Construction Method and Application of a High-Efficiency Ergothionein-Producing Bacillus Amyloliquefaciens Engineered Strain

Abstract

This invention provides a method for constructing a high-yield ergothionein-producing Bacillus amyloliquefaciens engineered strain and its application. The engineered strain uses the food-grade safe strain Bacillus amyloliquefaciens HZ12 as the starting strain. Through genetic engineering, a key enzyme gene for ergothionein biosynthesis is heterologously expressed in Bacillus amyloliquefaciens. An RBS co-expression strategy is employed to express the mutant gene NcEgt1 derived from Neurospora crassa. Y174F / E51A Co-expression of the EgtE gene derived from *Mycobacterium smegmatis* successfully achieved heterologous synthesis of ergothioneine in *Bacillus amyloliquefaciens*. Under these conditions, the yield of ergothioneine reached 402.68 mg / L, marking the achievement of ergothioneine production in *Bacillus amyloliquefaciens* from scratch. Subsequently, through chassis strain modification, the yield of ergothioneine was further increased to 690.43 mg / L.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering technology, specifically relating to a method for constructing a high-yield ergothionein-producing Bacillus amyloliquefaciens engineered strain and its application. Background Technology

[0002] Ergothioneine (EGT) is a natural sulfur-containing amino acid derivative with various physiological functions, including antioxidant, anti-inflammatory, neuroprotective, and anti-aging effects, and has broad application prospects in the food, pharmaceutical, and cosmetic fields. Currently, EGT production mainly relies on natural product extraction and microbial fermentation. Natural extraction primarily uses edible fungi as its source, but the long growth cycle of edible fungi (usually several weeks to months), demanding cultivation conditions, and complex extraction processes result in high production costs, making it difficult to meet the growing market demand. Therefore, there is an urgent need to develop an efficient, stable, and scalable alternative production process. Microbial fermentation, due to its green and sustainable characteristics, has become a major research direction in EGT production. In existing technologies, heterologous synthesis of EGT mainly relies on model microorganisms such as *Escherichia coli* and *Saccharomyces cerevisiae* as chassis strains, achieved by expressing a five-enzyme system (EgtA / EgtB / EgtC / EgtD / EgtE) derived from *Mycolicibacterium smegmatis* or a two-enzyme system (Egt1 / Egt2) derived from *Neurospora crassa*. However, all of these chassis strains have limitations to varying degrees. While *E. coli* offers abundant genetic manipulation tools, the presence of its endotoxins poses significant safety risks, requiring complex isolation and extraction processes, thus limiting its application in the food and pharmaceutical fields. Although *Saccharomyces cerevisiae* is a safe strain, its long growth cycle and relatively low protein expression levels make it difficult to meet the needs of industrial production.

[0003] Against this backdrop, *Bacillus amyloliquefaciens*, a recognized food-grade safe (GRAS) microorganism, has gradually become a promising new chassis strain for EGT heterologous synthesis. This strain does not produce endotoxins, exhibiting better biosafety, and possesses advantages such as rapid growth, strong protein secretion capacity, and good environmental adaptability. It has been successfully applied to the microbial synthesis of various functional food components, demonstrating its great potential as a chassis strain for EGT production. However, there are currently no successful reports of heterologous EGT synthesis in *Bacillus amyloliquefaciens*. Key technologies such as the screening of key enzymes for EGT synthesis suitable for this host, expression system adaptation, and metabolic network optimization still require breakthroughs. Therefore, developing a technical solution for efficient heterologous EGT synthesis in *Bacillus amyloliquefaciens* has significant theoretical importance and broad application prospects. Summary of the Invention

[0004] Technical problem to be solved: In view of the above-mentioned technical problems, the purpose of this invention is to provide a method for constructing a high-yield ergothionein-producing Bacillus amyloliquefaciens engineered strain and its application, aiming to screen for ergothionein synthases adapted to Bacillus amyloliquefaciens, so as to achieve heterologous synthesis of ergothionein in food-grade safe strains of Bacillus amyloliquefaciens.

[0005] Technical solution: A high-yield ergothionein-producing Bacillus amyloliquefaciens engineered strain, including HZ12-1, HZ12-2, and HZ12-3; the engineered strain HZ12-1 overexpresses the mutant NcEgt1 derived from Neurospora crassa. Y174F / E51A The engineered strain HZ12-2 overexpressed the C-terminal truncated protein NcEgt1-1 gene from Neurospora crassa, the CtEgtB gene from Agrophytes thermophila, and the MsEgtE gene from Mycobacterium smegmatis; the engineered strain HZ12-3 overexpressed the N-terminal truncated protein NcEgt1-2 gene from Neurospora crassa, and the MsEgtD and MsE genes from Mycobacterium smegmatis.

