CN116732064A - Sphingomonas ergothioneine synthetic gene or gene combination and application thereof - Google Patents
Sphingomonas ergothioneine synthetic gene or gene combination and application thereof Download PDFInfo
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- SSISHJJTAXXQAX-ZETCQYMHSA-N L-ergothioneine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC(=S)N1 SSISHJJTAXXQAX-ZETCQYMHSA-N 0.000 title claims abstract description 118
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Abstract
The invention discloses a sphingomonas ergothioneine synthetic gene or a gene combination and application thereof. The present invention provides any one or a combination of the following genes: (1) SPegtB; (2) SPegtD; (3) SPegtE; (4) SPegtB+SPegtD; (5) SPegtD+SPegtE; (6) SPegtB+SPegtD+SPegtE. According to the invention, the sphingomonas HBJ-193 is taken as an original strain, and the PBBR1MCS-2 is taken as a carrier to overexpress the sphingomonas HBJ-193 ergothioneine synthetic gene, so that the production capacity of the strain ergothioneine is improved, the yield can reach 73.46mg/L, and compared with the yield of the original strain ergothioneine, the yield is increased by 44.63%, a brand-new biosynthesis way is provided for producing the ergothioneine, and the disadvantages and the defects of the current ergothioneine producing strain are hopeful to be solved.
Description
Technical Field
The invention belongs to the technical field of biosynthesis, and particularly relates to a sphingomonas ergothioneine synthetic gene or a gene combination and application thereof.
Background
Ergothioneine (EGT) is a natural chiral amino acid strong antioxidant, also called 2-thio-L-histidine trimethyl inner salt, has strong antioxidant, anti-aging and anti-radiation activities, and is an important active substance for normal growth and metabolism of cells. Ergothioneine is very soluble in water, and its aqueous solution can remain active for a long period of time, is more stable than other antioxidants in performance, and does not undergo autoxidation under strong alkali and normal physiological pH conditions. Therefore, ergothioneine is widely used in the fields of medicine, cosmetics, foods and beverages, etc.
Ergothioneine synthesis genes are classified into fungal and bacterial sources. Among fungi, two synthetases Egt1, 2 are required for the synthesis of ergothioneine, and most non-yeast fungi such as Maitake Mushroom (Neurospora crassa), rhizopus oryzae (Rhizopus oryzae), boletus edulis (Boletus edulis), etc. contain ergothioneine. Bacterial synthesis of ergothioneine usually requires five synthetases EgtA, B, C, D, E, and only a small number of ergothioneine-synthesizing bacteria such as Mycobacterium tuberculosis (Mycobacterium tuberculosis), mycobacterium smegmatis (Mycobacterium smegmatis), cyanobacteria (Certain cyanobacteria), methylobacillus (Methylobacillus), actinomycetes (Actinomyces) are currently found.
The chemical synthesis method of the ergothioneine has the advantages of high difficulty, low yield, serious organic solvent residue and no guarantee on safety, so that the main source of the industrial ergothioneine is the deep fermentation extraction of edible fungus hyphae at present, but the natural yield is lower, and although the content of the ergothioneine in fruiting bodies of the edible and medicinal fungus is relatively higher, the method for extracting the ergothioneine from a large number of fruiting bodies in the industrial preparation is often not the most suitable, and the fruiting bodies cannot be suitable for industrial production due to long cultivation time and high cost.
From the technical application prospect, the biological method is the most potential production mode of the ergothioneine, and compared with fungi, the production method of the ergothioneine by utilizing bacteria has the advantages of short production period and simple extraction process. The strain with excellent ergothioneine synthesis capacity is selected, the key enzyme genes of the ergothioneine synthesis pathway are enhanced, and the construction of the ergothioneine microbial cell synthesis factory can lead the production of the ergothioneine to be more economical and efficient.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the primary object of the present invention is to provide a sphingomonas ergothioneine synthesis gene or gene combination.
