Construction method of recombinant escherichia coli for ergothioneine synthesis, strain and application thereof

Abstract

The invention provides a construction method of recombinant escherichia coli for synthesis of ergothioneine, a strain and application of the strain, and relates to the technical field of construction of strains related to synthesis of ergothioneine, an NcEgt1 * sequence and an Egt D * sequence are fused and connected with a ribosome binding site sequence, and after XbaI and NotI enzyme digestion, the mixture is connected to a carrier pET-Duet-1 to obtain a recombinant plasmid pET-Duet-1-NcEgt1 *-EgtD *; the EgtE is connected to a recombinant plasmid pET-Duet-1-NcEgt1 *-EgtD * through Nd < e > I / KpnI enzyme digestion, and finally a recombinant vector pET-Duet-1-NcEgt1 *-EgtD *-EgtE is obtained; the method comprises the following steps: transforming a recombinant vector pET-Duet-1-NcEgt1 *-EgtD *-EgtE into E.coli BL21 to obtain a bacterial strain for producing ergothioneine, and naming the bacterial strain as E.coli Egt116; then, the recombinant plasmid pRSFDuet-1-EgtD *-EgtE is converted into a bacterial strain E.coli Egt116, and a bacterial strain E.coli Egt117 capable of producing ergothioneine is obtained; and finally, constructing a mutant strain of double genes of the strain Ecoli Egt117sdaA / MetJ by using a Cre-lox system, naming the mutant strain as E.coli Egt118, and increasing the ergothioneine yield of the strain to 218.71% in comparison with the ergothioneine yield of the strain E.coli Egt116. The recombinant strain constructed by the invention can be used for mass production of the ergothioneine, and large-scale production of the ergothioneine is realized.

Description

Technical Field

[0001] The invention relates to the technical field of Escherichia coli construction for synthesizing ergothioneine, and more specifically to a construction method of a recombinant Escherichia coli for synthesizing ergothioneine, the Escherichia coli and applications. Background Art

[0002] Ergothioneine (mercaptohistidine trimethylol salt, ergothioneine, EGT) is a natural antioxidant that can protect cells in the human body and has multiple functions such as anti-oxidation, anti-inflammation, detoxification, maintaining DNA biosynthesis, normal cell growth and cell immunity. It has a wide range of uses and market prospects in the fields of medicine, food, animal feed, cosmetics, etc.

[0003] There are currently three methods for preparing thioneine, chemical synthesis, natural extraction and biosynthesis. The raw materials and reagents used in the chemical synthesis method are expensive, the reaction conditions are harsh, the product amount is low, and waste that is difficult to handle is generated, so it is difficult to industrialize. Natural extraction is to extract thioneine from the fruiting bodies of edible fungi, animal tissues and grains, and the disadvantages are low content, many raw material impurities and high extraction cost. The biosynthesis method is to catalyze the synthesis of thioneine by engineering bacteria catalyzing L-histidine and L-cysteine, with low fermentation cost, simple extraction process and easy large-scale production.

[0004] Therefore, how to provide a strain that can achieve large-scale production of ergothioneine is a technical problem that those skilled in the art need to solve urgently. Summary of the invention

[0005] In view of this, the present invention provides a construction method of a recombinant Escherichia coli for synthesizing ergothioneine, the Escherichia coli and applications.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The embodiment of the present invention provides a method for constructing a recombinant Escherichia coli for the synthesis of ergothioneine, the process comprising:

[0008] 1) The NcEgt1* sequence and the EgtD* sequence were fused and connected with the ribosome binding site sequence, and then connected to the vector pET-Duet-1 after being digested with XbaI and NotI to obtain the recombinant plasmid pET-Duet-1-NcEgt1*-EgtD*;

[0009] 2) EgtE was digested with NdeI / KpnI and connected to the recombinant plasmid pET-Duet-1-NcEgt1*-Egt D*, and finally the recombinant vector pET-Duet-1-NcEgt1*-EgtD*-EgtE was obtained;

[0010] 3) The recombinant vector pET-Duet-1-NcEgt1*-EgtD*-EgtE was transformed into E. coli BL21 to obtain an ergothioneine-producing strain, which was named E. coli Egt116;

[0011] Among them, the sequence of NcEgt1* is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2; the sequence of EgtD* is shown in SEQ ID NO.3, and the encoded protein is shown in SEQ ID NO.4;

[0012] The EgtE sequence is shown in SEQ ID NO.5, and the encoded protein is shown in SEQ ID NO.6;

[0013] The ribosome binding site sequence is shown in SEQ ID NO.7;

[0014] The sequence of pET-Duet-1-NcEgt1*-EgtD* is shown in SEQ ID NO.8;

[0015] The sequence of pET-Duet-1-NcEgt1*-EgtD*-EgtE is shown in SEQ ID NO.9.

