Method for synthesizing ergothioneine through extracellular biological catalysis of microbial cell membrane and application of ergothioneine

Abstract

The invention belongs to the field of biotechnology and fermentation, and particularly discloses a method for synthesizing ergothioneine through microbial cell membrane ectobiological catalysis and application. According to the invention, L-cysteine and N, N, N-trimethyl histidine are taken as substrates, two catalytic proteins are connected to a microbial cell membrane anchoring protein through a connexon for out-of-membrane expression and tandem catalytic biosynthesis of the ergothioneine, and cell catalytic production of the ergothioneine is successfully realized. Compared with reported organic and biosynthesis methods, the biosynthesis method disclosed by the invention overcomes the requirements that a substrate enters a cell membrane and a product is discharged out of the cell membrane in the traditional intracellular fermentation production, so that the substrate concentration and the product concentration in a cell catalysis process are easy to improve; meanwhile, the interference influence of a substrate and a product on the intracellular environment of microorganisms is reduced, and the strain can be applied to fermentation production, so that the strain is more economical and environment-friendly and has a great market development value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and fermentation, and particularly relates to a method and application of cell membrane extracellular biological catalytic synthesis of ergothioneine. Background Art

[0002] Ergothioneine is a rare natural chiral amino acid antioxidant. Due to its special redox properties, ergothioneine will not spontaneously oxidize and has good stability, and is regarded as one of the best natural antioxidant drugs. Studies have found that ergothioneine is an important physiologically active substance in the body. Ergothioneine ingested from food will be enriched in organs such as the liver, kidneys, eyeballs, semen, and red blood cells, and participate in multiple functions such as intracellular energy regulation and physiological protection. It has anti-aging, anti-radiation, and free radical scavenging effects. Due to the biological and pharmacological properties of ergothioneine and its safety and stability, ergothioneine has been widely used in cosmetics, functional foods, pharmaceuticals, therapeutics, biomedicine and other products and fields.

[0003] At present, the synthesis of thioneine is basically achieved by three methods: chemical synthesis, natural biological extraction and biosynthesis. However, the current organic synthesis of thioneine encounters the problem of difficult chiral synthesis or high raw material cost. And the natural extraction method has the problems of thioneine content bottom, many impurities and high extraction cost in the raw materials. Compared with chemical synthesis and natural biological extraction, biosynthesis is easy to amplify production, can reduce production costs, and can ensure the biosafety of thioneine products, which is the main direction of thioneine synthesis technology development.

[0004] The present application starts from the chemical structure of ergothioneine, connects two catalytic proteins (OvoA enzyme / Egt1 enzyme and EgtE enzyme) to the microbial cell membrane anchor protein through a linker for extracellular expression, and then performs a series catalytic biosynthesis of ergothioneine outside the cell membrane, successfully realizing the cell catalytic production of ergothioneine, overcoming the requirements for substrate entry into the cell membrane and product excretion from the cell membrane in traditional intracellular fermentation production, thereby facilitating the improvement of substrate concentration and product concentration in the cell catalytic process, while reducing the interference effect of substrate and product on the microbial intracellular environment.

[0005] There is no report on the tandem biosynthesis of ergothioneine using the extracellular expression catalytic protein proposed by us. This application provides a new and efficient method for large-scale production of ergothioneine. Summary of the invention

[0006] The invention aims to provide a cell catalysis method for biocatalytically synthesizing ergothioneine (L-(+)-Ergothioneine) by expressing a catalytic protein outside a cell membrane. The method is simple and suitable for large-scale promotion.

[0007] Another object of the present invention is to provide the application of the above method in preparing ergothioneine.