It is another object of the present invention to provide a sphingomonas ergothioneine synthetic protein or protein combination.
It is another object of the present invention to provide the use of the above-described sphingomonas ergothioneine synthesis gene, gene combination, protein or protein combination.
In order to achieve the above object, the present invention adopts the following technical scheme:
the sphingomonas ergothioneine synthesis gene or gene combination is any one of the following genes or gene combinations: (1) SPegtB; (2) SPegtD; (3) SPegtE; (4) SPegtB+SPegtD; (5) SPegtD+SPegtE; (6) SPegtB+SPegtD+SPegtE; wherein the nucleotide sequence of the SPegtB is shown as SEQ ID NO. 1, the nucleotide sequence of the SPegtD is shown as SEQ ID NO. 3, and the nucleotide sequence of the SPegtE is shown as SEQ ID NO. 5.
The related biological material of the sphingomonas ergothioneine synthetic gene or the gene combination is any one of the following biological materials:
1) The sphingomonas ergothioneine synthesis gene or the protein encoded by the gene combination;
2) An expression cassette containing said sphingomonas ergothioneine synthesis gene or combination of genes;
3) A recombinant vector comprising said sphingomonas ergothioneine synthetic gene or gene combination, or a recombinant vector comprising said expression cassette of 2);
4) Recombinant microorganisms comprising said sphingomonas ergothioneine synthesis gene or gene combination, or recombinant microorganisms comprising said expression cassette of 2), or recombinant microorganisms comprising said recombinant vector of 3).
Preferably, the protein of 1) is any one or a combination of the following proteins: (1) SPegtB; (2) SPegtD; (3) SPegtE; (4) SPegtB+SPegtD; (5) SPegtD+SPegtE; (6) SPegtB+SPegtD+SPegtE; wherein the amino acid sequence of SPegtB is shown as SEQ ID NO.2, the amino acid sequence of SPegtD is shown as SEQ ID NO. 4, and the amino acid sequence of SPegtE is shown as SEQ ID NO. 6.
Preferably, the recombinant vector in 3) is obtained by constructing the gene or the gene combination into a PBBR1MCS-2 plasmid; it is further preferred that the SPegtB+SPegtD or SPegtD+SPegtE is constructed into the PBBR1MCS-2 plasmid.
Preferably, the recombinant microorganism described in 4) is obtained by transforming the recombinant vector described in 3) into Sphingomonas; it is further preferred that the recombinant vector described in 3) is transformed into Sphingomonas sp HBJ-193.
The sphingomonas ergothioneine synthetic gene, gene combination or related materials are applied to synthesis of ergothioneine.
Preferably, said use is over-expression of said sphingomonas ergothioneine synthesis gene or combination of genes in sphingomonas; it is further preferred that SPegtB+SPegtD or SPegtD+SPegtE is overexpressed in Sphingomonas.
Preferably, the Sphingomonas is Sphingomonas sp HBJ-193.
A method of ergothioneine biosynthesis comprising the steps of:
(1) construction of expression plasmids PBBR1MCS-2-SPegtB-SPegtD or PBBR1 MCS-2-SPegtD-SPegtE:
carrying out homologous recombination on the SPegtD gene fragment with the homology arm and the SPegtB or SPegtE gene fragment with the homology arm and the linearized PBBR1MCS-2 carrier fragment simultaneously by utilizing homologous recombination enzyme to obtain an expression plasmid PBBR1MCS-2-SPegtB-SPegtD or PBBR1MCS-2-SPegtD-SPegtE;
(2) construction of recombinant bacteria HBJ-193-BD or HBJ-193-DE:
introducing plasmid PBBR1MCS-2-SPegtB-SPegtD or PBBR1MCS-2-SPegtD-SPegtE into Sphingomonas HBJ-193 by adopting a dielectric transformation method to obtain recombinant bacteria HBJ-193-BD or HBJ-193-DE;
(3) fermenting recombinant bacteria:
selecting recombinant bacteria HBJ-193-BD or HBJ-193-DE single colony to LB liquid culture medium, adding kanamycin, and culturing to obtain seed liquid; adding seed solution into fermentation medium, adding kanamycin, and culturing in dark place; adding equal amount of kanamycin again, continuing to culture in dark place, and ending fermentation;
(4) ergothioneine isolation and purification:
adding ethanol extract into the fermented culture solution, standing to obtain crude extract, centrifuging the crude extract, collecting supernatant, removing impurities, and filtering to obtain ergothioneine.
