrier DNA, 2-3 mu g of DNA fragment to be transformed and 1-1.5 mu g of gRNA plasmid containing Cas9 protein and hygromycin gene expression frame are added into the thalli. 600. Mu.L of PEG solution (40%PEG 4000,0.1M LiAc,10mM Tris-HCl pH 7.5,1mM EDTA) and a volume of DTT (final concentration of DTT 10 mM) were added. After thoroughly mixing, the mixture was subjected to a water bath at 30℃for 15min and a water bath at 47℃for 15min. Next, the supernatant was discarded, and the mixture was incubated with 800. Mu.L of YPD medium for 2 hours, followed by instantaneous separation at 8000rpm, and the supernatant was discarded, and 100. Mu.L of the sterilized aqueous suspension cells were applied to an antibiotic (hygromycin) plate and cultured at 30℃for two days.

Positive clone screening is carried out by adopting genome amplification, and the screening method comprises the following steps: single colonies were randomly picked from the transformation plate and incubated in liquid YPD medium in a 1.5mL centrifuge tube for 12h. mu.L per tube was used for genome extraction and the remaining glycerol was stored at-20 ℃. Numbering each tube, adding 100 mu L of 200mM LiAc 1%SDS,70 ℃ for 15min, adding 100% ethanol for 300 mu L for resuspension, centrifuging for 12000rpm for 5min, removing supernatant, reserving sediment, adding 70% ethanol for 200 mu L, washing for 2 times, 1200rpm for 2min, and naturally drying for 15-30 min. Adding 50 mu L of sterile water for dissolution, thus obtaining a crude extract solution containing genome. 1 mu L of each tube is used as a template for PCR detection, the transformant which is detected to be positive is sequenced and verified to be the correct recombinant Kluyveromyces marxianus strain, and the recombinant Kluyveromyces marxianus strain is named as KME1-WZ01. Fermentation was performed in YPD medium and its 3, 5 and 7 day ergothioneine synthesis capacity was measured by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry and the results are shown in FIG. 1.

As can be seen from FIG. 1, only the Kluyveromyces marxianus strain KME1-WZ01 with Neurospora crassa ergothioneine synthase gene Egt1 introduced can normally synthesize ergothioneine, and further proves that the endogenous Egt2 gene can normally express and function.

The recombinant yeast strain KME1-WZ01 described above was passaged 9 times in a liquid YPD medium without resistance to remove hygromycin-containing resistant plasmids in order to continue yeast transformation and transformant selection. The endogenous Egt2 position is obtained from chromosome six, 1000bp upstream and 1000bp downstream are taken, a knockout box is formed through fusion PCR, the endogenous Egt2 in KME1-WZ01 is knocked out through yeast transformation, and the influence on ergothioneine synthesis is detected, and the result is shown in figure 2.

As can be seen from fig. 2, the endogenous Egt2 gene was knocked out in KME1-WZ01 strain, and the yield of ergothioneine was significantly reduced, further demonstrating its ability to participate in synthesis of ergothioneine. The synthesis of small amounts of ergothioneine in cells, which may be an isozyme for Egt2 also present in the strain, was also in further investigation.

EXAMPLE 3 Effect of different temperatures on the Synthesis of ergothioneine by KME1-WZ01

This example measures the effect of KME1-WZ01 on ergothioneine synthesis at 30℃and 37 ℃.

The preparation method comprises inoculating 20 μL of KMEl-WZ01 strain preserved in-80deg.C refrigerator into 1mL YPD liquid culture medium, culturing in shaking table at 30deg.C and 200rpm for 24 hr, transferring into 100mL YPD seed culture medium (i.e. YPD liquid culture medium), culturing in shaking table at 37deg.C for 18 hr, and adjusting to initial OD ₆₀₀ YPD (glucose 20g/L, yeast powder 10g/L, peptone 20 g/L) and YPD (xylose 20g/L, yeast powder 10g/L, peptone 20 g/L) were inoculated into each of 0.1 and YPD (glucose 20g/L, yeast powder 10g/L, peptone 20 g/L), and sealed with a gas-permeable sealing film at 200rpm, and the results of the measurement are shown in FIG. 3 after fermentation at 30℃for 3 days, fermentation at 5 days and fermentation at 7 days were detected in a 2mL centrifuge tube using a 1mL sampling apparatus.

As can be seen from FIG. 3, KME1-WZ01 can produce ergothioneine at 30℃and 37℃and can synthesize ergothioneine at a higher yield than 37℃in YPD medium, more preferably at 30℃as the most suitable fermentation temperature. There was no significant difference in ergothioneine content between 30℃and 37℃in YPX medium. The fermentation temperature in the fermentation production is not limited to 30℃and 37℃and is suitably between them.

EXAMPLE 4 KME1-WZ01 production of ergothioneine Using glucose, xylose and Glycerol

The present example determines the yield of ergothioneine from KME1-WZ01 under fermentation conditions of the carbon sources glucose (20 g/L), xylose (20 g/L) and glycerol (20 g/L).

The preparation method comprises inoculating 20 μL of KME1-WZ01 strain preserved in-80deg.C refrigerator into 1mL YPD liquid culture medium, culturing in shaking table at 30deg.C and 200rpm for 24 hr, transferring into 100mL YPD seed culture medium (i.e. YPD liquid culture medium), culturing in shaking table at 30deg.C for 18 hr, and adjusting to initial OD ₆₀₀ YPD (glucose 20g/L, yeast powder 10g/L, peptone 20 g/L), YPG (xylose 20g/L, yeast powder 10g/L, peptone 20 g/L) and YPG (glycerol 20g/L, yeast powder 20g/L, peptone 20 g/L) were inoculated into each of 0.1, sealed with a gas-permeable sealing film, fermented at 30℃for 7 days at 200rpm, 1mL of the fermented liquid was sampled, and the apparatus was tested in a 2mL centrifuge tube, and the results were shown in FIG. 4.

