Amino acid permease involved in transmembrane transport of ergothioneine and application thereof

Abstract

This invention discloses an amino acid permeabilizing enzyme involved in ergothioneine transmembrane transport and its applications, belonging to the fields of microbial metabolic engineering and biomanufacturing technology. The permeabilizing enzyme AAP-encoding gene of *Rhodococcus etherealis* N1- was constructed using homologous recombination technology. Δaap And a replacement strain N1-Δ was constructed using shuttle plasmids. aap -Repaired, the changes in ergothioneine levels inside and outside cells were measured through fermentation, demonstrating that the permease AAP is involved in ergothioneine transport. Further, this gene was introduced into *Rhodococcus etherealis* N1, increasing the extracellular ergothioneine content of the engineered strain by 24.87%. Using engineered strain N1- aap To optimize the fermentation process of ergothioneine-producing strains, we further improved the fermentation yield of extracellular ergothioneine while reducing the growth inhibition caused by intracellular ergothioneine accumulation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the application of an amino acid permease derived from Rhodococcus etherealis N1 and its encoding gene in the biomanufacturing of ergothionein. Background Technology

[0002] Ergothioneine (ERG) is a natural sulfur-containing amino acid derivative with excellent antioxidant, anti-inflammatory, and cytoprotective functions. Studies have shown that ergothioneine can effectively scavenge reactive oxygen species and reactive nitrogen species, maintain cellular redox homeostasis, and play important roles in neuroprotection, anti-aging, cardiovascular protection, and immune regulation. Due to its good bioactivity and safety, ergothioneine has been widely used in food, health products, cosmetics, and pharmaceuticals, and market demand continues to grow.

[0003] Currently, ergothioneine production methods mainly include natural extraction, chemical synthesis, and microbial fermentation. Compared to natural extraction and chemical synthesis, microbial fermentation offers advantages such as lower production costs, environmental friendliness, and ease of large-scale production, making it an important direction for the industrial production of ergothioneine. In recent years, some progress has been made in increasing ergothioneine yield by modifying microbial hosts through metabolic engineering, but existing production systems still face limitations in yield improvement.

[0004] During ergothioneine biosynthesis, the product mainly accumulates inside the cell. When the intracellular ergothioneine concentration continues to rise, it may, on the one hand, exert feedback inhibition on related synthetic enzymes, reducing metabolic flux; on the other hand, excessive accumulation of the product increases the osmotic pressure burden and storage pressure on the cell, thereby affecting cell growth and further synthesis of the target product. Furthermore, since most ergothioneine remains intracellularly, subsequent production processes typically require cell disruption and solvent extraction for product recovery, which not only increases production costs but also raises the difficulty of separation and purification.

[0005] The transmembrane transport of ergothioneine is closely related to its intracellular accumulation and extracellular secretion, making the identification of transport proteins involved in this process crucial. However, current research on the transmembrane transport mechanism of ergothioneine is relatively limited. Most reported ergothioneine biosynthetic strains focus on enhancing the synthetic pathway, optimizing precursor supply, and regulating cofactors, while the development and utilization of product efflux and transport systems remain insufficient. Therefore, developing a method to enhance the efflux or transport capacity of ergothioneine in Rhodococcus bacteria, thereby improving the extracellular secretion level and overall production efficiency of ergothioneine, is of great significance for achieving efficient biomanufacturing of ergothioneine. Summary of the Invention

[0006] The purpose of this invention is to provide a protein involved in ergothioneine transmembrane transport and its encoding gene, thereby increasing the extracellular ergothioneine yield of microorganisms. To further enhance the ergothioneine production capacity of the engineered strain N1-aap, this invention adds exogenous amino acids to the fermentation medium and optimizes the amount and method of addition to coordinate the supply of ergothioneine synthesis precursors with the transmembrane transport of the product, thereby further increasing the fermentation yield of ergothioneine.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0008] An amino acid permeabilizer involved in the transmembrane transport of ergothioneine, the amino acid sequence of which is shown in SEQ ID No. 1.

[0009] The amino acid permease AAP was derived from Rhodococcus aetherivorans N1, with accession number CCTCC NO: M 20221270.

[0010] The nucleotide sequence encoding the amino acid permeabilizer involved in the transmembrane transport of ergothioneine is shown in SEQ ID No. 2.