Preferably, the homologous recombination described in (1) is effected using a ClonExpress Ultra One Step Cloning Kit kit.
Preferably, the 10. Mu.L reaction system for homologous recombination described in (1) consists of:
preferably, the reaction parameters of homologous recombination described in (1): incubate at 50℃for 30min and immediately cool.
Preferably, the seed liquid of (3) is inoculated in an amount of 2%.
Preferably, the conditions for the light-shielding culture described in (3) are 28℃at 150rpm for 72 hours.
Preferably, the kanamycin as described in (3) is added in an amount of 1mg in terms of its volume ratio to the medium: 40ml meter.
Preferably, the composition of the ethanol extract described in (4) is as follows: each 1L of the system contains 1.67g of Sodium Dodecyl Sulfate (SDS), 700mL of absolute ethyl alcohol and the balance of water.
Preferably, the ethanol extract described in (4) is added in an amount of 4 times by volume of the fermented culture broth.
Preferably, the conditions for standing described in (4) are 4℃for 12 hours.
Preferably, the centrifugation conditions described in (4) are 12000r/min and 10min.
The principle of the invention is as follows: sphingomonas sp. HBJ-193 was deposited on the China general microbiological culture Collection center, the national institutes of microbiology, national academy of sciences of China, no. 3, of the Korean area of Beijing, at 2022, 03, 29, accession number: CGMCC No.24613. Sphingomonas is a widely recognized and safe strain, is industrially used for producing extracellular polysaccharide, and the excavation of ergothioneine key enzyme synthesis genes of the food source strain is beneficial to the construction of a Sphingomonas ergothioneine synthesis plant.
Since the ergothioneine synthesis pathway of Sphingomonas is unknown, a local comparison nucleic acid library is established by utilizing genome data of Sphingomonas HBJ-193 under a Linux system, and BLAST comparison is carried out on a relatively near-edge and functional verified mycobacterium smegmatis (Mycobacterium smegmatis) serving as an input gene of the ergothioneine synthesis key gene of the Sphingomonas and the local nucleic acid library of the HBJ-193 genome. According to the comparison result of the local nucleic acid library and the annotation information of Pfam and Nr databases, the SPegtB, SPegtD, SPegtE genes of the sphingomonas HBJ-193 are respectively named as GE002087, GE002086 and GE001348 in the genome.
The DNA of the Sphingomonas HBJ-193 is extracted as a template, a specific primer is designed for cloning, the genes in the ergothioneine synthesis pathway are obtained, and partial genes are over-expressed in the Sphingomonas HBJ-193 by taking PBBR1MCS-2 as a carrier, so that the improvement of the ergothioneine yield is realized.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention uses the homologous comparison method to find out the ergothioneine synthase gene of the sphingomonas HBJ-193, and extracts the DNA of the sphingomonas HBJ-193 for cloning and obtaining. The comparison result of a local nucleic acid library is combined with the annotation information of Pfam and Nr databases, the ergothioneine synthase gene of the SpegtB gene HBJ-193 of the Sphingomonas is drawn, and the similarity of the amino acid sequence of the SpegtB gene GE002087 of the HBJ-193 of the Sphingomonas is 52.66% as compared with the key gene of the ergothioneine of the Mycobacterium smegmatis through SWISS-MODEL homology modeling; the similarity of the amino acid sequence of the SPegtD gene GE002086 of Sphingomonas HBJ-193 and the selected homologous modeling template is 40.72%; the amino acid sequence of the SPegtE gene GE001348 of Sphingomonas HBJ-193 has a 31.45% similarity to the selected homologous modeling template.
