A gene encoding EgtE-TS protein for improving ergothioneine biosynthesis yield, EgtE-TS protein, recombinant expression vector and recombinant bacteria and applications thereof

Abstract

This invention provides a gene encoding the EgtE-TS protein, the EgtE-TS protein itself, a recombinant expression vector, and recombinant bacteria for improving ergothioneine biosynthesis, belonging to the field of genetic engineering technology. This invention discovers that positions 331 to 337 of the C-terminal β10 domain of the C-S lyase have significant application value in ergothioneine biosynthesis. By replacing the C-terminal domain residue sequence 331TACELAR337 with 331TVSHAWS337, and utilizing the recombinant ergothioneine biosynthesis pathway in *E. coli*, the synthetic capacity of the recombinant bacteria was detected through induced expression. The results show that the replaced recombinant bacteria have better ergothioneine biosynthesis capacity; after 48 hours of induction, the ergothioneine yield increased by 1.64 times to 5.75 mg / L, significantly improving the ergothioneine biosynthesis yield.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a coding gene of an EgtE-TS protein for improving the biosynthesis yield of ergothioneine, an EgtE-TS protein, a recombinant expression vector and a recombinant bacterium and applications thereof. Background Art

[0002] Ergothioneine (ERG) is a natural amino acid derivative and a thiol compound derived from histidine. Studies have shown that ergothioneine has multiple biological functions. It not only has strong antioxidant activity: it can scavenge hydroxyl free radicals, chelate divalent metal ions, activate antioxidant enzymes, inhibit superoxide dismutase, etc., but also has anti-inflammatory and cell-protecting effects. Therefore, ergothioneine has been widely used in the fields of food, medicine and cosmetics.

[0003] The main methods for producing ergothioneine include extraction, chemical synthesis and microbial biosynthesis. Among them, the ergothioneine prepared by the extraction method has many impurities and low output ratio, which makes it difficult to achieve large-scale industrial production; the chemical synthesis method of ergothioneine has the disadvantages of high cost, high difficulty, and difficulty in ensuring product safety; in contrast, the microbial biosynthesis method has many advantages such as low cost, short production cycle and green environmental protection. Therefore, the use of microorganisms to synthesize ergothioneine has a very broad prospect. The prokaryotic biosynthesis pathway of ergothioneine includes 5 steps of enzyme-catalyzed reactions ( Figure 1 ), L-histidine methyltransferase EgtD transfers three methyl groups of S-adenosylmethionine to L-histidine to catalyze the formation of L-histidine betaine (HER). Subsequently, glutamylcysteine ​​synthetase EgtA connects glutamate to cysteine ​​to form γ-glutamylcysteine ​​(γ-GC). Next, ergothioneine synthase γ-glutamylacetylcysteine ​​sulfur-oxide synthase EgtB catalyzes the formation of a CS bond between γ-GC and HER to form hycine-glutamylcysteine ​​sulfoxide (γGC-HER). Then, γ-glutamylthiocysteine ​​S-oxide hydrolase EgtC removes the L-glutamate (L-Glu) part in γGC-HER to generate heptylcysteine ​​sulfoxide (Cys-HER). Finally, CS lyase EgtE cleaves the CS bond to ultimately generate ergothioneine.

[0004] At present, the production of ergothioneine by biosynthesis has gradually become a research hotspot, but the production performance of recombinant engineering strains is generally low, among which the accumulation of intermediate metabolites and the feedback inhibition of the product ERG are technical bottlenecks that hinder efficient synthesis. The development of rational / semi-rational metabolic engineering and construction strategies based on biological knowledge at the molecular, pathway and genomic levels has provided great help for the development of more economical and efficient ergothioneine production strains. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a kind of coding gene, EgtE-TS protein, recombinant expression vector and recombinant bacteria and application thereof of the EgtE-TS protein improving thioneine biosynthesis yield. The present invention significantly improves thioneine biosynthesis yield by optimizing key domains in thioneine synthesis key enzyme CS lyase EgtE, and provides efficient construction strategy for industrial production thioneine cell factory.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a gene encoding an EgtE-TS protein for improving the biosynthesis yield of ergothioneine. The nucleotide sequence of the gene encoding is shown in SEQ ID NO.1.

[0008] The invention provides an EgtE-TS protein for improving the biosynthesis yield of ergothioneine. The amino acid sequence of the EgtE-TS protein is shown in SEQ ID NO.2.

[0009] The present invention also provides a recombinant expression vector, which is obtained by recombining the coding gene into an empty vector; the empty vector is pBAD33.