[0008] In order to achieve the above object, the present invention adopts the following technical measures:

[0009] A cell catalytic method for biocatalytically synthesizing ergothioneine by expressing catalytic proteins outside cell membranes, comprising taking L-cysteine ​​and N,N,N-trimethylhistidine as substrates, adding sodium ascorbate, using microbial cells containing OvoA enzyme (or Egt1 enzyme) and EgtE enzyme expressed outside cell membranes as catalysts for catalytic reaction, and purifying to obtain the product;

[0010] The OvoA enzyme is a His-Cys synthetase homologue, which is shown in SEQ ID NO.4.

[0011] The Egt1 enzyme is a histidine-cysteine ​​synthetase homologue, and its SEQ ID NO.5 is shown.

[0012] The EgtE enzyme is: a CS lyase homologue, as shown in SEQ ID NO.6;

[0013] In the method described above, preferably, the method system contains a buffer, and the buffer is a mixture of KPi buffer (50mM KH2PO4, pH=8.0) and 50mM Tris-HCl, 20mM NaCl, 10% glycerol, pH=8.0 in a volume ratio of 1:0.5 to 1:2;

[0014] In the above method, preferably, the conditions of the catalytic reaction are: 25-37°C for 24-48h, and a rotation speed of 200-300rpm;

[0015] In the above method, preferably, the catalytic system includes: 0.2-20 mM L-cysteine, 0.2-20 mM N,N,N-trimethylhistidine, 0.1-1 mM sodium ascorbate, OD of microbial cells 600 is 5 to 50, and the concentration is the final concentration of the substance in the catalytic system;

[0016] Among the methods described above, preferably, the purification method used includes: a C18 reverse phase high performance liquid chromatography column, water (phase A, containing 0.1% formic acid (v / v)) and acetonitrile (phase B) as mobile phases, and a flow rate of 0.75 mL / min; 0-5 min 2% B, 5-20 min 2-5% B, 20-30 min 5% B, 30-35 min 5-2% B, 35-40 min 2% B.

[0017] Or HIIC column separation, acetonitrile / 5mM ammonium acetate buffer as the mobile phase for isocratic elution, flow rate of 0.5mL / min, column temperature of 30℃, detection wavelength of 254nm.

[0018] The method described above, preferably, the method for preparing microbial cells containing OvoA enzyme (or Egt1 enzyme) and EgtE enzyme expressed outside the cell membrane comprises: inserting the sequences of SEQ ID NO.1 or SEQ ID NO.2, and SEQ ID NO.3 into two gene expression frames of the pACYCDuet-1 vector, and transferring the obtained recombinant plasmid into Escherichia coli or yeast.

[0019] The application of the above method in preparing ergothioneine also belongs to the protection scope of the present invention.

[0020] In the present invention, the structural formula of described ergothioneine is:

[0021]

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The present invention proposes for the first time a method for biosynthesizing ergothioneine by expressing a fusion protein outside a microbial membrane. The method can directly use microbial cells as catalysts for biosynthesis of ergothioneine, and compared with an enzyme catalysis method, no protein purification is required. Moreover, cell catalysis can be separated from a liquid phase where a product is located after a biological reaction, which is beneficial to product purification.

[0024] Further, the applicant also constructs two fusion proteins (fusion protein of OvoA / Egt1-cell membrane anchor protein (such as OmpA) and fusion protein of EgtE-cell membrane anchor protein (such as OmpA)) double gene expression plasmid, successfully realizes the fermentation production of thioneine. The application of microbial membrane catalysis overcomes the requirement of high concentration substrate entering cell membrane and product discharge cell membrane in traditional intracellular fermentation production, so as to be easy to promote the substrate concentration and product concentration in cell catalysis process, and under the premise of ensuring enzyme catalytic activity and selectivity, it is easy to realize the separation of enzyme and reaction system, for low-cost realization of the industrialized production of thioneine provides potential possibility, while possessing economic environmental protection, the pollution to environment is also very small. DETAILED DESCRIPTION

[0025] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial channels.