(2) According to the invention, the sphingomonas HBJ-193 is taken as an original strain, and the PBBR1MCS-2 is taken as a carrier to overexpress the sphingomonas HBJ-193 ergothioneine synthetic gene, so that the production capacity of the strain ergothioneine is improved, the yield can reach 73.46mg/L, and compared with the yield of the original strain ergothioneine, the yield is increased by 44.63%, a brand-new biosynthesis way is provided for producing the ergothioneine, and the disadvantages and the defects of the existing ergothioneine producing bacteria are expected to be solved. Sphingomonas is a widely recognized safe strain, has the characteristics of short fermentation period and easy extraction of fermentation products, and has wide application prospect.
Drawings
FIG. 1 is an electrophoretogram of a recombinant plasmid; wherein m4 is Marker4, CK is PBBR1MCS-2, BD is PBBR1MCS-2-SPegtB-SPegtD, DE is PBBR1MCS-2-SPegtD-SPegtE;
FIG. 2 is a map of the PBBR1MCS-2-SPegtB-SPegtD expression plasmid in example 1;
FIG. 3 is a map of the PBBR1MCS-2-SPegtD-SPegtE expression plasmid in example 2;
FIG. 4 is a graph showing the results of HPLC ergothioneine detection of Sphingomonas HBJ-193 fermentation broth; wherein, the peak time of ergothioneine is 23.041min;
FIG. 5 is a graph showing the results of HPLC ergothioneine detection of recombinant Sphingomonas HBJ-193-BD fermentation broth in example 1; wherein, the peak time of ergothioneine is 23.025min;
FIG. 6 is a graph showing the results of HPLC ergothioneine detection of recombinant Sphingomonas HBJ-193-DE fermentation broth of example 2; wherein, the peak time of ergothioneine is 23.030min;
FIG. 7 is a map of the PBBR1MCS-2-SPegtB expression plasmid in comparative example 1;
FIG. 8 is a map of the PBBR1MCS-2-SPegtD expression plasmid in comparative example 2;
FIG. 9 is a map of the PBBR1MCS-2-SPegtE expression plasmid in comparative example 3;
FIG. 10 is a map of the PBBR1MCS-2-SPegtB-SPegtD-SPegtE expression plasmid in comparative example 4;
FIG. 11 is the results of HPLC ergothioneine detection of recombinant Sphingomonas HBJ-193-B fermentation broth of comparative example 1; wherein, the peak time of ergothioneine is 22.868min;
FIG. 12 is the results of HPLC ergothioneine detection of recombinant Sphingomonas HBJ-193-D fermentation broth of comparative example 2; wherein, the peak time of ergothioneine is 22.888min;
FIG. 13 is a HPLC ergothioneine assay result of recombinant Sphingomonas HBJ-193-E fermentation broth of comparative example 3; wherein, the peak time of ergothioneine is 22.917min;
FIG. 14 is the results of HPLC ergothioneine detection of recombinant Sphingomonas HBJ-193-BDE fermentation broth of comparative example 4; wherein, the peak time of ergothioneine is 22.937min.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The materials, reagents and the like used, unless otherwise specified, are those obtained commercially.
Sphingomonas sp HBJ-193: the culture of China general microbiological culture Collection center, deposit number: CGMCC No.24613.
LB medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl, distilled water to a volume of 1L, pH 7.4 and sterilizing by high-pressure steam at 121 ℃ for 30min. Adding 2% agar powder into the solid culture medium.
