Genetic engineering strain with high yield of ergothioneine as well as construction method and application of genetic engineering strain

Abstract

The invention belongs to the field of gene engineering, particularly relates to a gene engineering strain with high yield of ergothioneine, and further discloses a construction method of the gene engineering strain and application of the strain in synthesis of ergothioneine. According to the genetic engineering strain, escherichia coli is used as a chassis cell, pETDuet-1 is used as an expression vector, a coding gene of histidine methyltransferase NcEgt1-1 subjected to truncation, site-specific mutagenesis and combinatorial mutagenesis, a coding gene of sulfoxide synthase CtEgtB from Chlorbacillus thermophilus subjected to site-specific mutagenesis and a coding gene of histidine methyltransferase NcEgt1-1 subjected to site-specific mutagenesis are heterologously expressed in the escherichia coli, and pETDuet-1 is used as an expression vector. The invention relates to a gene engineering bacterium containing non-natural ergothioneine and a coding gene of PLP C-S lyase MsEgtE from Mycobacterium smegmatis, so that the gene engineering bacterium containing the non-natural ergothioneine synthesis route is constructed.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and particularly relates to a genetic engineering strain with high ergothioneine production, and further discloses a construction method thereof and application of the genetic engineering strain in synthesizing ergothioneine. Background Art

[0002] Ergothioneine (ERG), chemically known as 2-thiohistidine trimethylol salt, is the only natural 2-thioimidazole amino acid discovered so far. Compared with other thiol-containing antioxidants, ergothioneine has a special thioketone structure and a higher reduction potential (-60mV), so it has better antioxidant properties, and it can stably exist under physiological pH conditions and in strong alkaline solutions, and is not prone to self-oxidation. Therefore, ergothioneine, as a safe, non-toxic strong antioxidant, has many functions such as slowing down the aging rate of human cells, increasing the activity of skin cells, anti-inflammatory, maintaining redox homeostasis, and promoting the differentiation of neuronal stem cells; at the same time, ergothioneine, as a multifunctional cell physiological protective agent, is also very promising in the cosmetics, health care products, and pharmaceutical industries.

[0003] At present, the synthesis method of ergothioneine includes natural biological extraction method, chemical synthesis method and biosynthesis method. The natural biological extraction method generally extracts ergothioneine from the fruiting body of edible fungi, plant seeds and animal tissue cells, but due to the extremely low content of ergothioneine, there are problems such as low extraction efficiency, long time consumption and difficulty in large-scale preparation. The chemical synthesis method mainly introduces thiol into the imidazole ring, which usually has the problems of lengthy synthesis route and low yield, and has the disadvantages of difficult to ensure product safety, high synthesis raw materials and high cost.

[0004] Currently, there are three main common biosynthesis methods.

[0005] One is to utilize edible mushrooms with natural synthesis ability to carry out liquid deep fermentation, the method has low fermentation cost, is easy to scale, but the output of thioneine is low, and the fermentation process by-products are more, difficult to purify, therefore also can not prepare high-purity thioneine on a large scale.For example, the edible fungus white mushroom catalyzes histidine substrate to generate thioneine disclosed in Chinese patent CN102978121A, and its substrate conversion rate can reach 70%, and the high temperature resistant rough skin pleurotus ostreatus disclosed in Chinese patent CN110283856A ferments 10 days, and thioneine output is 300mg / L; For another example, the edible fungus rough skin pleurotus ostreatus disclosed in Chinese patent CN103734022A is fermented for 7-15 days, and thioneine output is the highest output 143.7mg / L.

[0006] The second method is to express ergothioneine synthases through plasmids, use histidine, cysteine ​​and methionine as substrates, and synthesize them in vitro through enzyme catalysis or whole-cell catalysis. This method has the advantages of high efficiency and high product purity, but there are problems such as long culture time of engineered bacteria and low protein expression; in particular, the enzyme activity is not high, resulting in low production efficiency, poor enzyme substrate tolerance and low conversion rate. For example, the composite enzyme for preparing thioneine disclosed in Chinese patent CN112301013A utilizes three enzymes to realize the synthesis of histidine to thioneine through two-step in vitro catalysis, and the yield reaches 72%; and the Ganoderma lucidum thioneine synthase disclosed in Chinese patent CN116949070A synthesizes thioneine by whole-cell catalysis in vitro, and the thioneine output is 3.2 mg / L; For another example, the composite enzyme composed of L-histidine methylase, L-histidine betaine sulfurylase and S-adenosylmethionine synthase disclosed in Chinese patent CN115851655A, after in vitro enzyme catalysis, the thioneine output is 300 mg / L.

[0007] The third is to use genetically engineered strains for fermentation. At present, thioneine genetic engineering bacteria are mainly based on Escherichia coli, yeast or other fungi as chassis cells, and cell factories are constructed by introducing thioneine heterologous synthase, and the method is generally low in fermentation cost, and the extraction process is relatively simple, easy to scale, and has industrial application potential. For example, Chinese patent CN111534535A discloses that torus red yeast is used as host to express exogenous genes, and the production of thioneine of engineering bacteria fermentation reaches 1.5g / L; and, Chinese patent CN114262702A discloses that Corynebacterium glutamicum is used as host to express exogenous genes, and the production of thioneine of engineering bacteria fermentation reaches 3g / L; For example, Chinese patent CN117511831A discloses that Escherichia coli is used as chassis bacteria through genetic modification, and exogenous mutants are expressed, and the production of thioneine of fermentation reaches 5.02g / L. Therefore, this area expects to develop more genetically engineered strains with high-yield thioneine, to improve the industry output of thioneine. Summary of the invention

[0008] To this end, the technical problem to be solved by the present invention is to provide a genetically engineered strain with high yield of ergothioneine;

[0009] The second technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned genetically engineered strain with high ergothioneine yield;

[0010] The third technical problem to be solved by the present invention is to provide the application of the above-mentioned genetically engineered strain with high ergothioneine yield in synthesizing ergothioneine.