[0016] Preferably, it also includes:

[0017] 4) The genes of EgtD* and EgtE were respectively digested by NcoI / Hind III and NdeI / KpnI and connected to pRSFDuet-1 to obtain the recombinant plasmid pRSFDuet-1-EgtD*-EgtE; and the recombinant plasmid pRSFDuet-1-EgtD*-EgtE was transformed into the strain E.coli Egt116 to obtain the ergothioneine-producing strain E.coli Egt117, and the sequence of pRSFDuet-1-EgtD*-EgtE is shown in SEQ ID NO.10.

[0018] Preferably, it also includes:

[0019] 5) The mutant strain of strain Ecoli Egt117 sdaA / MetJ double gene was constructed by CRISPR / Cas9 gene editing technology and named E.coli Egt118. Change to "The mutant strain of strain Ecoli Egt117 sdaA / MetJ double gene was constructed by cre-lox system (reference Broad-Host-Range cre-lox system for antibiotic marker recycling in Gram-negative bacteria) and named E.coli Egt118".

[0020] Preferably, the mutation of the sdaA / MetJ double gene is that the coding genes of the double gene are completely deleted.

[0021] The cre-lox system was used to construct a mutant strain of the sdaA / MetJ double gene of strain Ecoli Egt117, which was named E. coli Egt118. Here, the sdaA / MetJ double gene mutant refers to a complete deletion of the ORFs of these two genes.

[0022] The second aspect of the embodiments of the present invention provides a strain constructed by the method described above.

[0023] The third aspect of the embodiment of the present invention provides the use of the strain in fermentation to produce ergothioneine.

[0024] A fourth aspect of the embodiment of the present invention provides a method for producing thioneine by fermentation, the process comprising: adding 2ml activated bacterial solution to 100ml fermentation medium, 4ml 75% glycerol, 50 μL, and 100mg / ml ampicillin, then placing it in a 37°C shaking table, culturing for 3h at 200rpm, then adding 100 μL, 1M IPTG and placing it in a 28°C shaking table, culturing for 20h at 200rpm; then incubating at 95°C for 10min, centrifuging at 4000rpm for 10min, sucking the supernatant through a 0.22um water filter membrane, and obtaining thioneine.

[0025] Preferably, the fermentation medium comprises per liter: 24 g yeast extract, 12 g tryptone, 3 g NaCl, 2 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.06 g ammonium ferric citrate, 1 g cysteine ​​hydrochloride, 1 g histidine and 1 g methionine.

[0026] It can be known from the above technical scheme that the present invention obtains three strains that can produce thioneine by means of genetic recombination, and the output of E.coli Egt116 is 194.93mg / L, the output of E.coli Egt117 is 308.33mg / L, and the output of E.coli Egt118 is 426.34mg / L. The output of thioneine of E.coli Egt117 is increased to 158.76% compared with E.coli Egt116, and the output of thioneine of E.coli Egt118 is increased to 138.27% compared with E.coli Egt117, realizing the large-scale production of thioneine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results are shown in the graph of shake flask fermentation assay results. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1 Construction of strain E. coli Egt116

[0030] 1) The NcEgt1* sequence and the EgtD* sequence were fused, and the fusion site was connected by the ribosome binding site sequence. The restriction enzyme sites XbaI and NotI were added to both ends of the fused sequence respectively. The fusion sequence was directly synthesized by a sequence synthesis company. The fused sequence was double-digested with XbaI and NotI and then connected to the vector pET-Duet-1, and transformed into E. coli DH5α. After verification, the recombinant plasmid pET-Duet-1-NcEgt1*-EgtD* was obtained;