Fermentation medium: 10g of tryptone, 7.5g of glycerol, 10g of NaCl, 40mM of cysteine, distilled water to a volume of 1L, pH 5.4 and high-pressure steam sterilization at 121 ℃ for 30min. 6.25. Mu.l kanamycin mother liquor was added before fermentation.
Kanamycin mother liquor (100 mg/ml): 100mg of kanamycin powder was weighed, dissolved in 1ml of ultrapure water, filtered and sterilized in a sterile environment and stored at-20 ℃ for later use.
Ethanol extract: 1.67g of Sodium Dodecyl Sulfate (SDS) was weighed, dissolved in 700mL of absolute ethanol, and the volume was adjusted to 1000mL using ultrapure water.
Marker3: the DNA fragments from top to bottom were 4500bp, 300bp, 2000bp, 1200bp, 800bp, 500bp, 200bp in length, respectively, purchased from Tongsheng Biotechnology Co., guangzhou.
Marker4: the DNA fragments from top to bottom were 15000bp, 8000bp, 5000bp, 2500bp, 1000bp, 500bp, respectively, purchased from Guangzhou Dong Biotechnology Co.
The PBBR1MCS-2 plasmid was purchased from Shanghai sea Ji Haoge Biotechnology Co.
DNA polymerase 2X Rapid Taq Master Mix (P222), DNA polymeraseMax Super-Fidelity DNA Polymerase (P505), kit ClonExpress Ultra One Step Cloning Kit for homologous recombination (C115) were purchased from Nanjinouzan Biotechnology Co., ltd.
Plasmid extraction kit HiPure Plasmid Mini Kit, nucleic acid purification recovery kit HiPure Gel Pure DNA Mini Kit, bacterial DNA extraction kit HiPureBacterial DNA Kit are purchased from meibased organisms.
Primers used in the following examples (Table 1):
TABLE 1 primer sequences
Primers were synthesized by Beijing engine biotechnology Co.
Example 1: cloning of key genes for synthesizing ergothioneine by Sphingomonas
A local comparison nucleic acid library is established by utilizing genome data of Sphingomonas HBJ-193 under a Linux system, and BLAST comparison is carried out on a relatively near and functional verification mycobacterium smegmatis (Mycobacterium smegmatis) of ergothioneine synthesis key genes of the near and functional verification mycobacterium smegmatis serving as an input gene of the local comparison and the local nucleic acid library of the HBJ-193 genome. According to the comparison result of the local nucleic acid library and the annotation information of Pfam and Nr databases, the SPegtB, SPegtD, SPegtE genes of the sphingomonas HBJ-193 are respectively named as GE002087, GE002086 and GE001348 in the genome.
The sphingomonas DNA is obtained through a HiPure Bacterial DNA Kit bacterial DNA extraction kit, the sphingomonas DNA is used as a template, primers are designed according to GE002087, GE002086 and GE001348 sequences in the genome of the sphingomonas HNJ-193, and the specificity of the primers is verified by means of Nucleolide BLAST (website: https:// BLAST. NCBI. Lm. Nih. Gov/BLAST. Cgi) in NCBI, and DNA polymerase is utilizedMax Super-Fidelity DNA Polymerase amplified SPegtB, SPegtD, SPegtE target genes respectively, and the reaction system and reaction parameters are as follows:
PCR reaction system:
PCR reaction parameters:
pre-denaturation at 95℃for 3min, denaturation at 95℃for 15ec, SPegtBu55℃annealing for 15sec, extension at 72℃for 1min, then back to the annealing step for a total of 35 cycles, and thorough extension at 72℃for 5min; pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, SPegtD 60℃annealing for 15sec, extension at 72℃for 45sec, then back to the annealing step for a total of 35 cycles, and thorough extension at 72℃for 5min; pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, SPegtE60℃annealing for 15sec, extension at 72℃for 1min, followed by a total of 35 cycles of annealing and thorough extension at 72℃for 5min.