[0026] Embodiment 1:

[0027] The microorganism (Escherichia coli) cell membrane extracellular catalytic biosynthesis preparation method of ergothioneine comprises:

[0028] In a 50 mL biosynthesis reaction system, add 5 mM L-cysteine, 5 mM N,N,N-trimethylhistidine, 2 mM sodium ascorbate, and OD 600 =30 of the recombinant microbial engineering bacteria, and the rest was buffer; the reaction was carried out at 30°C and 200 rpm for 36 hours.

[0029] The recombinant microbial engineering bacteria are microorganisms expressing OvoA enzyme (shown in SEQ ID NO.4) and EgtE enzyme (shown in SEQ ID NO.6), and these two enzymes are anchored on the microbial cell membrane. In this embodiment, the microorganism is Escherichia coli BL21. The microorganism of the scheme of the present invention can also be yeast, that is, yeast expresses these two enzymes outside the membrane. In view of the length of the text, the present invention specification will not be repeated.

[0030] After the reaction was completed, the mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a membrane and separated and purified by HPLC to obtain thioneine with a yield of 200 mg / L.

[0031] The buffer solution is a mixture of the following two buffer solutions in a volume ratio of 1:1: KPi buffer solution (50mM KH2PO4 / K2HPO4, pH=8.0) and buffer solution (50mM Tris-HCl, 20mM NaCl, 10% glycerol, pH=8.0).

[0032] The separation and purification method used is: C18 reverse phase high performance liquid chromatography column, water (phase A, containing 0.1% formic acid (v / v)) and acetonitrile (phase B) as mobile phases, flow rate of 0.75 mL / min; 0-5 min 2% B, 5-20 min 2-5% B, 20-30 min 5% B, 30-35 min 5-2% B, 35-40 min 2% B.

[0033] Alternatively, separation was performed by HIIC column, with acetonitrile / 5 mM ammonium acetate buffer as the mobile phase for isocratic elution, a flow rate of 0.5 mL / min, a column temperature of 30°C, and a detection wavelength of 254 nm.

[0034] The hydrogen spectrum data of the isolated ergothioneine were consistent with the literature values ​​after structural verification.

[0035] 1 H NMR (400MHz, MeOD) δ = 7.63 (d, J = 8.8, 1H), 6.69 ( d, J = 2.1, 1H), 6.47 ( dd, J = 8.7, 2.1, 1 H), 3.90 (dd, J = 8.9, 2.6, 1H), 3.58 (dd, J = 18.6, 2.8, 1H), 3.40 (dd, J = 18.7, 9.0, 1H).

[0036] The molecular structural formula of the product ergothioneine obtained is:

[0037]

[0038] Embodiment 2:

[0039] Method for constructing microorganisms that express extracellular anchored OvoA enzyme and EgtE enzyme:

[0040] 1) Construction of double fusion protein gene expression plasmid:

[0041] The double fusion protein gene expression plasmid is obtained by inserting the OvoA-OmpA fusion protein gene (shown in SEQ ID NO.1) and the EgtE-OmpA fusion protein gene (shown in SEQ ID NO.3) into two gene expression frames of the pACYCDuet-1 vector respectively;

[0042] In this embodiment, the OvoA enzyme can also be replaced with the Egt1 enzyme (the amino acid sequence is shown in SEQ ID NO.5, and the Egt1-OmpA fusion protein gene is shown in SEQ ID NO.2). The construction method is the same, and due to the length of the article, the description of the present invention will not be repeated.

[0043] 2) Biological fermentation:

[0044] The double fusion protein gene expression plasmid was transferred into BL21 competent cells and plated. After 12 hours, a single colony was picked and placed in 50 mL LB broth. Chloramphenicol antibiotics (50 μg / mL) were added and cultured overnight at 37°C and 220 rpm. The culture was then inoculated and amplified until OD 600 When the OD value of the recombinant E. coli reached 2-3, the inducer IPTG (0.2 mM) was added and the culture was continued at 37°C and 220 rpm for 6-8 hours until the OD value of the recombinant E. coli reached 1. 600 =30, stop fermentation.