[0006] The above coding is derived from the mutant NcEgt1 of Neurospora crassa. Y174F / E51A The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] The nucleotide sequence encoding the MsEgtE gene from Mycobacterium smegmatis is shown in SEQ ID NO.2.

[0008] The nucleotide sequence of the above-mentioned gene, which is derived from the C-terminal truncated protein NcEgt1-1 of Neurospora crassa, is shown in SEQ ID NO. 3.

[0009] The nucleotide sequence of the CtEgtB gene, which is encoded by the above-mentioned thermophilic green bacteria, is shown in SEQ ID NO.4.

[0010] The nucleotide sequence encoding the N-terminal truncated protein NcEgt1-2 gene of Neurospora crassa is shown in SEQ ID NO. 5.

[0011] The above encoding is derived from the MsEgtD gene of Mycobacterium smegmatis, as shown in SEQ ID NO. 6.

[0012] The aforementioned engineered strains of Bacillus amyloliquefaciens that produce high levels of ergothionein also include HZ / S-1, HZ / SA-1, AM1-1, AM1-2, AM3-1, AM4-1, AM5-1, and AM6-1.

[0013] The engineered strain HZ / S-1 integrated and expressed the SAM2 gene in Bacillus amyloliquefaciens HZ12 and overexpressed the mutant NcEgt1 derived from Neurospora crassa. Y174F / E51A The engineered strains included the MsEgtE gene derived from Mycobacterium smegmatis; the engineered strain HZ12 / SA-1 integrated and expressed the Escherichia coli metA gene based on the HZ12 / S-1 strain; the engineered strain AM1-1 integrated and expressed the Escherichia coli metB gene based on the HZ12 / SA-1 strain; the engineered strain AM1-2 lacked the mccA gene based on the AM1-1 strain; the engineered strain AM3-1 lacked the sucC gene based on the AM2-1 strain; the engineered strain AM4-1 lacked the thrB gene based on the AM3-1 strain; the engineered strain AM5-1 lacked the srfAC gene based on the AM4-1 strain; the engineered strain AM6-1 lacked the ituD gene based on the AM5-1 strain; and the engineered strain AM6-1 lacked the ituD gene based on the AM5-1 strain.

[0014] The nucleotide sequence encoding the SAM2 gene is shown in SEQ ID NO.7.

[0015] The nucleotide sequence encoding the Escherichia coli metA gene is shown in SEQ ID NO.8.

[0016] The nucleotide sequence encoding the Escherichia coli metB gene is shown in SEQ ID NO.9.

[0017] The nucleotide sequence encoding the mccA gene is shown in SEQ ID NO.10.

[0018] The nucleotide sequence encoding the sucC gene is shown in SEQ ID NO.11.

[0019] The nucleotide sequence encoding the thrB gene is shown in SEQ ID NO.12.

[0020] The nucleotide sequence encoding the srfAC gene is shown in SEQ ID NO.13.

[0021] The nucleotide sequence encoding the ituD gene is shown in SEQ ID NO.14.

[0022] The application of the above-mentioned high-yield ergothionein-producing Bacillus amyloliquefaciens engineered strain in ergothionein production.

[0023] The above method for producing ergothioneine includes the following steps:

[0024] (1) After activating the engineered strain HZ12-1 or HZ12-2 or HZ12-3 or engineered strain HZ / S-1 or HZ / SA-1 or AM1-1 or AM1-2 or AM3-1 or AM4-1 or AM5-1 or AM6-1, pick a single colony and inoculate it into LB liquid medium. Incubate at 35~37℃ and 200~220 rpm / min until OD. 600 Achieving a pH of 3.0-4.0 is suitable for use as seed solution;

[0025] (2) The seed liquid was transferred to the ergothioneine fermentation medium at an inoculation rate of 3%, and fermented at 35~37℃ and 200~220 rpm / min for 60 h to produce ergothioneine.

[0026] Beneficial effects:

[0027] The technical effect of this invention is to achieve heterologous synthesis of ergothionein in Bacillus amyloliquefaciens, with a yield of 690.43 mg / L. This invention involves genetically engineering the starting strain Bacillus amyloliquefaciens HZ-12 to obtain engineered strains HZ12-1, HZ12-2, and HZ12-3. Shake-flask fermentation experiments showed that all engineered strains successfully synthesized ergothionein, with yields ranging from 149.97 to 402.68 mg / L after 60 h of culture. Among them, engineered strain HZ12-1 contains the complete bifunctional enzyme NcEgt1. Y174F / E51A The system expressing MsEgtE in tandem achieved the highest yield, reaching 402.68 mg / L. Further analysis of the SAM synthesis module led to the construction of chassis strains HZ / S-1, HZ / SA-1, AM1-1, AM2-1, AM3-1, AM4-1, AM5-1, and AM6-1. Chassis strains adapted for ergothioneine synthesis were screened, further increasing ergothioneine yield to 503.01–690.43 mg / L. Among these, the engineered strain AM4-1 integrated the SAM2 gene, *E. coli* metA gene, and *E. coli* metB gene into the HZ-12 strain, while deleting the mccA and sucC genes. Simultaneously, NcEgt1… Y174F / E51A When expressed in tandem with MsEgtE, the highest yield of ergothioneine was observed, at 690.43 mg / L. Attached Figure Description

