Ergothioneine synthetic fusion enzyme, coding gene thereof, recombinant vector, recombinant strain and application of ergothioneine synthetic fusion enzyme and coding gene thereof

Abstract

The invention relates to the fields of genetic engineering and microbial fermentation, and discloses an ergothioneine synthetic fusion enzyme, and a coding gene, a recombinant vector and a recombinant strain thereof, and applications of the ergothioneine synthetic fusion enzyme and the coding gene, the recombinant vector and the recombinant strain. The ergothioneine synthetic fusion enzyme is an enzyme with an amino acid sequence as shown in SEQ ID NO: 1. The original strain of the recombinant strain is Yarrowia lipolytica, and the original strain of the recombinant strain is Yarrowia lipolytica; the method for producing the ergothioneine through fermentation comprises the following steps: inoculating a recombinant strain containing the ergothioneine synthesis fusion enzyme into a fermentation culture medium for fermentation. The ergothioneine synthesis fusion enzyme can promote a substrate channel while exerting the efficient expression and catalysis effects of ergothioneine synthetase, reduces the accumulation of intermediate metabolites, relieves the cell burden, and further improves the fermentation yield of ergothioneine.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and microbial fermentation, and particularly relates to an ergothioneine synthetic fusion enzyme, its encoding gene, recombinant vector, recombinant strain, and their applications. Background Art

[0002] Ergothioneine (ERG), also known as thiotriazolylalanine, is a thiol compound synthesized from histidine as a precursor. It has extremely high water solubility and mainly exists in the form of thione in aqueous solution. Ergothioneine has a relatively high redox potential, higher thermal stability and better pH stability compared to other thiol compounds such as glutathione, and is not easily oxidized spontaneously. It is a natural amino acid-based strong antioxidant with a variety of unique biological functions and pharmacological activities, including antioxidant, free radical scavenging, cell protection, anti-radiation damage, regulation of inflammation, and adjuvant treatment of various diseases, etc. It has broad market application prospects in the fields of medicine, cosmetics, and functional foods.

[0003] In nature, it is known that microorganisms such as mycobacteria, methylobacteria, cyanobacteria, and mushrooms can naturally synthesize ergothioneine, but there is no literature report that animals and plants can synthesize it by themselves. They can only absorb and accumulate it in tissues or cells from the environment. The human body absorbs ergothioneine from food through the high-affinity transporter for ergothioneine, organic cation transporter type I OCTN1 (encoded by the gene SLC22A4).

[0004] The production methods of ergothioneine include extraction method, chemical synthesis method, and microbial fermentation method. The extraction method mainly obtains it from natural raw materials such as fruiting bodies of mushrooms and grains. However, the raw material consumption is large, there are many impurities, the yield ratio is low, and the extraction cost is high, resulting in low extraction efficiency and difficulty in meeting large-scale production. The chemical synthesis method has cumbersome synthesis steps, high processing difficulty, high synthesis cost, and the by-products generated during the synthesis process cause serious environmental pollution, making it difficult to ensure the safety of the target product, thus affecting the commercial production of ergothioneine. The microbial fermentation method for synthesizing ergothioneine is the most promising production method. It has been verified in microorganisms such as Escherichia coli, Saccharomyces cerevisiae, Aspergillus oryzae, and Methylobacterium aquaticum 22A. It has advantages such as simple cultivation, short fermentation cycle, and low production cost. It is the most effective method for future production of ergothioneine. Furthermore, through metabolic engineering strategies such as metabolic regulation and pathway modification, the production efficiency of ergothioneine can be effectively enhanced, making it the best choice for realizing large-scale industrial production of ergothioneine.

[0005] With the rapid development of synthetic biology tools, some microorganisms have been reconstructed for the biosynthesis of ergothioneine. Currently, the heterologous expression of ERG synthase has been successfully verified in microorganisms such as Escherichia coli, Saccharomyces cerevisiae, Aspergillus oryzae, and Methylobacterium aquaticum. Although the heterologous expression of ERG biosynthetic genes in different chassis microorganisms can improve the synthesis efficiency, there are still problems such as excessive accumulation of intermediates, resulting in a heavy cell burden. In addition, the catalytic efficiency of ERG biosynthetic enzymes is low, leading to low ERG production and inability to meet the requirements of large-scale production. Summary of the Invention

[0006] The object of the present invention is to overcome the problems existing in the prior art, such as excessive accumulation of intermediates in the engineering bacteria for the production of ergothioneine, resulting in a heavy cell burden, low yield, and inability to meet the requirements of large-scale production. The present invention provides an ergothioneine synthetic fusion enzyme, its encoding gene, recombinant vector, recombinant strain, and their applications. The ergothioneine synthetic fusion enzyme can promote substrate channeling while exerting the high-efficiency expression and catalytic efficacy of ergothioneine synthase, reduce the accumulation of intermediate metabolites, relieve the cell burden, and further improve the fermentation yield of ergothioneine.

[0007] To achieve the above object, the first aspect of the present invention provides an ergothioneine synthetic fusion enzyme, which is the enzyme described in any one of (a)-(e):

[0008] (a) An enzyme having the amino acid sequence shown in SEQ ID NO: 1;

[0009] (b) An enzyme having an amino acid sequence in which one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, or added, and still having the activity of ergothioneine synthetic fusion enzyme;

[0010] (c) An enzyme having an amino acid sequence with more than 90% homology to the amino acid sequence shown in SEQ ID NO: 1 and having the activity of ergothioneine synthetic fusion enzyme;

[0011] (d) An enzyme having an amino acid sequence with a tag linked to the amino terminus and / or carboxyl terminus of the amino acid sequence described in (a), (b), or (c);

[0012] (e) An enzyme having an amino acid sequence with a signal sequence linked to the amino terminus of the amino acid sequence described in (a), (b), or (c).