[0031] 2) Restriction endonucleases NdeI and KpnI were added to the two ends of the EgtE gene sequence, and the sequence was directly synthesized by a synthesis company. After double digestion with NdeI and KpnI, the sequence was connected to the recombinant plasmid pET-Duet-1-NcEgt1*-EgtD* and transformed into E. coli DH5α. After verification, the recombinant vector pET-Duet-1-NcEgt1*-EgtD*-EgtE was obtained;

[0032] 3) The recombinant vector pET-Duet-1-NcEgt1*-EgtD*-EgtE was transformed into E.coli BL21 to obtain an ergothioneine-producing strain, and the recombinant strain was obtained by verification and named E.coli Egt116;

[0033] Among them, the sequence of NcEgt1* is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2; the sequence of EgtD* is shown in SEQ ID NO.3, and the encoded protein is shown in SEQ ID NO.4;

[0034] The EgtE sequence is shown in SEQ ID NO.5, and the encoded protein is shown in SEQ ID NO.6;

[0035] The ribosome binding site sequence is shown in SEQ ID NO.7;

[0036] The sequence of pET-Duet-1-NcEgt1*-EgtD* is shown in SEQ ID NO.8;

[0037] The sequence of pET-Duet-1-NcEgt1*-EgtD*-EgtE is shown in SEQ ID NO.9.

[0038] Example 2 Construction of strain E. coli Egt117

[0039] NcoI / Hind III and NdeI / KpnI restriction endonuclease sites were added to both ends of the gene sequence of EgtD* and the gene sequence of EgtE, and the sequence was synthesized by a synthesis company. The synthesized products were respectively digested by NcoI / Hind III and NdeI / KpnI, and then connected to the vector pRSFDuet-1. After transformation verification, the recombinant plasmid pRSFDuet-1-EgtD*-EgtE was obtained; and the recombinant plasmid pRSFDuet-1-EgtD*-Egt E was transformed into the strain E.coli Egt116 to obtain the ergothioneine-producing strain E.coli Egt117, and the pRSFDuet-1-EgtD*-EgtE sequence was shown in SEQ ID NO.10.

[0040] Example 3 Construction of strain E. coli Egt118

[0041] The cre-lox system (reference: Broad-Host-Range cre-lox system for antibiotic marker recycling in Gram-negative bacteria) was used to construct a strain Ecoli Egt117 sda A / MetJ double gene deletion strain, named E. coli Egt118. The construction process is as follows:

[0042] 1) Design the genes SdaA and MetJ to be 500 upstream and downstream of the open reading frame on the genome respectively.

[0043] - Primers for 600 bp homology fragments: SdaAup-F / R and SdaAdown-F / R, MetJup-F / R and MetJdown-F / R

[0044] 2) Using E. coli BL21 total DNA as a template, the homologous fragments were amplified with the above primers, and the upstream and downstream of the SdA homologous fragments were double-digested with restriction endonucleases KpnI / NdeI and ApaI / SacI, respectively, and then connected to the vector pCM184 to obtain the recombinant vector pCM1

[0045] 84-SdaA up-down.

[0046] 3) The recombinant vector pCM184-SdaAup-down was transformed into E. coli BL21, and after screening, the plasmid pCM157 expressing the recombinase was introduced into the correct strain to obtain a mutant with gene SdaA deletion. Based on the SdaA deletion mutant, the mutant with double gene deletion of SdaA and MetJ was obtained by the same method.

[0047] Example 4

[0048] The bacterial strain constructed with embodiment 1-3 is fermented respectively, and fermentation condition is 100ml fermentation medium, 4ml75% glycerol, 50 μ L, 100mg / ml ampicillin, add 2ml activated rear bacterial liquid, then be placed in 37 DEG C of shaking tables, 200rpm cultivate 3h, then add 100 μ L, 1M IPTG and be placed in 28 DEG C of shaking tables, 200rpm cultivate 20h;Then after 95 DEG C incubation 10min, 4000rpm centrifugal 10min, draw supernatant and cross 0.22um water system filter membrane, obtain ergothioneine. Every liter of fermentation medium includes: 24g yeast extract, 12g tryptone, 3g Nacl, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 0.06g ammonium ferric citrate, 1g cysteine ​​hydrochloride, 1g histidine and 1g methionine.

[0049] The fermented samples were subjected to HPLC analysis.