[0028] Figure 1 This section describes the specific genes involved in the modification of different SAM chassis strains based on the SAM (S-adenosylmethionine) synthesis module. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments:

[0030] Biological material source description: Bacillus amyloliquefaciens HZ12 is currently preserved in the Microbial Engineering Laboratory of Huazhong Agricultural University.

[0031] The components and ratio of LB medium are: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0032] Components and ratios of the growth medium: 0.5 mol / L sorbitol was added to LB medium.

[0033] The composition and ratio of the washing medium: LB medium with 0.5 mol / L sorbitol, 0.5 mol / L mannitol and 10% (v / v) glycerol added.

[0034] The composition and ratio of the recovery medium: LB medium with 0.5 mol / L sorbitol and 0.38 mol / L mannitol added.

[0035] The components and proportions of the ergothionein fermentation medium are as follows: 40 g / L xylose, 10 g / L peptone, 2 g / L urea, 6.3 g / L (NH4)2SO4, 2.5 g / L NaCl, 4.2 g / L MgSO4, 3 g / L KH2PO4, 1 g / L methionine, 1 g / L histidine, and 1 g / L L-cysteine.

[0036] Genetic origin: NcEgt1 from Neurospora crassa Y174F / E51A (Genebank accession number: XP_956324.3); CtEgtB from Chlorobaculum tepidum (Genebank accession number: WP_014099806.1); MsEgtE from Mycolicibacterium smegmatis (Genebank accession number: WP_011731155.1); MsEgtD from Mycolicibacterium smegmatis (Genebank accession number: WP_011731156.1); all of the above genes underwent codon optimization and gene synthesis by a biotechnology company to obtain plasmid templates containing the target genes.

[0037] Example 1

[0038] This example uses the overexpression vector pHT254-P43-NcEgt1. Y174F / E51A The construction method of RBS-MsEgtE and engineered strain HZ12-1 includes the following steps:

[0039] S1. Using the primers shown in Table 1, with the Bacillus subtilis 168 genome as a template and P43-F and P43-R as primers, the P43 promoter (SEQ ID NO. 15) was amplified by PCR; using PUC-NcEgt1 Y174F / E51A As a template, NcEgt1 Y174F / E51A -F and NcEgt1 Y174F / E51A -R is the primer, and PCR amplification yields NcEgt1. Y174F / E51A (SEQ ID NO.1); Using PUC-MsegtE as a template, and RBS+MsEgtE-F and RBS+MsEgtE-R as primers, the RBS+MsEgtE fragment (SEQ ID NO.16) was obtained by PCR amplification; the PCR amplification system is shown in Table 2, and the PCR amplification conditions are: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 34 cycles, 72℃ final extension for 5 min, and 4℃ incubation.

[0040] S2. PCR products were recovered, and the linearized vector pHT254 was double-digested with restriction endonucleases XbaI and BamHI. The double-digested vector was recovered, and homologous recombination of the PCR products and double-digested products was performed using the ClonExpress II One Step Cloning Kit according to Table 3 (50℃, ligation for 20 min). The ligation product was transformed into E. coli DH5α competent cells. Transformants were verified by colony PCR using pHT254-F / R primers. After preliminary confirmation of positive transformants, plasmids were extracted for double digestion with XbaI and BamHI and DNA sequencing verification. After successful verification, the overexpression vector pHT254-P43-NcEgt1 was obtained. Y174F / E51A -RBS- MsEgtE;

[0041] S3. Streak Bacillus amyloliquefaciens HZ12 strain on a plate, pick an appropriate amount of cells and inoculate into 5 mL of liquid LB medium, incubate at 37°C and 180 r / min for 8 h; transfer the culture to 50 mL of growth medium, incubate at 37°C and 180 r / min for 3 h, and allow OD to develop. 600 The culture medium was increased to 1.5-2.5. The culture medium was poured into a sterile 50 mL centrifuge tube, pre-cooled on ice for 10 min, and centrifuged at 6500 r / min for 5 min to collect the bacterial cells. The bacterial cells were washed 3-4 times with 20 mL of pre-cooled washing medium, centrifuged at 6500 r / min for 5 min, the supernatant was discarded, and the bacterial cells were resuspended in 800 μL of washing medium. 100 μL of the resuspended cells were dispensed into 1.5 mL centrifuge tubes and stored at -80°C for later use to obtain Bacillus amyloliquefaciens HZ12 competent cells.