[0013] Preferably, the ergothioneine synthetic fusion enzyme is an enzyme having an amino acid sequence with more than 95%, preferably more than 98% homology to the amino acid sequence shown in SEQ ID NO: 1 and having the activity of ergothioneine synthetic fusion enzyme.

[0014] In the second aspect of the present invention, a gene encoding ergothioneine synthetic fusion enzyme is provided, and the gene has a nucleotide sequence encoding the ergothioneine synthetic fusion enzyme as described above.

[0015] Preferably, the gene is a nucleotide sequence encoding an enzyme having the amino acid sequence shown in SEQ ID NO: 1.

[0016] More preferably, the gene has the nucleotide sequence shown in SEQ ID NO: 2.

[0017] In the third aspect of the present invention, a recombinant vector is provided, and the recombinant vector contains the gene as described above.

[0018] Preferably, the expression vector of the recombinant vector is pUC-HUH-TEF plasmid.

[0019] In the fourth aspect of the present invention, a recombinant strain is provided, and the recombinant strain expresses the gene as described above or contains the recombinant vector as described above; the starting strain of the recombinant strain is Yarrowia lipolytica.

[0020] Preferably, the starting strain of the recombinant strain is Yarrowia lipolytica in which the coding gene ku70 for non-homologous recombination has been knocked out, and the nucleotide sequence of the coding gene ku70 is as shown in SEQ ID NO: 3.

[0021] In the fifth aspect of the present invention, provided is the use of at least one of the ergothioneine synthetic fusion enzyme as described above, the gene as described above, the recombinant vector as described above, and the recombinant strain as described above in the preparation of ergothioneine.

[0022] In the sixth aspect of the present invention, a method for fermentatively producing ergothioneine is provided, and the method includes: inoculating the recombinant strain as described above into a fermentation medium for fermentation.

[0023] Preferably, the fermentation medium contains: 20 - 40 g / L of glucose, 0.1 - 0.3 g / L of calcium chloride, 0.4 - 0.8 g / L of potassium sulfate, 0.6 - 1 g / L of sodium chloride, 6 - 10 mg / L of ferrous sulfate heptahydrate, 4 - 6 mg / L of calcium pantothenate, 3 - 5 mg / L of manganese chloride tetrahydrate, 0.06 - 0.1 mg / L of sodium molybdate dihydrate, 6 - 10 mg / L of vitamin B6, 12 6 - 10 mg / L of vitamin B6, 8 - 12 g / L of yeast powder, and 15 - 25 g / L of tryptone.

[0024] Preferably, the fermentation conditions include: an inoculum amount of 2 - 10%, a temperature of 25 - 35 °C, a rotation speed of 200 - 250 rpm, and a time of 4 - 6 d.

[0025] Through the above technical solution, the beneficial effects of the present invention are as follows:

[0026] The ergothioneine synthetic fusion enzyme provided by the present invention has both the high expression and catalytic activities of the ergothioneine biosynthetic enzymes Egt1 and Egt2, and can form a direct path between Egt1 and Egt2 and promote substrate channeling, prevent unwanted competing pathways, bypass unfavorable equilibria, reduce the accumulation of intermediate metabolites, relieve the cell burden, effectively promote the synthesis of ergothioneine in the cell metabolism process, improve the yield of ergothioneine produced by microbial fermentation, and provide a theoretical reference and research idea for the subsequent modification and optimization of the key enzymes for ergothioneine synthesis.

[0027] Further preferably, using the lipolytic yeast strain with the non-homologous recombination encoding gene ku70 knocked out as the host strain can further improve the yield of ergothioneine and is conducive to realizing large-scale production. Description of the Drawings

[0028] Figure 1 It is the map of the recombinant plasmid pUC-HUH-GS2 in Example 1. Detailed Embodiments

[0029] The endpoints and any values disclosed in this article for a range are not limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0030] The first aspect of the present invention provides an ergothioneine synthetic fusion enzyme, and the ergothioneine synthetic fusion enzyme is the enzyme described in any one of (a)-(e):

[0031] (a) The enzyme having the amino acid sequence shown in SEQ ID NO: 1;

[0032] (b) The enzyme shown by the amino acid sequence in which one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted or added and still have the activity of the ergothioneine synthetic fusion enzyme;

[0033] (c) The enzyme shown by the amino acid sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO: 1 and having the activity of the ergothioneine synthetic fusion enzyme;

[0034] (d) The enzyme shown by the amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the amino acid sequence described in (a), (b) or (c);

[0035] (e) An enzyme represented by an amino acid sequence having a signal sequence linked to the amino terminus of the amino acid sequence described in (a), (b), or (c).