[0010] The present invention also provides a recombinant bacterium, which is obtained by transferring the recombinant expression vector and the pBAD-EgtABCD expression vector into an empty vector bacterium; the empty vector bacterium is Escherichia coli BW25113.

[0011] Preferably, the pBAD-EgtABCD expression vector is obtained by recombining the ergothioneine synthesis pathway gene EgtABCD into an empty vector, and the empty vector is pBAD-hisA.

[0012] Preferably, the nucleotide sequence of the ergothioneine biosynthesis pathway gene EgtABCD is as SEQ ID NO.3.

[0013] Preferably, the ergothioneine synthesis pathway gene EgtABCD is recombined in the empty vector pBAD-hisA Nco Ⅰ and Hind III restriction sites.

[0014] The present invention also provides the use of the encoding gene, the EgtE-TS protein, the recombinant expression vector or the recombinant bacteria in improving the biosynthesis yield of ergothioneine.

[0015] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a coding gene of an EgtE-TS protein for improving the biosynthesis yield of ergothioneine, an EgtE-TS protein, a recombinant expression vector, a recombinant bacterium and its application. The present invention finds that the threonine residue at position 331 to the arginine residue at position 337 of the C-terminal domain β10 of the CS lyase EgtE CS lyase has important application value in the biosynthesis of the active product ergothioneine. The present invention replaces the threonine residue at position 331 to the arginine residue at position 337 of the C-terminal domain β10 of the key enzyme CS lyase for ergothioneine synthesis with 331TVSHAWS337, utilizes the Escherichia coli recombinant ergothioneine synthesis pathway, and detects the synthesis ability of the recombinant bacterium by inducing expression. The results showed that the replaced recombinant bacteria had better ergothioneine synthesis ability. After 48 hours of induction synthesis, the ergothioneine production was 5.75 mg / L, an increase of 1.64 times, which significantly increased the ergothioneine biosynthesis yield and provided an efficient construction strategy for the industrial production of ergothioneine cell factories. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 for the ergothioneine biosynthesis pathway;

[0017] Figure 2 This is the result of LC-MS detection of ergothioneine of the recombinant strain BW-E1;

[0018] Figure 3 This is the homology modeling structure diagram of the Mycobacterium smegmatis CS lyase EgtE, where A is the tertiary structure prediction of EgtE; B is the EgtE protein surface prediction;

[0019] Figure 4 The secondary structure and sequence analysis of CS lyase EgtE;

[0020] Figure 5 This is a mutation analysis of the CS lyase EgtE motif 331TACELAR337; A is the structure near the EgtE motif 331TACELAR337; B is the protein surface structure near the EgtE motif 331TACELAR337; C is the structure near the EgtE-TS motif 331TVSHAWS337; D is the protein surface structure near the EgtE-TS motif 331TVSHAWS337;

[0021] Figure 6 Analysis of ergothioneine synthesis yield of the recombinant strain after optimization of the 331TACELAR337 domain of CS lyase EgtE. DETAILED DESCRIPTION

[0022] The present invention provides a gene encoding an EgtE-TS protein for improving the biosynthesis yield of ergothioneine, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1, and is specifically as follows:

[0023]

[0024] The present invention provides an EgtE-TS protein for improving the biosynthesis yield of ergothioneine, wherein the amino acid sequence of the EgtE-TS protein is shown in SEQ ID NO.2, and is specifically as follows:

[0025] MMLAQQWRDARPKVAGLHLDSGACSRQSFAVIDATTAHARHEAEVGGYVAAEAATPALDAGRAAVASLIGFAASDVVYTSGSNHAIDLLLSSWPGKRTLACLPGEYGPNLSAMAANGFQVRALPVDDDGRVLVDEASHELSAHPVALVHLTALASHRGIAQPAAELVEACHNAGIPVVIDAAQALG HLDCNVGADAVYSSSRKWLAGPRGVGVLAVRPELAERLQPRIPPSDWPIPMSVLEKLELGEHNAAARVGFSVAVGEHLAAGPTAVRERLAEVGRLSRQVLAEVDGWRVVEPVDQPTAITTLESTDGADPASVRSWLIAERGIVTTVSHAWSAPFEMRTPVLRISPHVDVTVDELEQFAAALREAP.

[0026] The invention provides a recombinant expression vector, which is obtained by recombining the coding gene into an empty vector; the empty vector is pBAD33.

[0027] In the present invention, the coding gene is recombined into the empty vector pBAD33 Sac Ⅰ and Hind III restriction sites.

[0028] The present invention also provides a recombinant bacterium, which is obtained by transferring the recombinant expression vector and the pBAD-EgtABCD expression vector into an empty vector bacterium; the empty vector bacterium is Escherichia coli BW25113.