[0042] S4. Add 10 μL of the overexpression vector pHT254-P43-NcEgt1 Y174F / E51A The RBS-MsEgtE plasmid (50 ng / μL) was added to Bacillus amyloliquefaciens HZ12 competent cells, gently mixed, and then transferred to a pre-cooled 2 mm electroporation cuvette. After incubating on ice for 10 min, the cells were electroporated once at 2.4 KV using an electroporator. 800 μL of recovery medium was quickly added, and the cells were incubated at 37°C and 100 r / min for 3 h. The cells were then plated on a plate containing Chl antibiotics and incubated statically at 37°C for 16 h. Positive transformants were screened, and single colonies of the transformants were picked and streaked on a plate containing Chl antibiotics. After incubation for 12 h, single colonies were picked as templates for colony PCR. Colony PCR was performed using primers pHT254-F and pHT254-R to verify the results, and the engineered strain HZ12-1 was obtained.

[0043] Table 1 PCR Primers

[0044]

[0045] Table 2 PCR amplification system

[0046]

[0047] Table 3 Homologous recombination system

[0048]

[0049] Example 2

[0050] This embodiment describes the construction method of the overexpression vector pHT254-P43-NcEgt1-1-RBS+CtEgtB-RBS-MsEgtE and the engineered strain HZ12-2, including the following steps:

[0051] S1. Using the primers shown in Table 4, with pHT254-P43-NcEgt1 Y174F / E51AUsing RBS-MsEgtE plasmid as a template, and 254-Egt1B-AF and Egt1-B-EgtB-AR as primers, PCR amplification yielded the P43+NcEgt1-1 fragment (SEQ ID NO.17); using PUC-CtEgtB plasmid as a template, and RBS+CtEgtB-F and RBS+CtEgtB-R as primers, PCR amplification yielded the RBS+CtEgtB fragment (SEQ ID NO.18); using PUC-MsegtE plasmid as a template, and RBS+MsEgtE-F and RBS+MsEgtE-R as primers, PCR amplification yielded the RBS+MsEgtE fragment (SEQ ID NO.16). The PCR amplification system is shown in Table 2. The PCR amplification conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 1 minute. min, cycle 34 times, final extension at 72℃ for 5 min, hold at 4℃;

[0052] S2. Same as Example 1 S2, to obtain the overexpression vector pHT254-P43-NcEgt1-1-RBS+CtEgtB-RBS-MsEgtE;

[0053] S3. Same as Example 1 S3, obtain Bacillus amyloliquefaciens HZ12 competent cells;

[0054] S4. Same as Example 1 S4, to obtain engineered strain HZ12-2.

[0055] Table 4 PCR products

[0056]

[0057] Example 3

[0058] This embodiment describes the construction method of the overexpression vector pHT254-P43-MsEgtD-RBS-NcEgt1-2-RBS-MsEgtE and the engineered strain HZ12-3, including the following steps:

[0059] S1. Using the primers shown in Table 5, with PUC-MsEgtD as a template and P43+MsEgtD-F and P43+MsEgtD-R as primers, PCR amplification was performed to obtain the P43+MsEgtD fragment (SEQ ID NO.19); using PUC-Ncegt1... Y174F / E51AUsing plasmids as templates and RBS+NcEgt1-2-F and RBS+NcEgt1-2-R as primers, PCR amplification was performed to obtain the RBS+NcEgt1-2 fragment (SEQ ID NO.20); using PUC-MsEgtE as templates and RBS+MsEgtE-F and RBS+MsEgtE-R as primers, PCR amplification was performed to obtain the RBS+MsEgtE fragment (SEQ ID NO.16); the PCR amplification system is shown in Table 2, and the PCR amplification conditions are: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 34 cycles, 72℃ final extension for 5 min, and incubation at 4℃;

[0060] S2. Same as Example 1 S2, to obtain the overexpression vector pHT254-P43-MsEgtD-RBS-NcEgt1-2-RBS-MsEgtE;

[0061] S3. Same as Example 1 S3, obtain Bacillus amyloliquefaciens HZ12 competent cells;

[0062] S4. Same as Example 1 S4, to obtain engineered strain HZ12-3

[0063] Table 5 PCR Products

[0064]

[0065] Example 4

[0066] This embodiment describes a method for producing ergothioneine using engineered bacterial strains through fermentation, including the following steps:

[0067] S1. The engineered strains HZ12-1, HZ12-2, and HZ12-3 prepared in Examples 1-3 were activated on LB plates, and single colonies were picked and inoculated into 50 mL of LB liquid medium and cultured at 37°C and 143 rpm / min until OD. 600 The culture medium was 4.0, which was used as a seed culture. Then, 3% of the culture medium was transferred to 10 mL of ergothioneine fermentation medium and fermented at 37°C and 143 rpm / min for 60 h. Each group was repeated in triplicate.