[0036] The 20 amino acid residues that make up proteins can be divided into four categories according to the polarity of the side chains, namely non-polar amino acids, polar uncharged amino acids, positively charged amino acids, and negatively charged amino acids (see "Biochemistry" (Second Edition), Volume I, Shen Tong, Wang Jingyan, pp. 82-83, Higher Education Press, December 1990). If an amino acid residue substitution within the same category occurs in a protein, for example, arginine (Arg) replaces lysine (Lys) or leucine (Leu) replaces isoleucine (Ile), the role played by the residue in the protein domain (such as providing a positive charge or forming a hydrophobic pocket structure) remains unchanged, and the protein function can still be achieved. Such amino acid residue substitutions within the same category can occur at any amino acid residue position of the above-mentioned ergothioneine synthase fusion enzyme.

[0037] In addition to the above-mentioned amino acid residue substitutions, the ergothioneine synthase fusion enzyme provided by the present invention also includes proteins having one or more amino acid residue deletions and / or additions at any amino acid residue position compared to the amino acid sequence shown in SEQ ID NO: 1, or both.

[0038] As described above, the ergothioneine synthase fusion enzyme provided by the present invention can also be modified or mutated to obtain a derived protein. A derived protein refers to a protein having a difference in amino acid sequence from the ergothioneine synthase fusion enzyme having the above-mentioned amino acid sequence, and may also have a difference in the form of a modification that does not affect the sequence, or both. Forms of modification (usually without changing the primary structure, i.e., without changing the amino acid sequence) include: chemically derived forms of proteins in vivo or in vitro, such as acetylation or carboxylation; glycosylation forms, such as those proteins that undergo glycosylation modification during protein synthesis and processing or further processing steps; sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine), etc.

[0039] In the present invention, the enzyme may also be an enzyme represented by an amino acid sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO: 1 and having ergothioneine synthase fusion enzyme activity. Preferably, the ergothioneine synthase fusion enzyme is an enzyme represented by an amino acid sequence having more than 95%, more preferably more than 98% homology with the amino acid sequence shown in SEQ ID NO: 1 and having ergothioneine synthase fusion enzyme activity.

[0040] For the convenience of purification, common tags in the art can also be used to modify (a), (b), or (c). For example, it can be obtained by connecting an amino acid sequence with a tag (such as at least one of Poly-Arg, Poly-His, FLAG, Strep-tagⅡ, and c-myc) to the amino terminus and / or carboxyl terminus of (a). The tag does not affect the activity of the ergothioneine synthase fusion enzyme of the present invention, and whether to add a tag can be selected according to requirements during actual application.

[0041] In the present invention, a signal sequence can also be connected to the amino terminus of the ergothioneine synthase fusion enzyme. The signal sequence can be derived from Escherichia coli and / or Bacillus subtilis, but is not limited thereto.

[0042] In the present invention, still having enzyme activity means that under the same assay conditions, the enzyme derived from (a) still has enzyme activity, and the percentage (relative activity) of its enzyme activity to the enzyme activity of (a) is not less than 90% (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%).

[0043] The above-mentioned ergothioneine synthase fusion enzyme can be obtained by artificial synthesis, or its coding gene can be synthesized first and then obtained by biological expression.

[0044] The second aspect of the present invention provides a gene encoding an ergothioneine synthase fusion enzyme, and this gene has a nucleotide sequence encoding the ergothioneine synthase fusion enzyme as described above.

[0045] According to the present invention, preferably, the gene is a nucleotide sequence encoding an enzyme having the amino acid sequence shown in SEQ ID NO: 1.

[0046] As described above, correspondingly, the 5' end and / or 3' end of the nucleotide sequence can also be connected with a coding sequence of a tag.

[0047] According to the present invention, preferably, the gene has the nucleotide sequence shown in SEQ ID NO: 2.

[0048] The nucleotide sequence provided by the present invention can generally be obtained by polymerase chain reaction (PCR) amplification method, recombination method, or artificial synthesis method. For example, those skilled in the art can easily obtain templates and primers according to the nucleotide sequence provided by the present invention and use PCR to amplify the relevant sequence. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by the recombination method. Usually, the obtained nucleotide sequence is cloned into a vector, then transferred into a genetically engineered bacterium, and then the relevant nucleotide sequence is separated from the proliferated host cells by conventional methods.

[0049] In addition, the relevant nucleotide sequences can also be synthesized by known artificial chemical synthesis methods.

[0050] The third aspect of the present invention provides a recombinant vector, and the recombinant vector contains the gene as described above.

[0051] The "vector" used in the recombinant vector can be selected from various vectors known in the art, such as various commercially available plasmids, cosmids, phages, retroviruses, etc. The preferred expression vector of the present invention is the pUC-HUH-TEF plasmid.

[0052] Various methods for constructing recombinant vectors are known in the art to ligate the gene encoding ergothioneine synthase fusion enzyme to an expression vector to prepare a recombinant vector. For example, but not limited to, the classical "restriction-ligation" method, the Gateway cloning system developed by Invitrogen Corporation, and the ClonExpress cloning system developed by Novoprotein Scientific Inc. (such as the ClonExpress MultiS One Step Cloning Kit).

[0053] The fourth aspect of the present invention provides a recombinant strain, and the recombinant strain expresses the gene as described above or contains the recombinant vector as described above; the starting strain of the recombinant strain is Yarrowia lipolytica.

[0054] In the present invention, the recombinant vector can be transformed, transduced or transfected into the starting strain by conventional methods in the art, such as homologous recombination, heat shock transformation, calcium chloride chemical transformation, high-voltage electroporation transformation, and homologous recombination is preferably used.