[0029] In the present invention, the pBAD-EgtABCD expression vector is obtained by recombining the ergothioneine synthesis pathway gene EgtABCD into an empty vector, and the empty vector is pBAD-hisA.

[0030] In the present invention, the Mycobacterium smegmatis genome is used as a template, and a chemical biological method is used to synthesize the ergothioneine synthesis pathway gene EgtABCD, wherein the nucleotide sequence of the ergothioneine synthesis pathway gene EgtABCD is as shown in SEQ ID NO.3; the ergothioneine synthesis pathway gene EgtABCD is recombined in the empty vector pBAD-hisA Nco Ⅰ and Hind III restriction sites.

[0031] The present invention also provides the use of the encoding gene, the EgtE-TS protein, the recombinant expression vector or the recombinant bacteria in improving the biosynthesis yield of ergothioneine.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] The expression plasmids pBAD33 and pBAD-hisA were purchased from Wuhan Miaoling Biotechnology Co., Ltd. and are commercially available products;

[0034] Escherichia coli BW25113 was purchased from Beijing Novozyme Biotechnology Co., Ltd. and is a commercially available product.

[0035] Example 1 Biosynthesis of ergothioneine in an Escherichia coli chassis

[0036] 1. Experimental Methods

[0037] 1.1 Construction of ergothioneine synthesis strain

[0038] The ergothioneine synthesis pathway genes EgtABCD (gene ID: MSMEG_6250-MSMEG_6249- MSMEG_6248- MSMEG_6247), the sequence of which is shown in SEQ ID NO.3; and EgtE (gene ID: MSMEG_6246), the sequence of which is shown in SEQ ID NO.4, were synthesized by chemical synthesis, as follows:

[0039]

[0040]

[0041] The EgtABCD gene sequence and the EgtE gene sequence were amplified by PCR using the Mycobacterium smegmatis genome as a template. The PCR amplification system was 20 μL, including 10 μL 2×Phanta Max Master Mix, 1 μL DNA template, 1 μL upstream primer with an initial concentration of 10 μmol / L, 1 μL downstream primer with an initial concentration of 10 μmol / L, and 7 μL ddH2O. The PCR reaction program was as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 2 min, for a total of 35 cycles; 72°C extension for 10 min; 4°C storage. The amplified EgtABCD gene sequence was recovered by gel and connected to the recombinase after Nco Ⅰ / Hin The expression vector pBAD-EgtABCD was constructed on the pBAD-hisA vector containing sticky ends obtained by double digestion with d III.

[0042] The amplified EgtE gene sequence was recovered by gel ligation with the recombinase. Sac Ⅰ / Hin The expression vector pZH-EgtE was obtained by double digestion with d III on the pBAD33 vector containing sticky ends.

[0043] The PCR primers for the EgtABCD gene sequence are as follows:

[0044] GCTAACAGGAGGAATTAACCATGGATGGCCTTACCCGCCAGAAGTG (SEQ ID NO. 5);

[0045] CTCATCCGCCAAAACAGCCAAGCTTTCACCGCACCGCCAGCGACAACC (SEQ ID NO.6)

[0046] The PCR primers for the EgtE gene sequence are as follows:

[0047] GGGCTAGCGAATTCGAGCTCATGCTCGCGCAGCAGTGGCGTGA (SEQ ID NO. 7);

[0048] TCATCCGCCAAAACAGCCAAGCTTTCAGGGCGCCTCACGCAACGCT (SEQ ID NO. 8).

[0049] The constructed expression vectors pBAD-EgtABCD and pZH-EgtE were co-transformed into Escherichia coli BW25113, and the strain was named BW-E1. A single colony of Escherichia coli BW25113 was picked and inoculated into 5 mL of LB medium and cultured at 37°C and 220 rpm for 12 hours. The strain Escherichia coli BW25113 was inoculated into 100 mL of LB liquid medium at a 1% inoculation rate and cultured at 37°C and 220 rpm until the OD600 value reached about 0.4. The bacterial solution was transferred to a centrifuge tube and placed on ice for 10 minutes to cool. The cells were collected by centrifugation at 5000 rpm and 4°C for 10 minutes, and the cells were gently resuspended with 1 mL of pre-cooled 0.5 mol / L CaCl2 solution and 4 mL of ddH2O. Ice bath for 15 minutes, centrifuged to collect the bacteria, and 1 mL of pre-cooled 0.1 mol / L CaCl2 solution containing glycerol was added to suspend the cells to make competent cells.