[0068] S2. Add 1350 μL of methanol to 150 μL of fermentation broth, sonicate for 1 h, centrifuge at 12000 r / min for 10 min, collect the supernatant, and filter it through a 0.22 μm microporous membrane. Detection was performed using a Shimadzu SPD-20A high-performance liquid chromatography system equipped with an Agilent ZORBAX SB-Aq column (250 mm × 4.6 mm, 5 μm particle size), a detection wavelength of 254 nm, a column temperature of 30℃, and a mobile phase of water (solvent A) and acetonitrile (solvent B) eluted isocratically at a ratio of 98:2 (v / v). The flow rate was 1.0 mL / min, and the injection volume was 10 μL. Accurately weigh ergothioneine standard, dissolve it in ultrapure water, prepare a series of standard solutions, and establish a standard curve for quantitative analysis.

[0069] Table 6 Ergothioneine Production

[0070]

[0071] NcEgt1, a mutant derived from Neurospora crassa Y174F / E51A The gene possesses dual functions as both a methyltransferase and a sulfoxide synthase, catalyzing the first two key steps in ergothionein synthesis: first, histidine methylation to form HER; then, using L-cysteine ​​as a sulfur donor, a CS bond is formed between HER and L-cysteine ​​to generate Cys-HER. Due to NcEgt1... Y174F / E51A The large molecular weight and limited expression level of enzymes may affect the overall synthesis efficiency. Therefore, this invention utilizes NcEgt1... Y174F / E51A NcEgt1 was truncated at amino acid 358 (Met) to obtain the C-terminal truncated protein NcEgt1-1, which retains the complete putative methyltransferase domain. Simultaneously, to supplement the sulfoxide synthase function, CtEgtB from *Mycobacterium thermophilum* was introduced to catalyze the reaction of HER with L-cysteine ​​to produce Cys-HER. Meanwhile, the N-terminal truncated protein NcEgt1-2 was constructed at the same site, retaining the complete putative sulfoxide synthase domain, and combined with the methyltransferase MsEgtD from *Mycobacterium smegmatis* to provide sulfoxide synthesis and methyl transfer functions, respectively. Finally, using the PLP-dependent CS lyase MsEgtE from *Mycobacterium smegmatis*, Cys-HER was converted to the target product ergothioneine.

[0072] As shown in Table 6, all the modified engineered strains can ferment and produce ergothioneine, with yields ranging from 106.19 to 402.68 mg / L. The engineered strain HZ12-1 contains the complete bifunctional enzyme NcEgt1. Y174F / E51AThe system expressing MsEgtE in tandem yielded the highest output, reaching 402.68 mg / L. The optimal plasmid for ergothionein synthesis by Bacillus amyloliquefaciens was pHT254-P43-NcEgt1. Y174F / E51A -RBS-MsEgtE (Example 1).

[0073] Example 5

[0074] The difference between this embodiment and Example 1 is that in this embodiment, the overexpression vector pHT254-P43-NcEgt1 constructed in Example 1 is used. Y174F / E51A -RBS-MsEgtE was transferred into the chassis strain HZ / S to construct the engineered strain HZ / S-1. The remaining steps were the same as in Example 1. The construction method of the chassis strain HZ / S includes the following steps:

[0075] S1. The P43+SAM2+TamyL fragment (SEQ ID NO.21) along with its upstream and downstream homologous arms were homologously ligated with plasmid T2(2)-ori, and the ligation product was transformed into E. coli DH5α competent cells by calcium conversion method. The cells were plated on LB solid medium containing kanamycin resistance and screened at 37°C. Positive transformants were verified by colony PCR and DNA sequencing. After verification, the integration vector T2::SAM2 was obtained.

[0076] S2. The vector T2::SAM2 was electroporated into competent cells of Bacillus amyloliquefaciens HZ12, spread on LB solid medium containing kanamycin resistance, and screened at 37°C. Positive transformants were inoculated onto LB solid medium containing kanamycin resistance and cultured at 45°C for 12 h. The inoculation was repeated 3 times to obtain single-exchange strains. The single-exchange strains were inoculated onto LB solid medium without kanamycin resistance and cultured at 45°C for 12 h. The inoculation was repeated several times to obtain the chassis strain HZ / S with successful double exchange.