[0011] In order to solve the above-mentioned technical problems, a genetically engineered strain of high-yield ergothioneine of the present invention is provided, wherein the genetically engineered strain heterologously expresses the following features (1)-(3) in its chassis cells:

[0012] (1) A truncated histidine methyltransferase NcEgt1-1 from Neurospora crassa or a mutant thereof;

[0013] (2) a sulfoxide synthase CtEgtB derived from Chloracidobacterium thermophilum or a mutant thereof;

[0014] (3) A pyridoxal phosphate carbon-sulfur lyase MsEgtE from Mycobacterium smegmatis.

[0015] Specifically, the genetically engineered strain with high ergothioneine yield is:

[0016] The nucleotide sequence of the gene encoding the truncated histidine methyltransferase NcEgt1-1 is shown in SEQ ID NO.1; and / or,

[0017] The nucleotide sequence of the gene encoding the optimal mutant of the truncated histidine methyltransferase NcEgt1-1 is shown in SEQ ID NO.4; and / or,

[0018] The nucleotide sequence of the gene encoding the sulfoxide synthase CtEgtB from Chloracidobacterium thermophilum is shown in SEQ ID NO.2; and / or,

[0019] The nucleotide sequence of the coding gene of the optimal mutant of sulfoxide synthase CtEgtB from Chloracidobacterium thermophilum is shown in SEQ ID NO.5; and / or,

[0020] The nucleotide sequence of the gene encoding the pyridoxal phosphate carbon-sulfur lyase MsEgtE derived from Mycobacterium smegmatis is shown in SEQ ID NO.3.

[0021] Specifically, in the genetically engineered strain with high ergothioneine production, the chassis cells include Escherichia coli;

[0022] Preferably, the chassis cells include Escherichia coli DH5α or Escherichia coli BL21 (DE3). The present invention also discloses a fusion protein, which includes, from the N-terminus to the C-terminus:

[0023] (1) a histidine methyltransferase NcEgt1-1 having an amino acid sequence as shown in SEQ ID NO.8, or an optimal mutant of the histidine methyltransferase NcEgt1-1mut having an amino acid sequence as shown in SEQ ID NO.11; and

[0024] (2) A sulfoxide synthase CtEgtB having an amino acid sequence as shown in SEQ ID NO.9, or an optimal mutant of a sulfoxide synthase CtEgtBmut having an amino acid sequence as shown in SEQ ID NO.12.

[0025] Specifically, the fusion protein, the amino acid sequence of the fusion protein is shown as SEQ ID NO.13 or SEQ ID NO.14.

[0026] The present invention also discloses a gene encoding the fusion protein, which has a nucleotide sequence as shown in SEQ ID NO.6 or SEQ ID NO.7.

[0027] The present invention also discloses a recombinant expression vector, comprising the coding gene and a coding gene of pyridoxal phosphate carbon-sulfur lyase MsEgtE; wherein the coding gene of pyridoxal phosphate carbon-sulfur lyase MsEgtE has a nucleotide sequence as shown in SEQ ID NO.3;

[0028] Preferably, the recombinant expression vector includes pETDuet-1.

[0029] The present invention also discloses a genetically engineered strain expressing the fusion protein or comprising the recombinant expression vector according to claim 7;

[0030] Preferably, the genetically engineered strain comprises Escherichia coli.

[0031] The invention also discloses the genetic engineering strain with high ergothioneine production, or application of the genetic engineering strain in producing ergothioneine.

[0032] The invention also discloses a method for preparing high-yield ergothioneine, comprising the step of carrying out biosynthesis using the high-yield ergothioneine genetic engineering strain, or the genetic engineering strain as a fermentation strain.

[0033] The genetically engineered strain of the invention uses Escherichia coli as a chassis cell and pETDuet-1 as an expression vector, heterologously expresses in the Escherichia coli a coding gene of a histidine methyltransferase NcEgt1-1 that has undergone truncation, site-directed mutation and combined mutation, a coding gene of a sulfoxide synthase CtEgtB that has undergone site-directed mutation from Chloracidobacterium thermophilum and a coding gene of a PLP CS lyase MsEgtE from Mycobacterium smegmatis, and constructs a genetically engineered bacterium containing a non-natural ergothioneine synthesis pathway, effectively solving the problems of a large number of steps in the egtABCDE synthesis pathway and competition with glutathione synthesis.

[0034] The invention retains the methyltransferase activity of NcEgt1 from Neurospora crassa by truncation and modification, and modifies the truncated methyltransferase by means of site-directed mutagenesis and combined mutagenesis for the first time, thereby screening and obtaining mutants with further improved yield, so that the ergothioneine generation rate is effectively improved, and the yield of ergothioneine is increased by 5.5 times to 478.79±13.21 mg / L, thereby opening up a new way for producing ergothioneine by an Escherichia coli fermentation method and facilitating the industrialized production of ergothioneine. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0036] Figure 1 It is a schematic diagram of the structure of the recombinant plasmid pETDuet-1-T7-NcEgt1-1 / mutant+CtEgtB-T7-MsEgtE constructed by the present invention;

[0037] Figure 2 is an SDS-PAGE image, lane 1 is a marker, lane 2 is the supernatant of BL21 (DE3), lane 3 is the supernatant of BL21 (DE3) transformed with empty pETDuet-1, and lane 4 is the supernatant of BL21 (DE3) transformed with the recombinant plasmid pETDuet-1-T7-NcEgt1-1-CtEgtB-T7-MsEgtE;

[0038] Figure 3 It is a diagram of the synthesis pathway of ergothioneine;

[0039] Figure 4 It is the HPLC collection of illustrative plates detecting thioneine product in the embodiment of the present invention;

[0040] Figure 5This is the standard curve diagram of ergothioneine detected by high performance liquid chromatography. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention.