[0055] Preferably, in the present invention, the starting strain of the recombinant strain is Yarrowia lipolytica in which the coding gene ku70 responsible for non-homologous recombination is knocked out, and the nucleotide sequence of the coding gene ku70 is as shown in SEQ ID NO: 3. The inventors found during the research process that using Yarrowia lipolytica in which the coding gene ku70 responsible for non-homologous recombination is knocked out as the host strain can further improve the high-level expression and catalytic activity of ergothioneine synthase fusion enzyme, reduce the flux of unwanted competing pathways during metabolism, and better improve the yield of ergothioneine.

[0056] Exemplarily, using the Yarrowia lipolytica strain MYA2613 purchased from the American Type Culture Collection (ATCC) as the original strain, the coding gene ku70 responsible for non-homologous recombination was knocked out, and the nucleotide sequence of the coding gene ku70 is as shown in SEQ ID NO: 3, to obtain Yarrowia lipolytica Po1fΔku70, which can be used as the starting strain of the present invention.

[0057] In the present invention, when using a Yarrowia lipolytica strain with the coding gene ku70 of non-homologous recombination knocked out as the starting strain, after introducing the recombinant vector into the starting strain, positive clones can be screened out through a selection marker (such as a resistance gene), and verified by genomic PCR or by sequencing the genomic DNA, thereby obtaining the recombinant strain for producing terpenoids. For example, the Frozen-EZ Yeast Transformation II Kit yeast transformation kit produced by Zymo Research can be used for the process of preparing competent cells and transformation.

[0058] Based on the high catalytic activity of the ergothioneine synthesis fusion enzyme in the present invention, the fifth aspect of the present invention provides the use of at least one of the aforementioned ergothioneine synthesis fusion enzyme, the aforementioned gene, the aforementioned recombinant vector, and the aforementioned recombinant strain in the preparation of ergothioneine.

[0059] The sixth aspect of the present invention provides a method for fermentatively producing ergothioneine, which includes: inoculating the aforementioned recombinant strain into a fermentation medium for fermentation.

[0060] In the present invention, the fermentation process can adopt a conventional culture process. Preferably, the recombinant strain is first inoculated into a seed medium for seed culture to obtain a seed solution, and then the seed solution is inoculated into a fermentation medium for fermentation culture.

[0061] In the present invention, the seed medium contains the nutrient components required for the growth of the recombinant strain, so that the recombinant strain can be activated and propagated. Preferably, the seed medium contains: yeast extract 8 - 12 g / L, peptone 15 - 25 g / L, glucose 15 - 25 g / L; exemplarily, the seed medium adopts YPD liquid medium.

[0062] In the present invention, the condition parameters such as the temperature and time of seed culture only need to be able to meet the growth requirements of the recombinant strain. Preferably, the conditions of seed culture at least include: the temperature is 25 - 35 °C, specifically it can be 25 °C, 27 °C, 29 °C, 31 °C, 33 °C, 35 °C, or any value between the above two values; the rotation speed is 200 - 250 rpm, specifically it can be 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, or any value between the above two values; the time is 20 - 30 h, specifically it can be 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, or any value between the above two values.

[0063] According to the present invention, the fermentation medium contains the nutrients required for the growth of the recombinant strain, so that the recombinant strain can multiply in large quantities. Preferably, when the starting strain of the recombinant strain is Yarrowia lipolytica with the non-homologous recombination encoding gene ku70 knocked out, the fermentation medium contains: glucose 50 - 70 g / L, calcium chloride 0.1 - 0.3 g / L, potassium sulfate 0.4 - 0.8 g / L, sodium chloride 0.6 - 1 g / L, ferrous sulfate heptahydrate 6 - 10 mg / L, calcium pantothenate 4 - 6 mg / L, manganese chloride tetrahydrate 3 - 5 mg / L, sodium molybdate dihydrate 0.06 - 0.1 mg / L, vitamin B6 6 - 10 mg / L, vitamin B 12 6 - 10 mg / L, yeast powder 8 - 12 g / L, and tryptone 15 - 25 g / L. Under this preferred embodiment, the fermentation yield of ergothioneine can be further improved.

[0064] According to the present invention, preferably, the fermentation conditions include: the inoculum amount is 2 - 10% (v / v), specifically it can be 2% (v / v), 4% (v / v), 6% (v / v), 8% (v / v), 10% (v / v), or any value between the above two values; the temperature is 25 - 35 °C, specifically it can be 25 °C, 27 °C, 29 °C, 31 °C, 33 °C, 35 °C, or any value between the above two values; the rotation speed is 200 - 250 rpm, specifically it can be 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, or any value between the above two values; the time is 4 - 6 d, specifically it can be 4 d, 4.5 d, 5 d, 5.5 d, 6 d, or any value between the above two values.

[0065] Since the recombinant strain provided by the present invention contains the gene encoding the ergothioneine synthesis fusion enzyme, it can efficiently express the ergothioneine synthesis fusion enzyme. After fermentation and separation and purification, high-purity ergothioneine synthesis fusion enzyme can be obtained. Preferably, the separation process of the ergothioneine synthesis fusion enzyme includes: performing solid-liquid separation on the fermentation broth obtained by fermentation to obtain a bacterial sludge, and using the glass bead crushing method in a homogenizer to separate and obtain the ergothioneine synthesis fusion enzyme.