[0077] Example 6

[0078] The difference between this embodiment and Example 1 is that in this embodiment, the overexpression vector pHT254-P43-NcEgt1 constructed in Example 1 is used. Y174F / E51A -RBS-MsEgtE was transferred into the chassis strain HZ / SA to construct the engineered strain HZ / SA-1. The remaining steps were the same as in Example 1. The construction method of chassis strain HZ / SA includes the following steps:

[0079] S1. The P43+metA+TamyL fragment (SEQ ID NO.22) along with its upstream and downstream homologous arms were homologously ligated with plasmid T2(2)-ori, and the ligation product was transformed into E. coli DH5α competent cells by calcium conversion method. The cells were plated on LB solid medium containing kanamycin resistance and screened at 37°C. Positive transformants were verified by colony PCR and DNA sequencing. After verification, the integration vector T2::metA was obtained.

[0080] S2. Same as in Example 1 S3, obtain Bacillus amyloliquefaciens HZ / S competent cells;

[0081] S3. Following step S2 of Example 5, the integration vector T2::metA was electroporated into Bacillus amyloliquefaciens HZ / S competent cells to obtain the chassis strain HZ / SA.

[0082] Example 7

[0083] The difference between this embodiment and Example 1 is that in this embodiment, the overexpression vector pHT254-P43-NcEgt1 constructed in Example 1 is used. Y174F / E51A -RBS-MsEgtE was transferred into the chassis strain AM1 to construct the engineered strain AM1-1. The remaining steps were the same as in Example 1. The construction method of chassis strain AM1 includes the following steps:

[0084] S1. The P43+metB+TamyL fragment (SEQ ID NO.23) along with its upstream and downstream homologous arms were homologously ligated with plasmid T2(2)-ori, and the ligation product was transformed into E. coli DH5α competent cells by calcium conversion method. The cells were plated on LB solid medium containing kanamycin resistance and screened at 37°C. Positive transformants were verified by colony PCR and DNA sequencing. After verification, the integration vector T2::metB was obtained.

[0085] S2. Same as in Example 1 S3, obtain Bacillus amyloliquefaciens HZ / SA competent cells;

[0086] S3. Following step S2 of Example 5, the integration vector T2::metB was electroporated into Bacillus amyloliquefaciens HZ / SA competent cells to obtain chassis strain AM1.

[0087] Example 8

[0088] The difference between this embodiment and Example 1 is that in this embodiment, the overexpression vector pHT254-P43-NcEgt1 constructed in Example 1 is used. Y174F / E51A-RBS-MsEgtE was transferred into the chassis strain AM2 to construct the engineered strain AM2-1. The remaining steps were the same as in Example 1. The construction method of chassis strain AM2 includes the following steps:

[0089] S1. Homologous recombination ligation of the upstream and downstream homologous arms of gene mccA (SEQ ID NO.10) with plasmid T2(2)-ori was performed, and the ligation product was transformed into E.coli DH5α competent cells by calcium conversion method. The cells were plated on LB solid medium containing kanamycin resistance and screened at 37°C. Positive transformants were verified by colony PCR and DNA sequencing. After verification, the knockout vector T2ΔmccA was obtained.

[0090] S2. Same as in Example 1 S3, obtain Bacillus amyloliquefaciens AM1 competent cells;

[0091] S3. Following step S2 of Example 5, the knockout vector T2ΔmccA was electroporated into Bacillus amyloliquefaciens AM1 competent cells to obtain chassis strain AM2.

[0092] Example 9

[0093] The difference between this embodiment and Example 1 is that in this embodiment, the overexpression vector pHT254-P43-NcEgt1 constructed in Example 1 is used. Y174F / E51A -RBS-MsEgtE was transferred into the chassis strain AM3 to construct the engineered strain AM3-1. The remaining steps were the same as in Example 1. The construction method of chassis strain AM3 includes the following steps:

[0094] S1. Homologous recombination ligation was performed between the upstream and downstream homologous arms of gene sucC (SEQ ID NO.11) and plasmid T2(2)-ori, respectively. The ligation product was then transformed into E. coli DH5α competent cells by calcium conversion method. The cells were plated on LB solid medium containing kanamycin resistance and screened at 37°C. Positive transformants were verified by colony PCR and DNA sequencing. After verification, the knockout vector T2ΔsucC was obtained.

[0095] S2. Same as in Example 1 S3, obtain Bacillus amyloliquefaciens AM2 competent cells;

[0096] S3. Following step S2 of Example 5, the knockout vector T2ΔsucC was electroporated into Bacillus amyloliquefaciens AM2 competent cells to obtain chassis strain AM3.