[0011] A recombinant expression vector comprising a nucleotide molecule encoding an amino acid permease involved in ergothioneine transmembrane transport.

[0012] A genetically engineered host cell in which the recombinant expression vector is introduced, or the nucleic acid molecule is integrated into the genome.

[0013] The application of the amino acid permeabilizing enzyme in mediating ergothioneine transmembrane transport.

[0014] A method for increasing the yield of extracellular ergothionein fermentation by microorganisms includes the following steps:

[0015] (1) Overexpressing the gene encoding the amino acid permease in ergothioneine-producing host strains;

[0016] (2) The obtained recombinant engineered bacteria were subjected to liquid fermentation culture;

[0017] (3) Ergothionein was detected in the fermentation broth.

[0018] The host strain that produces ergothionein is Rhodococcus etherein N1.

[0019] In step (1), the host strain is Rhodococcus aetherivorans N1, with accession number CCTCC NO: M 20221270. The Rhodococcus shuttle plasmid pNV-aap was constructed by expressing plasmid pNV18.1 and introduced into Rhodococcus aetherivorans N1 to obtain engineered strain N1-aap. The engineered strain N1-aap overexpresses amino acid permease.

[0020] The fermentation conditions are as follows:

[0021] 1) Plate culture: The engineered strain N1-aap was streaked onto LB solid medium and cultured at 30℃ for 48 h.

[0022] 2) Seed culture: Colonies on the plate medium were inoculated into LB seed medium and cultured at 30℃ for 30 h.

[0023] 3) Fermentation culture: The seed culture solution is inoculated into the fermentation medium, the fermentation temperature is 30℃, and the fermentation time is 120h to produce ergothioneine.

[0024] The fermentation medium was formulated as follows: glucose 30 g / L, yeast powder 5 g / L, K2HPO4·3H2O 1.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, and trace elements 1 ml / L (trace element formulation: FeSO4·7H2O 80 mg / L, ZnSO4·7H2O 22.5 mg / L, MnSO4·4H2O 40 mg / L, CuSO4·5H2O 5 mg / L, NaCl 15 mg / L, Na2MoO4·2H2O 0.3 mg / L, KCl 0.3 mg / L, CoCl2·6H2O 0.03 mg / L, CaCl2·2H2O 300 mg / L).

[0025] Different concentrations of methionine, cysteine, and histidine were added to the basic fermentation medium to ferment the engineered strain N1-aap. The cell growth and the amount of ergothionein were used as evaluation indicators to determine the appropriate conditions for adding exogenous amino acids.

[0026] The concentration of histidine added is 0.8~1.2 g / L, preferably 1 g / L.

[0027] The concentration of methionine added is 0.4~1 g / L, preferably 0.4 g / L;

[0028] The concentration of cysteine ​​added is 0.4~1 g / L, preferably 0.4 g / L.

[0029] Preferably, histidine, methionine and cysteine ​​are added simultaneously, with histidine concentration of 1 g / L, methionine concentration of 0.4 g / L and cysteine ​​concentration of 0.4 g / L.

[0030] The Rhodococcus aetherivorans N1 strain has the accession number CCTCC NO: M 20221270.

[0031] A knockout plasmid was constructed using pK18mobsacB to knock out the Aap gene, resulting in the knockout strain N1-Δaap.

[0032] The Rhodococcus shuttle plasmid pNV-aap was constructed using expression plasmid pNV18.1 and introduced into the knockout strain N1-Δaap. Gene replacement was then performed to obtain the replaced strain N1-Δaap-Repaired.

[0033] The Rhodococcus shuttle plasmid pNV-aap was constructed using expression plasmid pNV18.1 and introduced into Rhodococcus ether-eating ether-eating N1 to obtain the engineered strain N1-aap.

[0034] The plasmid contains a kanamycin marker gene.

[0035] This invention constructs a knockout plasmid using pK18mobsacB to knock out the Aap gene, resulting in the knockout strain N1-Δaap. Then, a Rhodococcus shuttle plasmid pNV-aap is constructed using the expression plasmid pNV18.1 and introduced into the knockout strain N1-Δaap. Gene complementation is then performed to obtain the complemented strain N1-Δaap-Repaired. Gene knockout and complementation experiments confirm that the protein is involved in the transmembrane transport of ergothioneine.