[0050] Detection conditions: Balance system according to acetonitrile: water = 3:97, flow rate 0.8ml / min, balance for 20min

[0051] The detection procedure was isocratic elution, i.e., acetonitrile: water = 3:97, flow rate 0.8 ml / min, column temperature 30 °C, detection wavelength 254 nm

[0052] The results of shake flask fermentation showed that ( Figure 1 ), the yield of E.coli Egt116 was 194.93 mg / L, the yield of E.coli Egt117 was 308.33 mg / L, the yield of E.coli Egt118 was 426.34 mg / L, the yield of ergothioneine in E.coli Egt117 was increased by 158.76% compared with E.coli Egt116, and the yield of ergothioneine in E.coli Egt118 was increased by 138.27% compared with E.coli Egt117.

[0053] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0054] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a recombinant Escherichia coli for the synthesis of ergothioneine, characterized in that the process include: 1) The NcEgt1* sequence and the EgtD* sequence were fused and connected with the ribosome binding site sequence, and then connected to the vector pET-Duet-1 after being digested with XbaI and NotI to obtain the recombinant plasmid pET-Duet-1-NcEgt1*-EgtD*; 2) EgtE was digested with NdeI / KpnI and connected to the recombinant plasmid pET-Duet-1-NcEgt1*-Egt D*, and finally the recombinant vector pET-Duet-1-NcEgt1*-EgtD*-EgtE was obtained; 3) The recombinant vector pET-Duet-1-NcEgt1*-EgtD*-EgtE was transformed into E. coli BL21 to obtain an ergothioneine-producing strain, which was named E. coli Egt116; Among them, the sequence of NcEgt1* is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2; the sequence of EgtD* is shown in SEQ ID NO.3, and the encoded protein is shown in SEQ ID NO.4; The EgtE sequence is shown in SEQ ID NO.5, and the encoded protein is shown in SEQ ID NO.6; The ribosome binding site sequence is shown in SEQ ID NO.7; The sequence of pET-Duet-1-NcEgt1*-EgtD* is shown in SEQ ID NO.8; The sequence of pET-Duet-1-NcEgt1*-EgtD*-EgtE is shown in SEQ ID NO.

9.

2. the construction method of a strain for the synthesis of recombinant Escherichia coli of thioneine according to claim 1, is characterized in that, Also includes: 4) The genes of EgtD* and EgtE were respectively digested by NcoI / Hind III and NdeI / KpnI and connected to pRSFDuet-1 to obtain the recombinant plasmid pRSFDuet-1-EgtD*-EgtE; and the recombinant plasmid pRSFDuet-1-EgtD*-EgtE was transformed into the strain E.coli Egt116 to obtain the ergothioneine-producing strain E.coli Egt117, and the sequence of pRSFDuet-1-EgtD*-EgtE is shown in SEQ ID NO.

10.

3. the construction method of a strain for the synthesis of recombinant Escherichia coli of thioneine according to claim 2, is characterized in that, Also includes: 5) The mutant strain of strain Ecoli Egt117 sdaA / MetJ double gene was constructed using the cre-lox system. The mutant strain of strain Ecoli Egt117 sdaA / MetJ double gene was constructed using editing technology and named E.coli Egt118.

4. A strain constructed according to any one of claims 1 to 3.

5. application of the bacterial strain described in claim 4 in fermentation to produce ergothioneine.

6. A method for producing ergothioneine by fermentation, characterized in that, The process comprises: adding 2 ml of activated bacterial solution to 100 ml of fermentation medium, 4 ml of 75% glycerol, 50 μL, and 100 mg / ml of ampicillin, and then placing the mixture in a shaker at 37°C and culturing at 200 rpm for 3 hours; then adding 100 μL of 1M IPTG and placing the mixture in a shaker at 28°C and culturing at 200 rpm for 20 hours; then incubating the mixture at 95°C for 10 minutes, centrifuging the mixture at 4000 rpm for 10 minutes, and aspirating the supernatant to filter through a 0.22 um water filter membrane to obtain ergothioneine.

7. A method for producing ergothioneine by fermentation according to claim 6, characterized in that, Each liter of fermentation medium includes: 24 g yeast extract, 12 g tryptone, 3 g NaCl, 2 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.06 g ammonium ferric citrate, 1 g cysteine ​​hydrochloride, 1 g histidine and 1 g methionine.

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