[0097] Example 10

[0098] The difference between this embodiment and Example 1 is that in this embodiment, the overexpression vector pHT254-P43-NcEgt1 constructed in Example 1 is used. Y174F / E51A -RBS-MsEgtE was transferred into the chassis strain AM4 to construct the engineered strain AM4-1. The remaining steps were the same as in Example 1. The construction method of chassis strain AM4 includes the following steps:

[0099] S1. Homologous recombination ligation of the upstream and downstream homologous arms of the gene thrB (SEQ ID NO.12) with plasmid T2(2)-ori was performed, and the ligation product was transformed into E.coli DH5α competent cells by calcium conversion method. The cells were plated on LB solid medium containing kanamycin resistance and screened at 37°C. Positive transformants were verified by colony PCR and DNA sequencing. After verification, the knockout vector T2ΔthrB was obtained.

[0100] S2. Same as Example 1 S3, obtain Bacillus amyloliquefaciens AM3 competent cells;

[0101] S3. Following step S2 of Example 5, the knockout vector T2ΔthrB was electroporated into Bacillus amyloliquefaciens AM3 competent cells to obtain chassis strain AM4.

[0102] Example 11

[0103] The difference between this embodiment and Example 1 is that in this embodiment, the overexpression vector pHT254-P43-NcEgt1 constructed in Example 1 is used. Y174F / E51A -RBS-MsEgtE was transferred into the chassis strain AM5 to construct the engineered strain AM5-1. The remaining steps were the same as in Example 1. The construction method of chassis strain AM5 includes the following steps:

[0104] S1. Homologous recombination ligation of the upstream and downstream homologous arms of the gene srfAC (SEQ ID NO.13) with plasmid T2(2)-ori was performed, and the ligation product was transformed into E. coli DH5α competent cells by calcium conversion method. The cells were plated on LB solid medium containing kanamycin resistance and screened at 37°C. Positive transformants were verified by colony PCR and DNA sequencing. After verification, the knockout vector T2ΔsrfAC was obtained.

[0105] S2. Same as Example 1 S3, obtain Bacillus amyloliquefaciens AM4 competent cells;

[0106] S3. Following step S2 of Example 5, the knockout vector T2ΔsrfAC was electroporated into Bacillus amyloliquefaciens AM4 competent cells to obtain the chassis strain AM5.

[0107] Example 12

[0108] The difference between this embodiment and Example 1 is that in this embodiment, the overexpression vector pHT254-P43-NcEgt1 constructed in Example 1 is used. Y174F / E51A -RBS-MsEgtE was transferred into the chassis strain AM6 to construct the engineered strain AM6-1. The remaining steps were the same as in Example 1. The construction method of chassis strain AM6 includes the following steps:

[0109] S1. The gene ituD (SEQ ID NO.14) along with its upstream and downstream homologous arms were homologously ligated with plasmid T2(2)-ori, and the ligation product was transformed into E. coli DH5α competent cells by calcium conversion method. The cells were plated on LB solid medium containing kanamycin resistance and screened at 37°C. Positive transformants were verified by colony PCR and DNA sequencing. After verification, the knockout vector T2ΔituD was obtained.

[0110] S2. Same as in Example 1 S3, obtain Bacillus amyloliquefaciens AM5 competent cells;

[0111] S3. Following step S2 of Example 5, the knockout vector T2ΔituD was electroporated into Bacillus amyloliquefaciens AM5 competent cells to obtain chassis strain AM6.

[0112] Table 7. Chassis strains from Examples 5 to 13

[0113]

[0114] like Figure 1 As shown, this illustrates the specific genes involved in the modification of different SAM chassis strains based on the SAM (S-adenosylmethionine) synthesis module.

[0115] Example 13

[0116] The difference between this embodiment and embodiment 4 is that the engineered strains used for fermentation in this embodiment are HZ / S-1, HZ / SA-1, AM1-1, AM2-1, AM3-1, AM4-1, AM5-1, and AM6-1, respectively, while the remaining steps are the same as in embodiment 4.

[0117] Table 8 Ergothioneine Production

[0118]

[0119] As shown in Table 8, the ergothioneine yields produced by fermentation of engineered strains HZ / S-1, HZ / SA-1, M1-1, AM2-1, AM3-1, AM4-1, AM5-1, and AM6-1 reached 503.01 mg / L, 533.01 mg / L, 616.58 mg / L, 632.27 mg / L, 675.43 mg / L, 690.43 mg / L, 651.17 mg / L, 657.28 mg / L, and 276.38 mg / L, respectively.