[0036] The method for constructing the engineered Rhodococcus ether-eating bacteria first involves constructing the knockout plasmid pK-Δaap using pK18mobsacB. The pK-Δaap plasmid contains 1000bp sequences upstream and downstream of Aap. Then, the expression plasmid pNV18.1 is used to construct the Rhodococcus shuttle plasmid pNV-aap, which contains the amino acid permease AAP.

[0037] The plasmid pK-Δaap was introduced into Rhodococcus etherealis N1 by electroporation, and the Aap-knockout strain N1-Δaap was obtained by sucrose plate reverse screening. Then, the plasmid pNV-aap was introduced into Rhodococcus etherealis N1-Δaap by electroporation to obtain the complemented strain N1-Δaap-Repaired.

[0038] The construction method of Rhodococcus ethereatus N1-aap involves first constructing the knockout plasmid pK-Δaap using pK18mobsacB. Plasmid pK-Δaap contains 1000 bp upstream and downstream sequences of Aap. Then, the Rhodococcus shuttle plasmid pNV-aap, containing the amino acid permease Aap, is constructed using the expression plasmid pNV18.1. Finally, plasmid pNV-aap is introduced into Rhodococcus ethereatus N1 via electroporation to obtain the engineered Rhodococcus ethereatus N1-aap overexpressing the amino acid permease Aap.

[0039] The application of Rhodococcus ethereans N1-aap in the production of ergothioneine.

[0040] Ergothionein was produced by fermenting and culturing Rhodococcus etherealis under aerobic conditions.

[0041] This invention provides an ergothioneine transport-related protein derived from *Rhodococcus etherealis*, its encoding gene, and its applications. Gene knockout and gene complementation experiments confirmed that the protein participates in the transmembrane transport of ergothioneine, providing a new protein resource for improving ergothioneine production efficiency and constructing high-efficiency production strains through transport engineering. Overexpression of the functional amino acid permease Aap led to the construction of an engineered strain N1-aap, which enhances ergothioneine transport efficiency and increases extracellular ergothioneine production. To further enhance the ergothioneine production capacity of the engineered strain N1-aap, exogenous amino acids were added to the fermentation medium, coordinating the supply of ergothioneine precursors with transmembrane product transport, thereby further increasing the fermentation yield of ergothioneine.

[0042] Beneficial effects

[0043] 1. This invention is the first to discover and identify an amino acid permease involved in the transmembrane transport of ergothioneine, enriching the gene resources related to ergothioneine metabolism; it is expected to improve the transport efficiency and extracellular secretion capacity of ergothioneine, thereby promoting the performance optimization of ergothioneine-producing strains.

[0044] 2. This invention constructs an engineered strain N1-aap to improve the efficiency of ergothioneine transport, thereby increasing the extracellular ergothioneine yield of microorganisms.

[0045] 3. This invention further optimizes the fermentation conditions of engineered strain N1-aap by adding exogenous amino acids related to ergothioneine biosynthesis to the fermentation medium, thereby increasing the extracellular ergothioneine yield of microorganisms. Attached Figure Description

[0046] Figure 1 This is the plasmid map of pK-Δaap.

[0047] Figure 2This is a plasmid map of pNV-aap.

[0048] Figure 3 Liquid chromatography-masses of extracellular products of Rhodococcus ethereans N1, knockout strain N1-Δaap, replenishment strain N1-Δaap-Repaired, engineered strain N1-aap, and ergothioneine standard samples.

[0049] Figure 4 Extracellular ergothionein production of *Rhodococcus ethereinus* N1, knockout strain N1-Δaap, replenishment strain N1-Δaap-Repaired, engineered strain N1-aap, and engineered strain N1-aap.

[0050] Figure 5 The intracellular and extracellular distribution ratio of ergothionein in Rhodococcus ethereans N1 and engineered strain N1-aap.

[0051] Figure 6 Results of amino acid concentration optimization.

[0052] Figure 7 The yield graph shows the results after optimizing the amino acid concentration for engineered bacteria. Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0055] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0056] The strain used in this invention is *Rhodococcus aetherivorans*, strain number N1, deposited at the China Center for Type Culture Collection (CCTCC) on August 11, 2022, with accession number CCTCC NO: M20221270. The deposit address is Wuhan, China. This strain is the same strain reported in the prior art (CN116855401A) that degrades various lignin-derived aromatic compounds.

[0057] Example 1

[0058] The Aap gene was discovered in the ergothionein synthesis gene cluster of Rhodococcus through genomic analysis, and its function was then identified.