[0042] In the field of biology, Escherichia coli is known for its fast generation speed and is one of the free-living bacteria with the shortest known generation time. Therefore, it is considered to be an excellent chassis cell that can be used to construct an expression system. Although a variety of microorganisms (such as mycobacteria, streptomyces, molds and yeasts) have the ability to synthesize ergothioneine, Escherichia coli is generally unable to synthesize ergothioneine. Therefore, if Escherichia coli is to be used to produce ergothioneine, the heterologous protein gene required for synthesizing ergothioneine must be introduced.

[0043] Studies have shown that one of the significant advantages of using E. coli as a producer of ergothioneine is that most of the ergothioneine produced during the fermentation process is secreted into the fermentation liquid. This makes ergothioneine, as an extracellular product, unnecessary for the fermentation bacteria to undergo cell wall breaking, which greatly facilitates the extraction and purification of ergothioneine and eliminates the need for complex post-processing steps such as separation and extraction, thereby effectively reducing production costs.

[0044] In view of the above situation, the first aspect of the present application provides a truncated histidine methyltransferase (NcEgt1-1), whose amino acid sequence is shown in SEQ ID NO.8, and its truncated amino acid sequence is the N-terminal sequence obtained by truncating NcEgt1 from Neurospora crassa at position 358.

[0045] The second aspect of the present invention provides a fusion protein, which comprises a truncated histidine methyltransferase (NcEgt1-1) or a mutant thereof described in the present invention and the sulfoxide synthase (CtEgtB) or a mutant thereof;

[0046] Specifically, the fusion protein gene includes, from the N-terminus to the C-terminus, the coding gene of the truncated histidine methyltransferase (NcEgt1-1) or its mutant and the coding gene of the sulfoxide synthase (CtEgtB) or its mutant.

[0047] The amino acid sequence of the fusion protein is shown in SEQ ID NO.13 or SEQ ID NO.14; and the coding gene sequence of the fusion protein is shown in SEQ ID NO.6 or SEQ ID NO.7.

[0048] For example, the fusion protein includes a fusion of a truncated histidine methyltransferase and a sulfoxide synthase, and its amino acid sequence is shown as SEQ ID NO.13, and the coding gene sequence of the fusion protein is shown as SEQ ID NO.6.

[0049] For example, the fusion protein comprises a fusion of a truncated histidine methyltransferase optimal mutant NcEgt1-1mut and a sulfoxide synthase optimal mutant CtEgtBmut, whose amino acid sequence is shown in SEQ ID NO.14, and the coding gene sequence of the fusion protein is shown in SEQ ID NO.7.

[0050] In one embodiment, the present invention transforms an expression plasmid encoding a truncated histidine methyltransferase NcEgt1-1 (amino acid sequence as shown in SEQ ID NO.8) gene (as shown in SEQ ID NO.1) or a mutant thereof NcEgt1-1mut (amino acid sequence as shown in SEQ ID NO.11) gene (as shown in SEQ ID NO.4), a sulfoxide synthase CtEgtB (amino acid sequence as shown in SEQ ID NO.9) from Chloracidobacterium thermophilum (as shown in SEQ ID NO.2) or a mutant thereof CtEgtBmut (amino acid sequence as shown in SEQ ID NO.12) gene (as shown in SEQ ID NO.5), and a pyridoxal phosphate carbon-sulfur lyase MsEgtE (amino acid sequence as shown in SEQ ID NO.10) from Mycobacterium smegmatis (as shown in SEQ ID NO.3) into Escherichia coli BL21 (DE3) competent cells.

[0051] As an exemplary embodiment, the nucleotide sequence of the gene encoding a truncated histidine methyltransferase NcEgt1-1 (wherein the GenBank accession number of NcEgt1 is XP_956324.3, and the amino acid sequence is shown in SEQ ID NO.15) of the present invention is shown in SEQ ID NO.1.

[0052] As an exemplary embodiment, the nucleotide sequence of the gene encoding a truncated histidine methyltransferase NcEgt1-1 mutant (amino acid sequence as shown in SEQ ID NO.11) of the present invention is shown in SEQ ID NO.4.

[0053] It should be noted here that the truncated histidine methyltransferase NcEgt1-1 mutant of the present invention may have an amino acid sequence as shown in SEQ ID NO.11, and may be named NcEgt1-1mut herein, which is an (E51M and Y174F) mutant of NcEgt1-1. It should be understood that the above-mentioned NcEgt1-1 mutant is not limited to the mutant NcEgt1-1mut having an amino acid sequence of SEQ ID NO.11, but also includes a polypeptide comprising one or more of the following amino acid differences (E51A / M; L68K / M / Q; Y84R; I95V; R154D; Q158F / L / V; T173D; Y174F; Q316V) compared to SEQ ID NO.8. In addition, the present invention also discloses a gene encoding the above-mentioned truncated histidine methyltransferase (NcEgt1-1) mutant NcEgt1-1mut. For example, the nucleotide sequence of the above-mentioned NcEgt1-1mut encoding gene is shown in SEQ ID NO.4.

[0054] As an exemplary embodiment, the nucleotide sequence of the gene encoding the sulfoxide synthase (CtEgtB, GenBank accession number: WP_014099806.1, amino acid sequence as shown in SEQ ID NO.9) derived from Chloracidobacteriumthermophilum of the present invention is shown in SEQ ID NO.2.

[0055] As an exemplary embodiment, the nucleotide sequence of the gene encoding the CtEgtB mutant (amino acid sequence as shown in SEQ ID NO.12) subjected to site-directed mutagenesis of the present invention is shown in SEQ ID NO.5.

[0056] It should be noted here that the CtEgtB mutant subjected to site-directed mutation of the present invention may have an amino acid sequence as shown in SEQ ID NO.12, and may be named CtEgtBmut herein, which is a (P417L) mutant of CtEgtB. It should be understood that the above-mentioned CtEgtB mutant is not limited to the mutant CtEgtBmut having an amino acid sequence of SEQ ID NO.12, but also includes a polypeptide comprising one or more of the following amino acid differences (D52L, L162F / M, L275Q / V, R269N, P417C / D / T / A / R / N / Q / E / G / H / I / L / K / M / F / S / W / Y / V, A420Y / K) compared to SEQ ID NO.9. In addition, the present invention also discloses a gene encoding the CtEgtB mutant CtEgtBmut subjected to site-directed mutation. For example, the nucleotide sequence of the above-mentioned CtEgtBmut encoding gene is shown in SEQ ID NO.5.