[0066] The present invention can also prepare the fermentation broth obtained by fermentation into a fermentation agent for easy application. The fermentation agent can specifically exist in liquid form or solid form. The fermentation agent can contain auxiliary materials commonly added in the preparation of bacterial agents in the art, and those skilled in the art can select according to needs. Preferably, relative to each gram of the fermentation agent, the content of the recombinant strain is 10 7 - 10 9 CFU.

[0067] According to a particularly preferred embodiment of the present invention, the method for fermentatively producing ergothioneine comprises: inoculating the recombinant strain as described above into a seed medium first, performing seed culture at a temperature of 25 - 35 °C and a rotation speed of 200 - 250 rpm for 20 - 30 h to obtain a seed solution, and then inoculating the seed solution into a fermentation medium and performing fermentation culture at a temperature of 25 - 35 °C and a rotation speed of 200 - 250 rpm for 4 - 6 d;

[0068] Wherein, the seed medium contains: 8 - 12 g / L of yeast extract, 15 - 25 g / L of peptone, and 15 - 25 g / L of glucose; the fermentation medium contains: 20 - 40 g / L of glucose, 0.1 - 0.3 g / L of calcium chloride, 0.4 - 0.8 g / L of potassium sulfate, 0.6 - 1 g / L of sodium chloride, 6 - 10 mg / L of ferrous sulfate heptahydrate, 4 - 6 mg / L of calcium pantothenate, 3 - 5 mg / L of manganese chloride tetrahydrate, 0.06 - 0.1 mg / L of sodium molybdate dihydrate, 6 - 10 mg / L of vitamin B6, 12 6 - 10 mg / L of vitamin B6, 8 - 12 g / L of yeast powder, and 15 - 25 g / L of tryptone.

[0069] The present invention will be described in detail below by way of examples.

[0070] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following examples can all be obtained from commercial channels unless otherwise specified.

[0071] In the following examples, the starting strain was Yarrowia lipolytica Po1fΔku70 (MatA, ura3-302, leu2-270, xpr2-322, axp2-delta, NU49, XPR2::SUC2 MYA2613Δku70::hisG), which was derived from the Yarrowia lipolytica strain MYA2613 purchased from the American Type Culture Collection (ATCC). Using the method described in the reference "Gao S, Tong Y, Zhu L, et al. Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous β-carotene production [J]. Metabolic Engineering, 2017:192", the coding gene ku70 responsible for non-homologous recombination was knocked out. The nucleotide sequence of the coding gene ku70 is shown in SEQ ID NO: 3, and Yarrowia lipolytica Po1fΔku70 was obtained; this strain has been described and disclosed in CN114525215A.

[0072] 1. Culture Media and Reagents

[0073] YPD liquid medium: Peptone 20 g / L, Yeast Extract 10 g / L, Glucose 20 g / L;

[0074] YPD solid medium: Peptone 20 g / L, Yeast Extract 10 g / L, Glucose 20 g / L, Agar Powder 20 g / L;

[0075] LB solid medium: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, Agar Powder 20 g / L;

[0076] YNB-Leu medium: Glucose 20 g / L, Yeast Nitrogen Base (YNB, yeast nitrogen base without amino acids) 1.7 g / L, (NH4)2SO4 5 g / L, Leu (leucine) 1.7 g / L, Agar Powder 3 g / L.

[0077] 2. Amplification of Gene Fragments (PCR Amplification Method)

[0078] The PCR enzyme used in PCR amplification was PrimeSTAR Max DNA Polymerase from TAKARA; the PCR amplification system is shown in Table 1 below.

[0079] Table 1 PCR Amplification System

[0080]

[0081]

[0082] 3. The content of ergothioneine was quantified by high performance liquid chromatography. Shimadzu LC-20AD liquid chromatography was used with an SPD-20A ultraviolet / visible light detector. The chromatographic column was Poroshell 120, HILIC (4.6x250mm, 4μm); Detection procedure: 80% acetonitrile was used as the mobile phase, the wavelength was 254nm, the column oven temperature was 35°C, and the flow rate was 0.8mL·min -1 , the injection volume was 10μL, and the analysis time was 20min; the standard product of ergothioneine was used for qualitative and quantitative analysis.

[0083] Example 1 Preparation of the target gene and construction of the recombinant plasmid

[0084] (I) Preparation of the target gene and gene elements

[0085] 1. According to the nucleotide sequence of the orotidine-5'-phosphate decarboxylase encoding gene ura in Yarrowia lipolytica (Y. lipolytica) provided on NCBI (genebank accession number AJ306421.1) and the hisG tag (genebank accession number AF324729.1), it was commissioned to Tsingke Biotechnology Co., Ltd. for synthesis. The two hisG tag encoding gene sequences were inserted into the plasmid pUC. Specifically, pUC57 was used as the backbone. After digestion with ECORI and HindIII enzymes, the backbone was recovered. The hisG tag encoding gene was subjected to one-step cloning using the ClonExpress MultiS One Step Cloning Kit to construct the plasmid pUC-hisG-hisG. Then, using this as the backbone, after digestion with HindIII enzyme, the backbone was recovered. The ura encoding gene was subjected to one-step cloning using the ClonExpressMultiS One Step Cloning Kit to construct the plasmid pUC-hisG-ura-hisG (i.e., pUC-HUH-TEF) by inserting the orotidine-5'-phosphate decarboxylase encoding gene into the two hisG tag encoding gene sequences to enable ura marker recovery.