[0059] The Aap gene of Rhodococcus etherealis N1 was knocked out using homologous recombination.

[0060] (1) Using the genome of strain N1 as a template, the homologous arms A1 and A2 flanking Aap were amplified. The length of each homologous arm was 1000 bp. Linearized pK18mobsacB was obtained by reverse PCR. Multiple fragments were ligated using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. The ratio of vector to fragments in the reaction system was 1:1. The ligated reaction solution was introduced into competent E. coli cells, incubated in an ice-water bath for 30 min, a water bath at 42 ℃ for 90 s, an ice-water bath for 2 min, and then 900 μL of LB liquid medium was added. The cells were incubated at 37 ℃ and 180 rpm for 45-60 min. The cells were then screened with kanamycin selection plates to obtain the Aap knockout plasmid pK-Δaap.

[0061] (2) The constructed and verified plasmid was introduced into the original Rhodococcus ethereinus N1 by point transformation. The voltage was 2000 V, the resistance was 400 Ω, the capacitance was 25 μF, the electroporation time was 5-6 ms, and 400 μL of resuscitation medium was added immediately after electroporation. The culture was carried out in a shaker at 30 ℃ for 3.5 h. The strain was screened with kanamycin screening plates and verified.

[0062] (3) The strains that were verified to have undergone homologous recombination were picked and cultured in LB test tubes at 30°C and 180 rpm for 10 h. They were then spread on LB agar medium containing 30% sucrose and cultured at 30°C for 4 days. The Aap knockout strain N1-Δaap was screened by comparison with the marker. All primer sequences are shown in Table 1.

[0063] Table 1. Primer sequences required for construction

[0064] PK-U aagcttggcactggccgtc PK-D ggatccccgggtaccgagctc A1-U gaattcgagctcggtacccggggatccgggacgcaccggcggaaac A1-D catgtcttcgtggttggaatgtacgcgcgttcacccgcatcctgtc A2-U ccggcgacaggatgcgggtgaacgcgcgtacattccaaccacgaagac A2-D gtaaaacgacggccagtgccaagcttctgtgttccttgccgaggtcg

[0065] Table 2 PCR System Configuration Table

[0066] Phanta Max Super-Fidelity DNA Polymerase 1 μL buffer 25 μL <![CDATA[dd-H20]]> 20 μL dNTP 1 μL forward primer and reverse primer 1 μL and 1 μL

[0067] Table 3. Conditions required for fragment amplification

[0068] A1 1000 bp A1-U A1-D 60 1.5 A2 1001 bp A2-U A2-D 61 1.5 pK18mobsacB 7696 bp PK-U PK-D 55 6.0

[0069] Example 2

[0070] Replacement and overexpression of the knockout gene Aap.

[0071] (1) The amino acid permease Aap was amplified using the genome of strain N1 as a template. The Aap gene sequence is shown in SEQ ID No. 1. Linearized pNV18.1 was obtained by reverse PCR and fragment ligation was performed using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. The ratio of vector to fragments in the reaction system was 1:1. The ligated reaction solution was introduced into competent E. coli cells, incubated in an ice-water bath for 30 min, a water bath at 42 ℃ for 90 s, an ice-water bath for 2 min, and then 900 μL of LB liquid medium was added. The cells were incubated at 37 ℃ and 180 rpm for 45-60 min. The cells were then screened using kanamycin selection plates to obtain the Aap complement plasmid pNV-aap.

[0072] (2) The constructed and verified plasmid pNV-aap was introduced into engineered Rhodococcus etherealis N1-Δaap and Rhodococcus etherealis N1 by point transformation. The voltage was 2000 V, the resistance was 400 Ω, the capacitance was 25 μF, and the electroporation time was 5-6 ms. Immediately after electroporation, 400 μL of LB medium was added, and the culture was carried out in a shaker at 30 ℃ for 3 h. The strains were screened with kanamycin screening plates and verified by testing to obtain the complemented strain N1-Δaap-Repaired and the overexpressed engineered strain N1-aap. All primer sequences are shown in Table 4.