[0057] As an exemplary embodiment, the nucleotide sequence of the gene encoding the pyridoxal phosphate carbon-sulfur lyase (MsEgtE, GenBank accession number: WP_011731155.1, amino acid sequence as shown in SEQ ID NO.10) derived from Mycobacterium smegmatis of the present invention is shown in SEQ ID NO.3.

[0058] As an exemplary embodiment, when a plasmid is used to express the above-mentioned NcEgt1-1, CtEgtB and MsEgtE, a fusion gene of the coding gene of the above-mentioned truncated histidine methyltransferase, i.e., NcEgt1-1 or its mutant, and the coding gene of the sulfoxide synthase, i.e., CtEgtB or its mutant, and the coding gene of the pyridoxal phosphate carbon-sulfur lyase, i.e., MsEgtE, are cloned on the same plasmid for co-expression.

[0059] As an exemplary embodiment, in the above plasmid, a fusion gene of a truncated histidine methyltransferase, NcEgt1-1 or its mutant encoding gene, and a sulfoxide synthase, CtEgtB or its mutant encoding gene, and a pyridoxal phosphate carbon-sulfur lyase, MsEgtE encoding gene are placed under the control of T7 promoter respectively.

[0060] In one embodiment, the above-mentioned co-expression plasmid uses the pETDuet-1 plasmid as a backbone vector and the T7 promoter as a promoter for gene expression, and constructs a fusion expression containing NcEgt1-1 / mutant and CtEgtB / mutant by gene fragment recombination, and co-expresses the plasmid pETDuet-1-T7-NcEgt1-1 / mutant+CtEgtB-T7-MsEgtE with MsEgtE.

[0061] The third aspect of the present invention provides the use of the above-mentioned truncated histidine methyltransferase mutant NcEgt1-1mut or the above-mentioned gene, such as SEQ ID NO.11, in promoting microbial production of ergothioneine; or provides the use of the above-mentioned truncated histidine methyltransferase mutant NcEgt1-1mut in enzymatic synthesis of ergothioneine.

[0062] A fourth aspect of the present invention provides the use of the above-mentioned sulfoxide synthase mutant CtEgtBmut subjected to site-directed mutagenesis or the above-mentioned gene, such as SEQ ID NO.12, in promoting microbial production of ergothioneine; or provides the use of the above-mentioned sulfoxide synthase mutant CtEgtBmut subjected to site-directed mutagenesis in enzymatic synthesis of ergothioneine.

[0063] The fifth aspect of the present invention provides the use of the above-mentioned high-yield ergothioneine genetic engineering bacteria in the production of ergothioneine by fermentation.

[0064] In one embodiment, since histidine, methionine and cysteine ​​are precursors for synthesizing ergothioneine, L-histidine may be added to the fermentation medium, and L-methionine and / or L-cysteine ​​may also be added.

[0065] The invention constructs an Escherichia coli engineering strain for producing thioneine by means of genetic engineering; a truncated histidine methyltransferase mutant NcEgt1-1mut capable of improving the thioneine level of Escherichia coli is obtained by truncation, directed mutation and combined mutation techniques for a NcEgt1 methyltransferase functional region, so that the thioneine yield is effectively improved, and the output of thioneine is improved by 3.7 times to 325.18±57.85 mg / L; and a sulfoxide synthase mutant CtEgtBmut capable of improving the thioneine level of Escherichia coli is obtained by site-directed mutation and saturation mutation techniques for a sulfoxide synthase CtEgtB, so that the thioneine yield is further improved, and the output of thioneine is improved by 5.5 times to 478.79±13.21 mg / L, thereby opening up a new approach for producing thioneine by an Escherichia coli fermentation method.

[0066] In the following examples of the present invention, Escherichia coli DH5α and Escherichia coli BL21 (DE3) were purchased from Sangon Biotech Co., Ltd. and used for gene cloning and protein expression, respectively.

[0067] In the following examples of the present invention, the construction of the recombinant plasmid was entrusted to a commercial company for full gene synthesis.

[0068] In the following examples of the present invention, as an exemplary embodiment, the culture medium involved includes:

[0069] LB medium (g / L): tryptone 10.0, yeast extract 5.0, NaCl 10.0, deionized water 1 L, pH 7.0;

[0070] Fermentation medium formula (g / L): yeast extract powder 24.0, tryptone 12.0, sodium chloride 3.0, glycerol 30.0, dipotassium hydrogen phosphate 2.0, magnesium sulfate heptahydrate 0.5, cysteine ​​1.0, histidine 1.0, methionine 1.0, ferric citrate ammonium 0.06, deionized water 1L, pH 7.0.

[0071] The following embodiment of the present invention relates to a method for processing a fermentation broth: 1 mL of the fermentation broth was taken, centrifuged at 12000 rpm for 10 min, the supernatant was diluted several times, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC.

[0072] In the following examples of the present invention, the HPLC analysis conditions are: the chromatographic column is Waters C18 column, 5μm, 4.6×250mm; mobile phase A is ultrapure water, phase B is methanol, A and B are gradient eluted; column temperature is 30℃; detection wavelength is 257nm; flow rate is 0.7mL / min; injection volume is 5μL; detection wavelength is 257nm.

[0073] Example 1

[0074] In this embodiment, the NcEgt1 enzyme is adaptively truncated and modified, wherein the histidine methyltransferase is NcEgt1-1 (NcEgt1, GenBank accession number: XP_956324.3), and the NcEgt1 protein contains 878 amino acids (as shown in SEQ ID NO.15), and has two domains, a methyltransferase domain and a CS bond synthetase domain. In view of the low activity of histidine methyltransferase, which leads to insufficient supply of intermediates, this embodiment refers to the method recorded in Chinese Patent 116515785A to truncate and modify it, and obtain the NcEgt1-1 gene containing only the methyltransferase domain for subsequent enzyme modification.