As can be seen from FIG. 4, KME1-WZ01 of the present example can accumulate a large amount of ergothioneine under the condition of three carbon sources, wherein glycerol is the best, and the highest yield is 104mg/L.

Example 5 KME1-WZ01 increased ergothioneine yield with precursor addition

This example measures the promotion of ergothioneine synthesis by KME1-WZ01 under precursor addition conditions.

The method comprises the following specific steps: inoculating 20 μL of KME1-WZ01 strain preserved in-80deg.C refrigerator into 1mL YPD liquid culture medium, culturing at 30deg.C in 200rpm shaker for 24 hr, transferring into 100mL YPD seed culture medium (i.e. YPD liquid culture medium), culturing in 30deg.C shaker for 18 hr, and adjusting to initial OD ₆₀₀ 0.1 was inoculated into YPD (glucose 20g/L, yeast powder 10g/L, peptone 20g/L,1g/L His+Met) and sealed with a gas-permeable sealing film, and fermented at 200rpm for 7After the day, the 1mL sample device was placed in a 2mL centrifuge tube, and the measurement results are shown in FIG. 5.

As can be seen from FIG. 5, KME1-WZ01 further improved the ability to synthesize ergothioneine in YPD medium when 1g/L His+Met two precursors were added.

EXAMPLE 6KME1-WZ01 Synthesis of ergothioneine content in 3L fermenter

Slant activation culture: scraping a ring of strain from a bacteria-preserving tube of a refrigerator at the temperature of minus 80 ℃, uniformly coating the strain on an activation inclined plane, culturing for 12 hours at the temperature of 30 ℃, transferring to a shake flask, and continuously culturing for 12 hours;

fermentation culture: and (3) taking a proper amount of sterile water, washing out thalli in the shake flask, and inoculating the bacterial suspension into a 3L fermentation tank containing 1L fermentation medium to start fermentation. The pH is controlled to be stable at about 5.5 in the fermentation process, the temperature is maintained at 30 ℃, the rotating speed is 600rpm, and the ventilation is 1L/min. After the glycerol in the medium had been consumed, the highly concentrated YPG medium was fed at a flow rate of 1 mL/h.

The composition of the fermentation medium is: 20g/L of glycerol, 20g/L of yeast powder, 20g/L of peptone and the balance of water.

The composition of the feed medium was: 100g/L of glycerol, 100g/L of yeast powder, 100g/L of peptone and the balance of water.

pH regulation: 2M NaOH and 2M H ₂ SO ₄ 。

The results of the fermentation are shown in FIG. 6, and the results show that the yield of ergothioneine can reach 630mg/L after 7 days of fermentation in 1L of fermentation medium. The yield is 6.1 times of that of the shake flask, which shows the potential and application value in producing ergothioneine as a microbial cell factory.

The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications and additions to the person skilled in the art, including combinations of the different embodiments, can be made without departing from the method of the invention, which modifications and additions are also to be considered as being within the scope of the invention.

Claims (10)

1. The Kluyveromyces marxianus (Kluyveromyces marxianus) strain for producing ergothioneine is characterized by comprising ergothioneine synthetic genes Egt1 and Egt2, wherein the Egt2 gene is an endogenous gene of the Kluyveromyces marxianus, the nucleotide sequence of the endogenous gene is shown as SEQ ID NO.1, the amino acid sequence of Egt2 gene expression is shown as SEQ ID NO.2, and the Egt1 gene is an exogenous gene;

the preservation number of the strain is CGMCC No.27310, and the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with the preservation date of 2023, 5 and 10.

2. The kluyveromyces marxianus strain for producing ergothioneine according to claim 1, wherein the ergothioneine synthesis gene Egt1 is derived from Neurospora crassa (Neurospora crassa) and has a nucleotide sequence shown in SEQ ID NO. 5.

3. The kluyveromyces marxianus strain for producing ergothioneine according to claim 2, wherein the strain is constructed by the following steps: transforming the Neurospora crassa ergothioneine synthesis gene Egt1 plasmid into an escherichia coli DH5 alpha clone strain, adopting colony PCR to carry out positive clone screening, transforming a target DNA fragment into a Kluyveromyces marxianus original strain, and obtaining the Kluyveromyces marxianus recombinant strain through sequencing verification of a transformant with positive PCR detection.

4. The ergothioneine synthetic gene Egt2 is characterized in that the Egt2 gene is derived from Kluyveromyces marxianus as described in claim 1, and the nucleotide sequence of the Kluyveromyces marxianus is shown as SEQ ID NO. 1.

5. An amplification primer pair of the ergothioneine synthesis gene Egt2 according to claim 4, wherein the forward primer of the amplification primer pair has a sequence shown in SEQ ID NO.3, and the reverse primer has a sequence shown in SEQ ID NO. 4.

6. An enzyme expressed by the ergothioneine synthesis gene Egt2 according to claim 4, wherein the amino acid sequence of the enzyme is shown in SEQ ID NO. 2.

7. Use of kluyveromyces marxianus as defined in claim 1 for the production of ergothioneine.

8. A process for producing ergothioneine by Kluyveromyces marxianus as claimed in claim 1, wherein the fermentation medium comprises 20g/L of carbon source selected from glucose, xylose or glycerol, 10-20g/L of yeast powder and 20g/L of peptone.

9. The method for producing ergothioneine by Kluyveromyces marxianus according to claim 8, wherein the carbon source is glycerol.

10. The method for producing ergothioneine by Kluyveromyces marxianus according to claim 8, wherein precursor histidine and methionine are added into the fermentation medium, and the addition amount of histidine and methionine is 1g/L.