[0073] Table 4 Primer sequences required for construction

[0074] PNV-U agctcgaattcgtaatcatggtc PNV-D cggtacccggggatcc Aap-U atgaccatgattacgaattcgagcttcacggcacggcccggc Aap-D gactctagaggatccccgggtaccggtgagcgaaccgacaccc

[0075] The PCR enzyme used for amplification was Phanta Max Super-Fidelity DNA Polymerase from Nanjing Novizan Biotechnology Co., Ltd. The system is shown in Table 5, and the expansion procedure is shown in Table 6.

[0076] Table 5 PCR System Configuration Table

[0077] Phanta Max Super-Fidelity DNA Polymerase 1 μL 2 × Phanta Max Buffer 25 μL <![CDATA[dd-H20]]> 20 μL forward primer 1 μL reverse primer 1 μL dNTP 1 μL template 1 μL

[0078] Table 6 Amplification Procedure

[0079]

[0080] Table 7 shows the corresponding fragment lengths, primers, reaction temperatures, and times for different plasmid templates.

[0081] Plasmid template fragment length, upstream primer, downstream primer, temperature (a), time (b)

[0082] Table 7. Conditions required for fragment amplification

[0083] Aap 1245 bp Aap-U Aap-D 58 1.0 pNV18.1 4410 bp PNV-U PNV-D 60 4.5

[0084] Multiple fragment ligation was performed using the ClonExpress MultiS One Step Cloning Kit, with a vector-to-fragment ratio of 1:1 in the reaction system. The ligated reaction solution was introduced into competent E. coli cells and incubated at 30 min on an ice-water bath, 42 ℃ for 90 s in a water bath, and then on an ice-water bath for 2 min. 900 μL of LB solution was then added, and the cells were incubated at 37 ℃ and 180 rpm for 45–60 min. The cells were then screened using kanamycin selection plates.

[0085] Example 4

[0086] Growth and fermentation of Rhodococcus ethereans N1, knockout strain N1-Δaap, complement strain N1-Δaap-Repaired, and engineered strain N1-aap overexpressing aap:

[0087] (1) Activation of strains: Rhodococcus ethereinus N1 and engineered strain N1-aap were streaked on LB solid medium; knockout strain N1-Δaap and replenished strain N1-Δaap-Repaired were streaked on LB solid medium containing 50 mg / L kanamycin and cultured at 30℃ for 48 h.

[0088] (2) Seed culture: Single colonies were picked from solid agar plates and inoculated into 5 mL LB liquid medium and 5 mL LB liquid medium containing 50 mg / L kanamycin, respectively, and cultured at 30℃ and 180 rpm for 30 h.

[0089] (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium and the fermentation medium containing 50 mg / L kanamycin at 5% v / v respectively, and fermented at 30℃ and 180 rpm for 144 h.

[0090] The fermentation medium formula is as follows: glucose 30 g / L, yeast powder 5 g / L, K2HPO4·3H2O 1.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, and trace elements 1 ml / L (trace element formula: FeSO4·7H2O 80 mg / L, ZnSO4·7H2O 22.5 mg / L, MnSO4·4H2O 40 mg / L, CuSO4·5H2O 5 mg / L, NaCl 15 mg / L, Na2MoO4·2H2O 0.3 mg / L, KCl 0.3 mg / L, CoCl2·6H2O 0.03 mg / L, CaCl2·2H2O 300 mg / L).

[0091] (4) Quantitative analysis of ergothionein

[0092] The liquid chromatograph used in this patent is an Agilent Technologies 1200 Infinity series; the chromatographic column is an Ultimate XB-NH2 amino column, 5 μm, 4.6 mm × 250 mm; a UV detector is used, with a detection wavelength of 254 nm; the mobile phase is acetonitrile:ultrapure water = 7:3; the injection volume is 10 μL; the flow rate is controlled at 1 mL / min; and the column temperature is 30℃. Figure 3 Liquid chromatography-masses of extracellular fermentation products of Rhodococcus ethereans N1, knockout strain N1-Δaap, replenishment strain N1-Δaap-Repaired, engineered strain N1-aap, and ergothioneine standard samples; Figure 4 The extracellular ergothionein production of *Rhodococcus ethereinus* N1, knockout strain N1-Δaap, replenishment strain N1-Δaap-Repaired, and engineered strain N1-aap is shown in the figure, which are 59.32 mg / L, 4.25 mg / L, 52.23 mg / L, and 111.38 mg / L, respectively. Figure 5 The intracellular and extracellular distribution ratio of ergothionein in *Rhodococcus etherealis* N1 and the engineered strain N1-aap was significantly altered compared to the wild-type strain. In the wild-type strain, extracellular ergothionein accounted for 64.46% of the total ergothionein, while intracellular ergothionein accounted for 35.54%. In the engineered strain N1-aap, the proportion of extracellular ergothionein increased to 83.99%, while the proportion of intracellular ergothionein decreased to 16.01%. These results indicate that overexpression of Aap promotes the translocation of ergothionein from intracellular to extracellular spaces, increasing the extracellular accumulation level of ergothionein.