[0075] Example 2

[0076] This embodiment carries out the construction of thioneine synthesis pathway.

[0077] Construction of recombinant plasmid pETDuet-1-T7-NcEgt1-1+CtEgtB-T7-MsEgtE

[0078] like Figure 1 According to the procedure shown, a gene encoding a truncated histidine methyltransferase NcEgt1-1 (such as SEQID NO.1), a gene encoding a sulfoxide synthase CtEgtB from Chloracidobacterium thermophilum (SEQID NO.2), and a gene encoding a pyridoxal phosphate carbon-sulfur lyase MsEgtE from Mycobacterium smegmatis (SEQ ID NO.3) were commissioned to a commercial company for full gene synthesis. After receiving the conventional genes, they were cloned into the pETDuet-1 vector to obtain the recombinant plasmid pETDuet-1-T7-NcEgt1-1-CtEgtB-T7-MsEgtE.

[0079] The plasmid was transferred into Escherichia coli BL21 (DE3) competent cells, spread on a resistance plate (containing 100 μg / mL ampicillin), and cultured overnight at 37° C. A positive transformation single colony was picked, cultured in LB, and the plasmid was extracted and sequenced to obtain a recombinant genetic engineering bacterium E1 containing an ergothioneine synthesis pathway.

[0080] The SDS-PAGE images of each sample are shown in the attached Figure 2 Lane 1 is the marker, lane 2 is the supernatant of BL21(DE3), lane 3 is the supernatant of BL21(DE3) transformed with empty pETDuet-1, and lane 4 is the supernatant of BL21(DE3) transformed with the recombinant plasmid pETDuet-1-T7-NcEgt1-1-CtEgtB-T7-MsEgtE.

[0081] Example 3

[0082] like Figure 3 The procedure shown in this example utilizes the genetically engineered strain E1 constructed above to ferment and produce ergothioneine.

[0083] The empty vector control strain and the single clone of the E1 engineering strain were picked into 5 mL LB test tubes (containing 100 μg / mL ampicillin), placed in a 37°C shaker, and cultured at 220 rpm for 10 h; then transferred to a 250 mL shake flask containing 25 mL fermentation medium (containing 100 μg / mL ampicillin) at a 2% inoculum, placed in a 37°C shaker, and cultured at 220 rpm for 2.5-3 h, and then added with isopropylthiogalactoside (IPTG) at a final concentration of 0.2 mM, and placed in a 20°C shaker, 220 rpm for induction culture for 48 h.

[0084] After the fermentation is completed, the fermentation liquid is processed according to the aforementioned sample processing method, and high-performance liquid phase detection is performed. The HPLC atlas for detecting thioneine product is attached. Figure 4 , according to the standard curve (such as Figure 5 ) calculated the corresponding thioneine content, and the thioneine content was 87.02 mg / L.

[0085] Example 4

[0086] This example constructs and screens a mutant library of NcEgt1-1 enzyme based on the aforementioned NcEgt1-1 enzyme.

[0087] In this embodiment, three mutation sites were screened out based on the multiple sequence alignment of the methyltransferase domain. According to the mutation site, the corresponding primers were designed, and the gene of NcEgt1-1 was used as a template. Overlap extension PCR was performed using the high-fidelity polymerase KOD. The PCR reaction system is shown in Table 1 below, and the PCR reaction program is set as follows: the initial pre-denaturation temperature is 98°C, 5min, followed by 25 cycles (98°C denaturation for 10s, 55°C annealing for 30s, 68°C extension for 1min), and finally 68°C extension for 5min. The template was eliminated and then transferred to the competent state of Escherichia coli DH5α, and cultured overnight at 37°C on LB solid medium containing 100μg / mL ampicillin.

[0088] Table 1 PCR reaction system (50 μL)

[0089] Components Volume (μL) Sterile water 21 High-fidelity polymerase KOD 25 Upstream primer (diluted 10 times) 0.5 Downstream primer (diluted 10 times) 0.5 template 3

[0090] Positive clones were picked and placed in 5 mL LB, cultured overnight at 37°C and 220 rpm, and sequenced for verification. The resulting strains were named M1, M2, and M3. Shake flask fermentation was performed, and the ERG content was detected by high performance liquid chromatography.

[0091] Example 5

[0092] Based on the above-mentioned Example 4, this example further discusses the construction and screening of the NcEgt1-1 enzyme combinatorial mutation library.

[0093] Based on the catalytic hotspot amino acids predicted by HotSpot Wizard, 11 mutation sites were screened. According to the mutation sites, corresponding primers were designed, and the genes of mutants M1, M2, and M3 were used as templates. Overlap extension PCR was performed using high-fidelity polymerase KOD (PCR system as shown in Table 1), the template was eliminated, and then transferred into Escherichia coli DH5α competent cells and cultured overnight at 37°C on LB solid medium containing 100 μg / mL ampicillin.

[0094] Positive clones were picked into 5 mL LB and cultured overnight at 37°C and 220 rpm. The strains were verified by sequencing and named M1-01, M1-02...M1-11; M2-01, M2-02...M2-11; M3-01, M3-02...M3-11. Shake flask fermentation was performed and the ERG content was detected by HPLC.

[0095] Example 6

[0096] This example utilizes the mutant strain containing the mutant gene screened in the aforementioned Example 5 to ferment and produce ergothioneine.

[0097] Single clones of mutant strains were picked out and placed in 5 mL LB test tubes (containing 100 μg / mL ampicillin), and placed in a 37°C shaker with shaking at 220 rpm for 10 h; then transferred to a 250 mL shake flask containing 25 mL fermentation medium (containing 100 μg / mL ampicillin) at a 2% inoculum, placed in a 37°C shaker with shaking at 220 rpm for 2.5-3 h, and then isopropylthiogalactoside (IPTG) was added at a final concentration of 0.2 mM, and placed in a 20°C shaker with induction culture at 220 rpm for 48 h.