[0086] 2. Acquisition of homologous arms of IntF-up and IntF-down: Using IntF-up-F and IntF-up-R as primers (nucleotide sequences are shown in SEQ ID NO: 23 and SEQ ID NO: 24), the genomic DNA of Yarrowia lipolytica Po1fΔku70 was used as a template to amplify the IntF-up gene, and its nucleotide sequence is shown in SEQ ID NO: 4; using IntF-down-F and IntF-down-R as primers (nucleotide sequences are shown in SEQ ID NO: 25 and SEQ ID NO: 26), the genomic DNA of Yarrowia lipolytica Po1fΔku70 was used as a template to amplify the IntF-down gene, and its nucleotide sequence is shown in SEQ ID NO: 5;

[0087] 3. Acquisition of the Egt1-Egt2 fusion enzyme expression cassette element: According to the ergothioneine biosynthesis genes from Trichoderma reesei provided on NCBI: Egt1 (Genebank accession number: XP_006968620) and Egt2 (Genebank accession number: XP_006968735), the nucleotide sequences were optimized according to the codon preference of Yarrowia lipolytica and synthesized by Tsingke Biotechnology Co., Ltd. The nucleotide sequence of Egt1 is shown in SEQ ID NO: 6, and the nucleotide sequence of Egt2 is shown in SEQ ID NO: 7; The promoter P TEF and the terminator P XPR2t After codon optimization, (GGGGS)2 was synthesized in the form of primer homologous arms. The specific nucleotide sequence of (GGGGS)2 is shown in Table 2.

[0088] The primer sequences used in this example are shown in Table 3.

[0089] Table 2 Table 3

[0090]

[0091] (2) Construction of recombinant plasmids

[0092] 1. Construction of recombinant plasmid pUC-HUH-GS1

[0093] The vector plasmid pUC-HUH-TEF was digested with the restriction endonuclease Pac I, verified by agarose gel electrophoresis and recovered by gel extraction; The digestion system (50 μL): 4000 μg of vector plasmid, 2 μL of Pac I enzyme, 5 μL of 10×QCut Buffer, and ddH2O was quantified to 50 μL.

[0094] Use Prime Star high-fidelity DNA polymerase to prepare a PCR reaction system to amplify the gene expression cassette of ergothioneine synthetic fusion enzyme [Egt1-(GGGGS)2-Egt2] (the amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2); the PCR reaction program is: 95°C for 5 min, 95°C for 30 s, 58°C for 30 s, 72°C for 40 s. Starting from the second step, set 33 cycles. After obtaining the PCR product, verify it by agarose gel electrophoresis, and then cut the gel, recover and purify it.

[0095] Use the Clon Express MultiS One Step CloningKit of Nanjing Novoprotein Scientific Inc. to achieve one-step cloning. The one-step cloning system (10 μL): 4 μL of the target gene fragment, 1 μL of the vector digested fragment, 5 μL of 2×Clon Express Mix, and connect at 50°C for 50 min to obtain the ligation product pUC-HUH-GS2; further transform the ligation product pUC-HUH-GS2 into Escherichia coli DH5α competent cells. The transformation method is as follows:

[0096] 1) Under sterile conditions, take 100 μL of competent cells, add the ligation product and mix well, and place on ice for 30 min;

[0097] 2) Heat shock at 42°C for 90 s, and quickly place on ice for 3 min;

[0098] 3) Add 1000 μL of LB medium, incubate at 37°C and 220 r / min for 40 min;

[0099] 4) Take 200 μL and spread it on an LB solid medium plate containing 100 μg / mL ampicillin, invert and culture overnight at 37°C, select positive transformants, extract plasmids, and the sequencing verification results show successful ligation, obtaining the recombinant plasmid pUC-HUH-GS2, and its map is as Figure 1 shown.

[0100] Example 2 Construction of recombinant Yarrowia lipolytica strains YE01 - YE05

[0101] Transform the recombinant plasmid obtained in Example 1 into Yarrowia lipolytica PolfΔku70 to obtain the recombinant Yarrowia lipolytica strain YErg04. The specific method is as follows:

[0102] (1) Culture Yarrowia lipolytica Polf Δku70 overnight in YPD liquid medium until OD 600When it grows to 0.8, competent cells are prepared (using the kit Zymogen Frozen EZ Yeast Transformation Kit II, manufactured by Zymo Research Corporation).

[0103] (2) The recombinant plasmid pUC-HUH-GS2 was transformed into Yarrowia lipolytica PolfΔku70 using the Zymogen Frozen EZ Yeast Transformation Kit II produced by Zymo Research Corporation to perform homologous recombination;

[0104] (3) Screening was carried out using the screening medium YNB-Leu, and the positive clones with correct PCR identification were named recombinant Yarrowia lipolytica YErg04.

[0105] Example 3 Fermentation and Detection of Recombinant Strains

[0106] The recombinant Yarrowia lipolytica YErg04 constructed in Example 2 was used for fermentative production of ergothioneine, and the obtained ergothioneine was quantitatively analyzed by HPLC detection.