[0050] Mix 5 μL of plasmid pBAD-EgtABCD and 5 μL of pZH-EgtE, add to 50 μL of competent cells, mix well, and place on ice for 30 min. Heat the competent cells at 42°C for 45 s, and place on ice for 3 min. Add 500 μL of LB liquid culture medium, and culture at 37°C and 200 rpm for 45 min.

[0051] Take 200 μL of bacterial solution and spread it evenly on LB plate containing chloramphenicol (34 μg / mL) and ampicillin (50 μg / mL). After the liquid is absorbed, put it upside down in a 37°C incubator and culture for 12 hours. Pick a single colony for PCR verification. The primer sequences are SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

[0052] 1.2 Induced expression and yield detection of ergothioneine engineering strains

[0053] Recombinant bacteria BW-E1 was activated, and a single colony was picked and inoculated into 50 mL of LB liquid medium containing chloramphenicol (34 μg / mL) and ampicillin (50 μg / mL), and cultured at 37°C with shaking at 220 rpm for 12 h to prepare seed solution. The seed solution was inoculated into a new 50 mL of M9Y liquid medium (10 g / L glucose, 1 g / L yeast extract, 1 g / L NH4Cl, 8 g / L Na2HPO4, 5 g / L KH2PO4, 0.5 g / L NaCl, 1.5 g / L MgSO4·7H2O, 0.02 g / L CaCl2·2H2O) without antibiotics at a final concentration of OD600 = 0.1, and cultured at 37°C with shaking at 220 rpm. During the culture period, when the OD600 light absorption value of the bacterial solution reaches 0.8, add L-arabinose with a final concentration of 3‰ and induce at 30℃, 220rpm for 24h, then add L-arabinose with a final concentration of 3‰ again for 24h. Centrifuge at 8,000rpm for 10min to collect the bacteria. Resuspend the bacteria with 2.5mL Bugbuster, add 0.3μL nuclease, incubate at 26℃, 80rpm for 20min. Centrifuge at 8,000rpm for 30min, transfer the supernatant through a 0.22μm filter membrane to a 5mL centrifuge tube, and store at -80℃ for later use.

[0054] UPLC-MS / MS detection conditions

[0055] Chromatographic column: HypersilGOLD aQ chromatographic column (100×2.1mm, 1.9μm, Thermo Scientific, USA); column temperature: 30℃; injector temperature: 4℃; mobile phase ratio: water / methanol = 99 / 1, flow rate: 0.3mL / min, detection time: 20min; injection volume: 5μL.

[0056] Mass spectrometry conditions: electrospray ionization source (ESI), positive ion mode scanning; ion source temperature: 250°C, nebulizer flow: 11L / min, nebulizer gas pressure: 35psi, sheath gas temperature: 250°C, sheath gas flow rate: 11L / min, capillary voltage: 4000V; ergothioneine in the recombinant strain was quantitatively analyzed by multiple reaction monitoring (MRM) mode.

[0057] 2. Experimental results

[0058] The recombinant strain BW-E1 expressing the ergothioneine biosynthesis gene EgtABCDE of Mycobacterium smegmatis was successfully constructed using the Escherichia coli dual-plasmid expression system. The strain BW-E1 was cultured in a shake flask, and the inducer L-arabinose was added to promote gene expression. The ergothioneine biosynthesis ability of the strain BW-E1 was verified by LC-MS. The results are shown in Figure 2As shown, the peak time of the extracted sample is the same as that of the ergothioneine standard, and the mass-to-charge ratio (m / z=230) of the sample is consistent with that of the standard. This result indicates that the recombinant strain BW-E1 successfully synthesizes ergothioneine. According to the standard curve, the yield of ergothioneine synthesized by strain BW-E1 is 3.51 mg / L.

[0059] Example 2 Mining and analysis of CS lyase EgtE motif 331TACELAR337

[0060] 1. Experimental Methods

[0061] Bioinformatics analysis

[0062] BLAST was used to perform similarity analysis and alignment on the database. Homology modeling was performed on the Swiss-model server to analyze the EgtE protein structure. COACH and I-TASSER-MTD were used to analyze possible special regions such as catalytic domains. SwissDock and CB-Dock2 were used to predict the amino acid residues in the active center of EgtE binding to the substrate. Pymol was used to visualize and edit the protein structure diagram from the PDB file. The phylogenetic tree was constructed using MEGA 7.0 software according to the neighbor-joining method. SCHEMA software was used to perform hotspot domain analysis and protein modularization, and the results are shown in Figure 2~Figure 5 .