[0098] After fermentation, the fermented liquid was processed according to the previous sample processing method, and high-efficiency liquid phase detection was performed, and the corresponding thioneine content was calculated according to the standard curve. The thioneine output obtained by different NcEgt1-1 mutants is as shown in Table 2 below, wherein, the thioneine content produced by mutant M3-02 is up to 325.18 ± 57.85 mg / L, which is 3.7 times that of E1.

[0099] Table 2 Ergothioneine production detected by NcEgt1-1 mutant strain

[0100]

[0101]

[0102] Example 7

[0103] Based on the above Example 6, this Example further explores the construction and screening of the CtEgtB enzyme combinatorial mutation library.

[0104] Based on the catalytic hotspot amino acids predicted by HotSpot Wizard, 9 mutation sites were screened. Combined with the two mutants D52L and A420Y with improved enzyme activity in the literature (Naowarojna et al., 2019), a total of 11 mutants were constructed. According to the mutation site, the corresponding primers were designed, and the CtEgtB gene was used as a template. The high-fidelity polymerase KOD was used for overlap extension PCR (PCR system as shown in Table 1), the template was eliminated, and then transferred into Escherichia coli DH5α competent cells and cultured overnight at 37°C on LB solid medium containing 100 μg / mL ampicillin.

[0105] Positive clones were picked into 5 mL LB and cultured overnight at 37°C and 220 rpm. The obtained strains were verified by shaking flask fermentation and the ERG content was detected by high performance liquid chromatography.

[0106] Based on the site-directed mutagenesis results, saturation mutation was performed on position 417 of CtEgtB. According to the mutation site, corresponding primers were designed, and the gene of CtEgtB was used as a template. Overlap extension PCR was performed using high-fidelity polymerase KOD (PCR system as shown in Table 1), the template was eliminated, and then transferred into Escherichia coli DH5α competent cells and cultured overnight at 37°C on LB solid medium containing 100 μg / mL ampicillin.

[0107] Positive clones were picked into 5 mL LB, cultured overnight at 37°C and 220 rpm, and verified by sequencing. The resulting strains were fermented in shake flasks, and the ERG content was detected by high performance liquid chromatography.

[0108] Example 8

[0109] This example utilizes the mutant strain containing the mutant gene screened in the aforementioned Example 7 to ferment and produce ergothioneine.

[0110] Single clones of mutant strains were picked out and placed in 5 mL LB test tubes (containing 100 μg / mL ampicillin), and placed in a 37°C shaker with shaking at 220 rpm for 10 h; then transferred to a 250 mL shake flask containing 25 mL fermentation medium (containing 100 μg / mL ampicillin) at a 2% inoculum, placed in a 37°C shaker with shaking at 220 rpm for 2.5-3 h, and then isopropylthiogalactoside (IPTG) was added at a final concentration of 0.2 mM, and placed in a 20°C shaker with induction culture at 220 rpm for 48 h.

[0111] After fermentation, the fermented liquid was processed according to the previous sample processing method, and high-efficiency liquid phase detection was performed, and the corresponding thioneine content was calculated according to the standard curve. The thioneine output obtained by different CtEgtB mutants is shown in Table 3 below, wherein, mutant M3-02 P417L The highest ergothioneine content produced was 478.79±13.21 mg / L, which was 5.5 times that of E1.

[0112] Table 3 Ergothioneine production detected by CtEgtB mutant strains

[0113]

[0114]

[0115] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

[0116] SEQUENCE LISTING

[0117] SEQ ID NO.1:

[0118]

[0119] SEQ ID NO.2:

[0120]

[0121] SEQ ID NO.3:

[0122]

[0123] SEQ ID NO.4:

[0124]

[0125] SEQ ID NO.5:

[0126]

[0127] SEQ ID NO.6:

[0128]

[0129] SEQ ID NO.7:

[0130]

[0131] SEQ ID NO.8:

[0132] MPSAESMTPSSALGQLKATGQHVLSKLQQQTSNADIIDIRRVAVEINLKTEITSMFRPKDGPRQLPTLLLYNERGLQLFERITYLEEYYLTNDEIKILTKHATEMASFIPSGAMIIELGSGNLRKVNLLLEALDNAGKAIDYYALDLSREELERTLAQVPSYKHVKCHGLLGTYDDGRDWLKAPENINKQKCILHLGSSIGNFNRSDAATFLKGFTDVLGPNDKMLIGVDACNDPARVYHAYNDKVGITHEFILNGLRNANEIIGETAFIEGDWRVIGEYVYDEEGGRHQAFYAPTRDTMVMGELIRSHDRIQIEQSLKYSKEESERLWSTAGLEQVSEWTYGNEYGLHLLAKSR

[0133] SEQ ID NO.9:

[0134] MEAARSHPEPIQSGEVSDRKAWQRHYRAVRAVSEAICQPLETEDYVVQPMPDVSPPKWHLGHTSWFFETFILKSGLADYRPFHPRYDYIFNSYYEAVGARHPRPQRGLLTRPTVSEVYAYRAHVDAAVERFIAHSDTRTWAALQPILELGLHHEQQHQELLLTDIKAILATNPLDPVYRPQPQPLPSPVEQLSPTGDWHIVEGGRYAIGHAGRGFAFDNEGPRHDVLLRPCRIAARPVTNGEFLAFMADGGYRRPELWLSDGWAAVTARGWEAPLYWRQAADGTWETLTLHGVQPVAPYEPVCHISFYEADAYARWAGKRLPTEAEWEVVAARLPVTGNFYESGVLHPRPVSVSAAFYGDVWVWTASPYVGYPGFRPVSGALGEYNGKFMCNQMVLRGGSCATSLTHIRSTYRNFFPPDARWQFTGVRLAEDMS

[0135] SEQ ID NO.10:

[0136] MMLAQQWRDARPKVAGLHLDSGACSRQSFAVIDATTAHARHEAEVGGYVAAEAATPALDAGRAAVASLIGFAASDVVYTSGSNHAIDLLSSWPGKRTLACLPGEYGPNLSAMAANGFQVRALPVDDDGRVLVDEASHELSAHPVALVHLTALASHRGIAQPAAELVEACHNAGIPVVIDAAQAL GHLDCNVGADAVYSSSRKWLAGPRGVGVLAVRPELAERLQPRIPPSDWPIPMSVLEKLELGEHNAAARVGFSVAVGEHLAAGPTAVRERLAEVGRLSRQVLAEVDGWRVVEPVDQPTAITLESTDGADPASVRSWLIAERGIVTACELARAPFEMRTPVLRISPHVDVDELEQFAAALREAP

[0137] SEQ ID NO.11:

[0138] MPSAESMTPSSALGQLKATGQHVLSKLQQQTSNADIIDIRRVAVEINLKT M ITSMFRPKDGPRQLPTLLLYNERGLQLFERITYLEEYYLTNDEIKILTKHATEMASFIPSGAMIIELGSGNLRKVNLLLEALDNAGKAIDYYALDLSREELERTLAQVPSYKHVKCHGLLGT F DDGRDWLKAPENINKQKCILHLGSSIGNFNRSDAATFLKGFTDVLGPNDKMLIGVDACNDPARVYHAYNDKVGITHEFILNGLRNANEIIGETAFIEGDWRVIGEYVYDEEGGRHQAFYAPTRDTMVMGELIRSHDRIQIEQSLKYSKEESERLWSTAGLEQVSEWTYGNEYGLHLLAKSR

[0139] SEQ ID NO.12:

[0140] MEAARSHPEPIQSGEVSDRKAWQRHYRAVRAVSEAICQPLETEDYVVQPMPDVSPPKWHLGHTSWFFETFILKSGLADYRPFHPRYDYIFNSYYEAVGARHPRPQRGLLTRPTVSEVYAYRAHVDAAVERFIAHSDTRTWAALQPILELGLHHEQQHQELLLTDIKAILATNPLDPVYRPQPQPLPSPVEQLSPTGDWHIVEGGRYAIGHAGRGFAFDNEGPRHDVLLRPCRIAARPVTNGEFLAFMADGGYRRPELWLSDGWAAVTARGWEAPLYWRQAADGTWETLTLHGVQPVAPYEPVCHISFYEADAYARWAGKRLPTEAEWEVVAARLPVTGNFYESGVLHPRPVSVSAAFYGDVWVWTASPYVGYPGFRPVSGALGEYNGKFMCNQMVLRGGSCATSLTHIRSTYRNFF L PDARWQFTGVRLAEDMS

[0141] SEQ ID NO.13:

[0142] MPSAESMTPSSALGQLKATGQHVLSKLQQQTSNADIIDIRRVAVEINLKTEITSMFRPKDGPRQLPTLLLYNERGLQLFERITYLEEYYLTNDEIKILTKHATEMASFIPSGAMIIELGSGNLRKVNLLLEALDNAGKAIDYYALDLSREELERTLAQVPSYKHVKCHGLLGTYDDGRDWLKAPENINKQKCILHLGSSIGNFNRSDAATFLKGFTDVLGPNDKMLIGVDACNDPARVYHAYNDKVGITHEFILNGLRNANEIIGETAFIEGDWRVIGEYVYDEEGGRHQAFYAPTRDTMVMGELIRSHDRIQIEQSLKYSKEESERLWSTAGLEQVSEWTYGNEYGLHLLAKSRMEAARSHPEPIQSGEVSDRKAWQRHYRAVRAVSEAICQPLETEDYVVQPMPDVSPPKWHLGHTSWFFETFILKSGLADYRPFHPRYDYIFNSYYEAVGARHPRPQRGLLTRPTVSEVYAYRAHVDAAVERFIAHSDTRTWAALQPILELGLHHEQQHQELLLTDIKAILATNPLDPVYRPQPQPLPSPVEQLSPTGDWHIVEGGRYAIGHAGRGFAFDNEGPRHDVLLRPCRIAARPVTNGEFLAFMADGGYRRPELWLSDGWAAVTARGWEAPLYWRQAADGTWETLTLHGVQPVAPYEPVCHISFYEADAYARWAGKRLPTEAEWEVVAARLPVTGNFYESGVLHPRPVSVSAAFYGDVWVWTASPYVGYPGFRPVSGALGEYNGKFMCNQMVLRGGSCATSLTHIRSTYRNFFPPDARWQFTGVRLAEDMS

[0143] SEQ ID NO.14:

[0144] MPSAESMTPSSALGQLKATGQHVLSKLQQQTSNADIIDIRRVAVEINLKTMITSMFRPKDGPRQLPTLLLYNERGLQLFERITYLEEYYLTNDEIKILTKHATEMASFIPSGAMIIELGSGNLRKVNLLLEALDNAGKAIDYYALDLSREELERTLAQVPSYKHVKCHGLLGTFDDGRDWLKAPENINKQKCILHLGSSIGNFNRSDAATFLKGFTDVLGPNDKMLIGVDACNDPARVYHAYNDKVGITHEFILNGLRNANEIIGETAFIEGDWRVIGEYVYDEEGGRHQAFYAPTRDTMVMGELIRSHDRIQIEQSLKYSKEESERLWSTAGLEQVSEWTYGNEYGLHLLAKSRMEAARSHPEPIQSGEVSDRKAWQRHYRAVRAVSEAICQPLETEDYVVQPMPDVSPPKWHLGHTSWFFETFILKSGLADYRPFHPRYDYIFNSYYEAVGARHPRPQRGLLTRPTVSEVYAYRAHVDAAVERFIAHSDTRTWAALQPILELGLHHEQQHQELLLTDIKAILATNPLDPVYRPQPQPLPSPVEQLSPTGDWHIVEGGRYAIGHAGRGFAFDNEGPRHDVLLRPCRIAARPVTNGEFLAFMADGGYRRPELWLSDGWAAVTARGWEAPLYWRQAADGTWETLTLHGVQPVAPYEPVCHISFYEADAYARWAGKRLPTEAEWEVVAARLPVTGNFYESGVLHPRPVSVSAAFYGDVWVWTASPYVGYPGFRPVSGALGEYNGKFMCNQMVLRGGSCATSLTHIRSTYRNFFLPDARWQFTGVRLAEDMS