[0093] Example 5

[0094] Optimization of the concentration of exogenous amino acids in the fermentation medium of engineered strain N1-aap.

[0095] To further improve the ergothioneine fermentation yield of engineered strain N1-aap, this example verifies the effects of exogenous addition of histidine, cysteine, and methionine on cell growth and ergothioneine synthesis, and determines the appropriate addition concentrations of each of the three amino acids.

[0096] Using the basic fermentation medium as a control, three amino acids were added separately to make the final concentrations of amino acids 0 g / L, 0.2 g / L, 0.4 g / L, 0.8 g / L, 1.0 g / L and 1.2 g / L.

[0097] The seed culture of the engineered strain N1-aap was inoculated into fermentation media with different amino acid concentrations at an inoculation rate of 5% (v / v) and cultured at 30℃ and 180 rpm for 120 h.

[0098] During fermentation, samples were taken periodically to measure the absorbance of the bacterial solution at 600 nm to evaluate cell growth. After fermentation, the total yield of ergothioneine was determined. Figure 6 To optimize the amino acid concentration, when three amino acids were added simultaneously—histidine at 1 g / L, methionine at 0.4 g / L, and cysteine ​​at 0.4 g / L—the engineered strain N1-aap exhibited good growth and the highest total ergothioneine yield of 192.42 mg / L, representing a 36.84% increase compared to the control group without histidine. Figure 7 The yield graph shows the optimized amino acid concentration for engineered bacteria.

[0099] SEQ ID No.1

[0100] MSEPTPLQRRRLGTGDAVAIGLGAMIGAGVFSAFAPAAAAAGAGLLVGLAVAAVVAYCNATSSAQLAAQYPTSGGTYVYGRERLGPWWGFVAGWGFVIGKTASCAAMALTFAAYAVVPEPWRRPVAVAAVLALTAVNYRGITRTARLARIIVALVLVALAVAVTASWAGPGAQPADPFGPVGGGAYGVLQAAGLLFFAFAGYARIATLG EEVREPRRTIPRAIVGALGFVVLLYAVVGVTLLACLGPDRVASSSAPLLDAVRAGGWAWAAPVVVAGAAVASLGALLALVTGVGRTALAMARQGDLPRPLAAVHPRFAVPHRAELAVAAVVCLLVATVDLRGVIGFSSFGVLVYYLVANLSAFTQRDEHRRYPRFLQVIGAVGCVALTLTLPPGAVGVGAAVLAVGVLARATRRAVP

[0101] SEQ ID No.2

[0102]

Claims

1. An amino acid permeabilizing enzyme involved in the transmembrane transport of ergothioneine, characterized in that, The amino acid sequence of the amino acid permease is shown in SEQ ID No.

1.

2. The nucleic acid molecule encoding the amino acid permeabilizer involved in ergothioneine transmembrane transport as described in claim 1, characterized in that, Its base sequence is shown in SEQ ID No.

2.

3. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 2.

4. A genetically engineered bacterium, characterized in that, The recombinant expression vector of claim 3 was introduced.

5. The application of the amino acid permeabilizing enzyme of claim 1 in the transmembrane transport of ergothionein.

6. The application of the amino acid permease described in claim 1 in the production of ergothionein by constructing an ergothionein-synthesizing strain.

7. A method for increasing the production of extracellular ergothionein by Rhodococcus etherealis N1, characterized in that, The engineered strain N1- was constructed by introducing the amino acid permease described in claim 1. aap Fermentation culture increases the accumulation of extracellular ergothionein.

8. A method for increasing the production of extracellular ergothionein by *Rhodococcus etherealis* N1 according to claim 7, characterized in that, In the fermentation culture, histidine, methionine and cysteine ​​were added, with histidine added at a concentration of 1 g / L, methionine added at a concentration of 0.4 g / L and cysteine ​​added at a concentration of 0.4 g / L.

Images