[0045] L-cysteine ​​(5mM), N,N,N-trimethylhistidine (5mM), sodium ascorbate (2mM) were added to the fermentation broth, the culture temperature was adjusted to 30°C and the culture was continued at a speed of 200-300rpm for 36h. After the reaction was completed, centrifugation was performed at 12000rpm for 10min, and the supernatant was filtered through a membrane and separated and purified by HPLC to obtain ergothioneine.

[0046] The separation and purification method includes: a C18 reverse phase high performance liquid chromatography column, water (phase A, containing 0.1% formic acid (v / v)) and acetonitrile (phase B) as mobile phases, a flow rate of 0.75 mL / min; 0-5 min 2% B, 5-20 min 2-5% B, 20-30 min 5% B, 30-35 min 5-2% B, 35-40 min 2% B.

[0047] Alternatively, separation was performed by HIIC column, with acetonitrile / 5 mM ammonium acetate buffer as the mobile phase for isocratic elution, a flow rate of 0.5 mL / min, a column temperature of 30°C, and a detection wavelength of 254 nm.

[0048] The output of ergothioneine prepared by the above system is 220 mg / L.

Claims

1. A cell catalysis method for biocatalytically synthesizing ergothioneine by expressing catalytic protein outside a cell membrane, comprising: L -cysteine ​​and N,N,N-trimethylhistidine are used as substrates, sodium ascorbate is added, and microbial cells containing extracellularly expressed OvoA enzyme or Egt1 enzyme and EgtE enzyme are used as catalysts for catalytic reaction, and then purified to obtain the product; The OvoA enzyme is: a similar enzyme to the histidine-cysteine ​​coupling enzyme, as shown in SEQ ID NO.4; The Egt1 enzyme is: the analogous enzyme of histidine-cysteine ​​coupling enzyme is shown in SEQ ID NO.5; The EgtE enzyme is an enzyme similar to CS bond cleavage enzyme, and is shown in SEQ ID NO.

6.

2. The method according to claim 1, characterized in that The catalytic reaction system in the method described herein contains a buffer solution, which is a mixture of KPi buffer solution and 50 mM Tris-HCl, 20 mM NaCl, 10% glycerol, pH = 8.0 in a volume ratio of 1:0.5 to 1:2; The Kpi buffer is 50 mM KH2PO4, pH = 8.

0.

3. The method according to claim 1, characterized in that: The conditions of the catalytic reaction are: 25~37 o C reaction for 24~48 hours, rotation speed 200-300rpm.

4. The method according to claim 1, characterized in that: The catalytic system includes: L -Cysteine ​​0.2~20 mM, N,N,N-trimethylhistidine 0.2~20 mM, sodium ascorbate 0.1~1 mM, microbial cell OD 600 5~50.

5. The method according to claim 1, characterized in that: The purification method includes: C18 reverse phase high performance liquid chromatography column, water and acetonitrile as mobile phase, flow rate of 0.75 mL / min; 0-5 min 2% B, 5-20 min 2-5% B, 20-30 min 5% B, 30-35 min 5-2% B, 35-40 min 2% B; The water is phase A, containing 0.1% formic acid; acetonitrile is phase B; Or HIIC column separation, acetonitrile / 5 mM ammonium acetate buffer as the mobile phase for isocratic elution, the flow rate is 0.5 mL / min, the column temperature is 30°C, and the detection wavelength is 254 nm.

6. The method according to claim 1, characterized in that: The preparation method of the microbial cells containing extracellularly expressed OvoA enzyme or Egt1 enzyme and EgtE enzyme comprises: inserting the sequences of SEQ ID NO.1 or SEQ ID NO.2 and SEQ ID NO.3 into two gene expression frames of pACYCDuet-1 vector, and transferring the obtained recombinant plasmid into Escherichia coli or yeast.

7. the application of the method described in claim 1 in preparing thioneine.

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