[0145] SEQ ID NO.15:

[0146] MPSAESMTPSSALGQLKATGQHVLSKLQQQTSNADIIDIRRVAVEINLKTEITSMFRPKDGPRQLPTLLLYNERGLQLFERITYLEEYYLTNDEIKILTKHATEMASFIPSGAMIIELGSGNLRKVNLLLEALDNAGKAIDYYALDLSREELERTLAQVPSYKHVKCHGLLGTYDDGRDWLKAPENINKQKCILHLGSSIGNFNRSDAATFLKGFTDVLGPNDKMLIGVDACNDPARVYHAYNDKVGITHEFILNGLRNANEIIGETAFIEGDWRVIGEYVYDEEGGRHQAFYAPTRDTMVMGELIRSHDRIQIEQSLKYSKEESERLWSTAGLEQVSEWTYGNEYGLHLLAKSRMSFSLIPSVYARSALPTLDDWEALWATWDVVTRQMLPQEELLEKPIKLRNACIFYLGHIPTFLIQLTKTTKQAPSEPAHFCK IFERGIDPDVDNPELCHAHSEIPDEWPPVEEILTYQETVRSRRLRGLYAHGIANIPRNVGRAIWVGFEHELMHIETLLYMMLQSDKTLIPTHIPRPDFDKLARKAESERVPNQWFKIPAQEITIGLDDDPEDGSDINKHYGWDNEKPPRRVQVAAFQAQGRPITNEEYAQYLLEKNIDKLPASWARLDNENISNGTTNSVSGHHSNRTSKQQLPSSFLEKT AVRTVYGLVPLKHALDWPVFASYDELAGCAAYMGGRIPTFEETRSIYAYADALKKKKEAERQLGRTVPAVNAHLTNNGVEITPPSSPSSETPAESSSPSDSNTTLITTEDLFSDLDGANVGFHNWHPMPITSKGNTLVGQGELGGVWEWTSSVLRKWEGFEPMELYPGYTADFDEKHNIVLGGSWATHPRIAGRKSVNWYQRNYPYAWVGARVVRDL

[0147] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A genetically engineered strain for high-yield ergothioneine, characterized in that, The genetically engineered strain heterologously expresses the following features (1)-(3) in its chassis cells: (1) A truncated histidine methyltransferase NcEgt1-1 from Neurospora crassa or a mutant thereof; (2) a sulfoxide synthase CtEgtB derived from Chloracidobacterium thermophilum or a mutant thereof; (3) A pyridoxal phosphate carbon-sulfur lyase MsEgtE from Mycobacterium smegmatis.

2. according to the genetically engineered strain of high-yield ergothioneine of claim 1, it is characterized in that: The nucleotide sequence of the gene encoding the truncated histidine methyltransferase NcEgt1-1 is shown in SEQ ID NO.1; and / or, The nucleotide sequence of the gene encoding the truncated histidine methyltransferase NcEgt1-1 mutant is shown in SEQ ID NO.4; and / or, The nucleotide sequence of the gene encoding the sulfoxide synthase CtEgtB from Chloracidobacterium thermophilum is shown in SEQ ID NO.2; and / or, The nucleotide sequence of the gene encoding the sulfoxide synthase CtEgtB mutant derived from Chloracidobacterium thermophilum is shown in SEQ ID NO.5; and / or, The nucleotide sequence of the gene encoding the pyridoxal phosphate carbon-sulfur lyase MsEgtE derived from Mycobacterium smegmatis is shown in SEQ ID NO.

3.

3. according to the genetically engineered strain of high-yield ergothioneine described in claim 1 or 2, it is characterized in that, The chassis cells include Escherichia coli; Preferably, the chassis cells include Escherichia coli DH5α or Escherichia coli BL21 (DE3).

4. A fusion protein, characterized in that The fusion protein comprises, from N-terminus to C-terminus: (1) a histidine methyltransferase NcEgt1-1 having an amino acid sequence as shown in SEQ ID NO.8, or a histidine methyltransferase mutant NcEgt1-1mut having an amino acid sequence as shown in SEQ ID NO.11; and (2) A sulfoxide synthase CtEgtB having an amino acid sequence as shown in SEQ ID NO.9, or a sulfoxide synthase mutant CtEgtBmut having an amino acid sequence as shown in SEQ ID NO.

12.

5. The fusion protein according to claim 4, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO.13 or SEQ ID NO.

14.

6. A gene encoding the fusion protein according to claim 5, characterized in that: It has a nucleotide sequence as shown in SEQ ID NO.6 or SEQ ID NO.

7.

7. A recombinant expression vector, characterized in that: Comprising the coding gene of claim 6, and a coding gene of pyridoxal phosphate carbon-sulfur lyase MsEgtE; wherein the coding gene of pyridoxal phosphate carbon-sulfur lyase MsEgtE has a nucleotide sequence as shown in SEQ ID NO.3; Preferably, the recombinant expression vector includes pETDuet-1.

8. A genetically engineered strain expressing the fusion protein of claim 4 or 5 or comprising the recombinant expression vector of claim 7; Preferably, the genetically engineered strain comprises Escherichia coli.

9. the genetically engineered strain of high-yield thioneine described in any one of claims 1-3, or the application of the genetically engineered strain described in claim 8 in producing thioneine.

10. A method for preparing high-yield ergothioneine, characterized in that, The method comprises the step of using the genetically engineered strain with high ergothioneine production according to any one of claims 1 to 3, or the genetically engineered strain according to claim 8 as a fermentation strain for biosynthesis.

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