[0120] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A high-yield ergothionein-producing Bacillus amyloliquefaciens engineered strain, characterized in that: The engineered strains include HZ12-1, HZ12-2 and HZ12-3; The engineered strain HZ12-1 overexpressed a mutant of Neurospora crassa, NcEgt1. Y174F / E51A The engineered strain HZ12-2 overexpressed the C-terminal truncated protein NcEgt1-1 gene from Neurospora crassa, the CtEgtB gene from Agrophytes thermophila, and the MsEgtE gene from Mycobacterium smegmatis; the engineered strain HZ12-3 overexpressed the N-terminal truncated protein NcEgt1-2 gene from Neurospora crassa, and the MsEgtD and MsEgtE genes from Mycobacterium smegmatis.

2. The engineered strain of Bacillus amyloliquefaciens with high ergothioneine production according to claim 1, characterized in that: The code is derived from the mutant NcEgt1 of Neurospora crassa. Y174F / E51A The nucleotide sequences of the genes are shown in SEQ ID NO.1; the nucleotide sequence encoding the MsEgtE gene from Mycobacterium smegmatis is shown in SEQ ID NO.2; the nucleotide sequence encoding the C-terminal truncated protein NcEgt1-1 gene from Neurospora crassa is shown in SEQ ID NO.3; the nucleotide sequence encoding the CtEgtB gene from Agrophytes thermophila is shown in SEQ ID NO.4; the nucleotide sequence encoding the N-terminal truncated protein NcEgt1-2 gene from Neurospora crassa is shown in SEQ ID NO.5; and the nucleotide sequence encoding the MsEgtD gene from Mycobacterium smegmatis is shown in SEQ ID NO.

6.

3. The engineered strain of Bacillus amyloliquefaciens with high ergothioneine production according to claim 1 or 2, characterized in that: The engineered strains also include HZ / S-1, HZ / SA-1, AM1-1, AM1-2, AM3-1, AM4-1, AM5-1, and AM6-1; the engineered strain HZ / S-1 integrates and expresses the SAM2 gene in Bacillus amyloliquefaciens HZ12 and overexpresses the mutant NcEgt1 derived from Neurospora crassa. Y174F / E51A The engineered strains included the MsEgtE gene derived from Mycobacterium smegmatis; the engineered strain HZ12 / SA-1 integrated and expressed the Escherichia coli metA gene based on the HZ12 / S-1 strain; the engineered strain AM1-1 integrated and expressed the Escherichia coli metB gene based on the HZ12 / SA-1 strain; the engineered strain AM1-2 lacked the mccA gene based on the AM1-1 strain; and the engineered strain AM3-1 lacked the sucC gene based on the AM2-1 strain. The engineered strain AM4-1 is derived from the AM3-1 strain by deleting the thrB gene; the engineered strain AM5-1 is derived from the AM4-1 strain by deleting the srfAC gene. The engineered strain AM6-1 is based on the AM5-1 strain but lacks the ituD gene.

4. The engineered strain of Bacillus amyloliquefaciens with high ergothioneine production according to claim 3, characterized in that: The nucleotide sequence encoding the SAM2 gene is shown in SEQ ID NO.7; the nucleotide sequence encoding the E. coli metA gene is shown in SEQ ID NO.8; the nucleotide sequence encoding the E. coli metB gene is shown in SEQ ID NO.9; the nucleotide sequence encoding the mccA gene is shown in SEQ ID NO.10; the nucleotide sequence encoding the sucC gene is shown in SEQ ID NO.11; the nucleotide sequence encoding the thrB gene is shown in SEQ ID NO.12; the nucleotide sequence encoding the srfAC gene is shown in SEQ ID NO.13; and the nucleotide sequence encoding the ituD gene is shown in SEQ ID NO.

14.

5. The application of a high-yield ergothionein-producing Bacillus amyloliquefaciens engineered strain according to any one of claims 1 to 4 in the production of ergothionein.

6. The application according to claim 5, characterized in that, The method for producing ergothioneine includes the following steps: (1) After activating the engineered strain HZ12-1 or HZ12-2 or HZ12-3 as described in claim 1 or 2, or the engineered strain HZ / S-1 or HZ / SA-1 or AM1-1 or AM1-2 or AM3-1 or AM4-1 or AM5-1 or AM6-1 as described in claim 3 or 4, pick a single colony and inoculate it into LB liquid medium, and culture it at 35~37℃ and 200~220 rpm / min until OD. 600 Achieving a pH of 3.0-4.0 is suitable for use as seed solution; (2) The seed liquid was transferred to the ergothioneine fermentation medium at an inoculation rate of 3%, and fermented at 35~37℃ and 200~220 rpm / min for 60 h to produce ergothioneine.

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