[0107] The process of fermentative production was as follows: The recombinant Yarrowia lipolytica YErg04 was cultured in YPD liquid medium at a temperature of 30 °C and a rotation speed of 220 rpm for 24 h to obtain a seed solution;

[0108] The seed solution was inoculated into 50 mL of fermentation medium (containing 40 g / L of glucose, 0.2 g / L of calcium chloride, 0.6 g / L of potassium sulfate, 0.8 g / L of sodium chloride, 8 mg / L of ferrous sulfate heptahydrate, 5 mg / L of calcium pantothenate, 4 mg / L of manganese chloride tetrahydrate, 0.08 mg / L of sodium molybdate dihydrate, 8 mg / L of vitamin B6, vitamin B 12 8 mg / L, 10 g / L of yeast extract, and 20 g / L of tryptone) at a temperature of 30 °C and a rotation speed of 220 rpm, and shaken and cultured for 5 days for fermentation;

[0109] After the fermentation was completed, 1 mL of the fermentation broth was boiled at 95 °C for 15 minutes, and then centrifuged at 12000 r for 15 min to collect the supernatant.

[0110] Quantitative analysis of ergothioneine: The supernatant was filtered through an aqueous phase filter membrane (0.22 μm), and the obtained filtrate was transferred to a liquid phase bottle. The content of ergothioneine was detected by HPLC. Each treatment was set with 3 replicates, and the average value was taken; the fermentation results are shown in Table 4.

[0111] The fermentation results are shown in Table 4. After 120 h of fermentation, the yield of ergothioneine by the recombinant strain YErg04 was 547.599 mg / L, which is the highest value reported for the production of ergothioneine by microbial shake-flask fermentation so far.

[0112] Example 4

[0113] The recombinant Yarrowia lipolytica YErg04 was cultured in YPD liquid medium at 25 °C and 200 rpm for 30 h to obtain a seed solution.

[0114] The seed solution was inoculated into 50 mL of fermentation medium (containing 30 g / L glucose, 0.3 g / L calcium chloride, 0.8 g / L potassium sulfate, 0.6 g / L sodium chloride, 6 mg / L ferrous sulfate heptahydrate, 6 mg / L calcium pantothenate, 5 mg / L manganese chloride tetrahydrate, 0.06 mg / L sodium molybdate dihydrate, 10 mg / L vitamin B6, vitamin B 12 6 mg / L, 12 g / L yeast extract, and 25 g / L tryptone) at 25 °C and 200 rpm, and shaken for 6 days for fermentation.

[0115] After fermentation, 1 mL of the fermentation broth was boiled at 95 °C for 15 minutes, and then centrifuged at 12000 r for 15 min to collect the supernatant.

[0116] The supernatant was filtered through an aqueous phase filter membrane (0.22 μm), and the obtained filtrate was transferred to a liquid phase bottle. The ergothioneine content was detected by HPLC. Each treatment was set with 3 replicates, and the average value was taken. The fermentation results are shown in Table 4.

[0117] Example 5

[0118] The recombinant Yarrowia lipolytica YErg04 was cultured in YPD liquid medium at 35 °C and 250 rpm for 20 h to obtain a seed solution.

[0119] The seed solution was inoculated into 50 mL of fermentation medium (containing 20 g / L glucose, 0.1 g / L calcium chloride, 0.4 g / L potassium sulfate, 1 g / L sodium chloride, 10 mg / L ferrous sulfate heptahydrate, 4 mg / L calcium pantothenate, 3 mg / L manganese chloride tetrahydrate, 0.1 mg / L sodium molybdate dihydrate, 6 mg / L vitamin B6, vitamin B 12 10 mg / L, 8 g / L yeast extract, and 15 g / L tryptone) at 35 °C and 250 rpm, and shaken for 4.5 days for fermentation.

[0120] After fermentation, 1 mL of the fermentation broth was boiled at 95 °C for 15 minutes, and then centrifuged at 12,000 r for 15 min to collect the supernatant.

[0121] The supernatant was filtered through an aqueous phase filter membrane (0.22 μm), and the obtained filtrate was transferred to a liquid phase bottle. The ergothioneine content was detected by HPLC. Three replicates were set for each treatment, and the average value was taken; the fermentation results are shown in Table 4.

[0122] Example 6

[0123] The method in Example 3 was used for the fermentation of the recombinant strain, except that the fermentation medium was replaced with: 40 g / L glucose, 10 g / L yeast powder, and 20 g / L tryptone; the fermentation results are shown in Table 4.

[0124] Example 7

[0125] The method in Example 3 was used for fermentation, except that the starting strain of the recombinant strain YErg04 was replaced with the Yarrowia lipolytica strain MYA2613; the fermentation results are shown in Table 4.

[0126] Comparative Example 1

[0127] The recombinant plasmids pUC-HUH-GS1, pUC-HUH-EK1, pUC-HUH-2A, and pUC-HUH-EK2 were constructed respectively using the method in Example 1. The construction method was similar to that of the recombinant plasmid pUC-HUH-GS2, except that the inserted gene (GGGGS)2 was replaced with (GGGGS)1, (EAAAK)1, P2A, and (EAAAK)2 respectively; the specific nucleotide sequences are shown in Table 2.

[0128] The recombinant Yarrowia lipolytica strains YErg01 (corresponding to the recombinant plasmid pUC-HUH-GS1), YErg02 (corresponding to the recombinant plasmid pUC-HUH-EK1), YErg03 (corresponding to the recombinant plasmid pUC-HUH-2A), and YErg05 (corresponding to the recombinant plasmid pUC-HUH-EK2) were constructed respectively using the method in Example 2, and the fermentation and detection of the recombinant strains were carried out using the method in Example 3. The results are shown in Table 4.

[0129] The fermentation results showed that after 120 h of fermentation, the yields of ergothioneine of the recombinant strains YErg01, YErg02, YErg03, and YErg05 were much lower than that of the recombinant strain YErg04.