[0063] 2. Experimental results

[0064] CS lyase EgtE is the last enzyme in the synthesis of ergothioneine and is also a key enzyme in the synthesis pathway. Bioinformatics analysis showed that CS lyase EgtE is composed of 371 amino acid residues, including 15 α-helices and 11 β-fold structures, and the active center can bind to PLP molecules and the intermediate product heptylcysteine ​​sulfoxide. The amino acid residues in the active center are Tyr48, Tyre106, Asp180, Gln183, Arg202, Lys203 and Arg348. The motif from the threonine residue 331 to the arginine residue 337 in the C-terminal domain β10 (331TACELAR337, nucleotide sequence: ACCGCGTGTGAACTCGCGCGG, SEQ ID NO.9) may be involved in the recognition process of the substrate in the active site center and play an important role in the synthesis of ergothioneine catalyzed by EgtE.

[0065] Example 3 Application of motif 331TACELAR337 in optimization of CS lyase EgtE

[0066] 1. Experimental Methods

[0067] By chemical synthesis, using Mycobacterium smegmatis EgtE as a template, the CS lyase EgtE motif 331TACELAR337 fragment replaced the homologous structure 331TVSHAWS337 (nucleotide sequence: ACTGTCTCTCACGCTTGGAGC, SEQ ID NO.10), synthesized gene fragment S1, and the recombinant protein was named EgtE-TS (amino acid sequence see SEQ ID NO.1). The synthesized gene fragment S1 was amplified by PCR method, and the amplification procedure and system were referred to the PCR amplification procedure and system in Example 1. The amplified gene fragment S1 was recovered by gel and connected to the recombinase by recombinase. Sac Ⅰ / Hind The pBAD33 vector containing sticky ends obtained by double digestion with III was cloned, and the PCR primers were SEQ ID NO.7 and SEQ ID NO.8 to construct the expression vector pZH-EgtE-TS.

[0068] 2. Induced expression and yield detection of ergothioneine engineering strain BW-E2

[0069] The constructed expression vectors pBAD-EgtABCD and pZH-EgtE-TS were co-transformed into Escherichia coli BW25113, and the constructed strain was named BW-E2. L-arabinose was used to induce the expression of ergothioneine synthesis genes in the recombinant strain, and the synthesis capacity was detected by liquid chromatography-mass spectrometry. The specific method is shown in Example 1.

[0070] 3. Experimental results

[0071] Using Mycobacterium smegmatis EgtE as a template, the CS lyase EgtE motif 331TACELAR337 fragment replaced the homologous structure 331TVSHAWS337, and the recombinant protein was named EgtE-TS. The ergothioneine synthesis pathway was recombined in Escherichia coli, and the synthesis ability of the recombinant strain was detected by induced expression. The results of LC-MS showed that ( Figure 6 ), the recombinant strain had better ergothioneine synthesis ability. After 48 days of induction, the ergothioneine production was 5.75 mg / L, which was 1.64 times higher than that of the strain before transformation.

[0072] The present invention finds that CS lyase EgtE is a key enzyme for thioneine synthesis. The optimization of the 331TACELAR337 fragment of its C-terminal domain β10 has the effect of significantly improving the catalytic function of the enzyme and increasing the synthesis yield of thioneine, and has important application value in improving the biosynthesis of the active product thioneine.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gene encoding an EgtE-TS protein that improves ergothioneine biosynthesis output, characterized in that, The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

2. An EgtE-TS protein that improves the biosynthetic yield of ergothioneine, characterized in that, The amino acid sequence of the EgtE-TS protein is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector is obtained by recombining the coding gene according to claim 1 into an empty vector; the empty vector is pBAD33.

4. A recombinant bacterium, characterized in that: The recombinant bacteria are obtained by transferring the recombinant expression vector described in claim 3 and the pBAD-EgtABCD expression vector into empty vector bacteria; the empty vector bacteria are Escherichia coli BW25113.

5. The recombinant bacterium according to claim 4, characterized in that The pBAD-EgtABCD expression vector is obtained by recombining the ergothioneine synthesis pathway gene EgtABCD into an empty vector, and the empty vector is pBAD-hisA.

6. The recombinant bacterium according to claim 5, characterized in that The nucleotide sequence of the ergothioneine synthesis pathway gene EgtABCD is as shown in SEQ ID NO.

3.

7. The recombinant bacterium according to claim 5, characterized in that The ergothioneine synthesis pathway gene EgtABCD is recombined into the empty vector pBAD-hisA Nco Ⅰ and Hind III restriction sites.

8. Application of the coding gene according to claim 1, the EgtE-TS protein according to claim 2, the recombinant expression vector according to claim 3 or the recombinant bacteria according to any one of claims 4 to 7 in improving the biosynthetic output of ergothioneine.

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