[0130] Comparative Example 2

[0131] Fermentation was carried out using the method in Example 3, except that the recombinant strain YErg04 was replaced with the starting strain Yarrowia lipolytica Po1fΔku70; the fermentation results are shown in Table 4.

[0132] Comparative Example 3

[0133] The recombinant strain was constructed using the method in Example 2, except that the starting strain was replaced with Escherichia coli BL21; specifically, the fusion enzyme [Egt1-(GGGGS)2-Egt2] was transferred into the plasmid vector of pET-28a, and Escherichia coli BL21 was introduced to obtain recombinant Escherichia coli. The recombinant Escherichia coli was inoculated into a test tube containing 5 mL of LB liquid medium and cultured at 37 °C and 220 rpm until OD 600 = 0.8 - 1 to obtain a seed solution; the seed solution was transferred and inoculated into 50 mL of fermentation medium at an inoculation amount of 1% by volume (the components of the fermentation medium are: peptone 4 g / L, yeast extract 6 g / L, glucose 20 g / L, KH2PO4 4 g / L, MgSO4·7H2O 1 g / L, (NH4)2SO4 6 g / L, Na2HPO4 9.916 g / L, citric acid 2 g / L, biotin 0.1 g / L, ammonium ferric citrate 0.01 g / L, trace elements 1 mL / L), induced with IPTG at a final concentration of 0.2 mM, and fermented at 25 °C for 72 h.

[0134] After fermentation, the ergothioneine content in the fermentation broth was detected, and the results are shown in Table 4.

[0135] Table 4

[0136] Number <![CDATA[OD 600 > Content of ergothioneine (mg / L) Example 3 11.84 547.599 Example 4 11.32 518.784 Example 5 10.44 500.474 Example 6 10.15 487.321 Example 7 10.55 434.135 YErg01 9.69 370.434 YErg02 9.34 416.831 YErg03 8.75 325.334 YErg05 9.89 420.382 Comparative Example 2 6.84 0 Comparative Example 3 8.75 55.257

[0137] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A ergothioneine synthetic fusion enzyme, characterized in that, The ergothioneine synthetic fusion enzyme is the enzyme described in any one of (a)-(e): (a) an enzyme having the amino acid sequence shown in SEQ ID NO: 1; (b) an enzyme shown by an amino acid sequence in which one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted or added and still has ergothioneine synthetic fusion enzyme activity; (c) an enzyme shown by an amino acid sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO: 1 and having ergothioneine synthetic fusion enzyme activity; (d) an enzyme shown by an amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the amino acid sequence described in (a), (b) or (c); (e) an enzyme shown by an amino acid sequence with a signal sequence connected to the amino terminus of the amino acid sequence described in (a), (b) or (c).

2. The ergothioneine-synthesizing fusion enzyme according to claim 1, wherein The ergothioneine synthetic fusion enzyme is an enzyme shown by an amino acid sequence having more than 95%, preferably more than 98% homology with the amino acid sequence shown in SEQ ID NO: 1 and having ergothioneine synthetic fusion enzyme activity.

3. A gene encoding ergothioneine synthetic fusion enzyme, characterized in that, This gene has a nucleotide sequence encoding the ergothioneine synthetic fusion enzyme described in claim 1 or 2.

4. The gene according to claim 3, wherein The gene is a nucleotide sequence encoding an enzyme having the amino acid sequence shown in SEQ ID NO: 1; More preferably, the gene has the nucleotide sequence shown in SEQ ID NO:

2.

5. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 3 or 4; Preferably, the expression vector of the recombinant vector is the pUC-HUH-TEF plasmid.

6. A recombinant strain, characterized in that, The recombinant strain expresses the gene described in claim 3 or 4 or contains the recombinant vector described in claim 5; the starting strain of the recombinant strain is Yarrowia lipolytica.

7. The recombinant strain according to claim 6, wherein The starting strain of the recombinant strain is Yarrowia lipolytica in which the coding gene ku70 for non-homologous recombination is knocked out, and the nucleotide sequence of the coding gene ku70 is as shown in SEQ ID NO:

3.

8. Use of at least one of the ergothioneine synthetic fusion enzyme described in claim 1 or 2, the gene described in claim 3 or 4, the recombinant vector described in claim 5, and the recombinant strain described in claim 6 or 7 in the preparation of ergothioneine.

9. A method for fermentatively producing ergothioneine, characterized in that, This method includes: inoculating the recombinant strain described in claim 6 or 7 into a fermentation medium for fermentation.

10. The method according to claim 9, characterized in that, The fermentation medium contains: 20 - 40 g / L of glucose, 0.1 - 0.3 g / L of calcium chloride, 0.4 - 0.8 g / L of potassium sulfate, 0.6 - 1 g / L of sodium chloride, 6 - 10 mg / L of ferrous sulfate heptahydrate, 4 - 6 mg / L of calcium pantothenate, 3 - 5 mg / L of manganese chloride tetrahydrate, 0.06 - 0.1 mg / L of sodium molybdate dihydrate, 6 - 10 mg / L of vitamin B6, vitamin B 12 6 - 10 mg / L, 8 - 12 g / L of yeast powder, and 15 - 25 g / L of tryptone; Preferably, the conditions of the fermentation include: the inoculum size is 2-10%, the temperature is 25-35 °C, the rotation speed is 200-250 rpm, and the time is 4-6 d.

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