After the PCR products were identified by 1% agarose gel electrophoresis, the PCR products with correct bands were sent to Beijing qing Biotech Co.
The sequencing result is identical with the sequences of GE002087, GE002086 and GE001348 in the genome of Sphingomonas HNJ-193, namely the Sphingomonas ergothioneine synthetase gene is successfully cloned.
Example 2: construction and transformation of PBBR1MCS-2-SPegtB-SPegtD expression plasmid
The method for constructing the vector is a homologous recombination method, a gene fragment with a homology arm is connected into an expression vector PBBR1MCS-2 by utilizing homologous recombinase, and then the recombinant bacterium is constructed by introducing Sphingomonas through dielectric transformation.
2.1 addition of homology arms to Gene fragments
Using SpegtB (GE 002087) and SpegtD (GE 002086) sequences in SpegtB HNJ-193 genome were designed based on SpegtB DNA as template&SPegtB-R、B-D&D-p, using DNA polymeraseMax Super-Fidelity DNA Polymerase amplified target genes SPegtB and SPegtD with homology arms, the reaction parameters were as follows:
adding a homology arm PCR reaction system:
PCR reaction parameters:
pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, SPegtBu55℃annealing for 15sec, extension at 72℃for 1min, followed by a total of 35 cycles of annealing and thorough extension at 72℃for 5min; pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, SPegtD 60℃annealing for 15sec, extension at 72℃for 45sec, then back to the annealing step for a total of 35 cycles, and thorough extension at 72℃for 5min.
After the PCR product is identified by 1% agarose gel electrophoresis, the HiPure Gel Pure DNA Mini Kit kit is adopted to recycle the nucleic acid, the detailed steps are shown in the specification, and the recycled product is stored at the temperature of minus 20 ℃ for standby.
2.2 double cleavage of vector
Shaking overnight, and extracting the PBBR1MCS-2 plasmid by using a plasmid extraction kit HiPure Plasmid Mini Kit. The PBBR1MCS-2 plasmid was then double digested with FastDigest EcoR I and FastDiest SacI, the reaction system and parameters were as follows:
double cleavage reaction system:
double cleavage reaction parameters: incubate 40min at 37℃and inactivate 5min at 80℃and ice-bath at 4℃for 5min.
After gel electrophoresis identification is carried out on the double enzyme digestion products by 1% agarose, the recovery of nucleic acid is carried out by adopting a HiPure Gel Pure DNA Mini Kit kit, the steps are detailed in the specification, and the recovered products are stored at the temperature of minus 20 ℃ for standby.
2.3 construction of expression plasmids
The vector is constructed by ClonExpress Ultra One Step Cloning Kit homologous recombination enzyme, and a plurality of gene fragments with homology arms and a linearized vector fragment are subjected to homologous recombination to construct a plasmid with a circular structure. The specific reaction system and parameters are as follows:
homologous recombination reaction system:
homologous recombination reaction parameters: incubate at 50℃for 30min and immediately cool on ice. The recombinant product may be stored at-20 ℃.
Ice bath thawing and preserving at-80 ℃ with 0.1M CaCl 2 The prepared E.coli DH5 alpha competent cells were added with 10. Mu.L of the recombinant product, gently stirred and mixed, and placed in an ice bath for 10min. The centrifuge tube is not shaken during the process of heat-shock in a water bath at 42 ℃ for 30sec and then immediately ice-bath for 2 min. Adding 500 mu L of sterile LB liquid medium, uniformly mixing, placing at 37 ℃ for culturing for 1h at 200rpm, and resuscitating the thalli. Resuscitating bacteria liquid is centrifugally concentrated and coated on LB solid plates containing 50 mug/ml kanamycin, and the plates are inverted and cultured overnight at 37 ℃. After single colonies were grown, the colonies were identified by DNA polymerase 2X Rapid Taq Master Mix, and the reaction system and reaction parameters were as follows:
colony identification reaction system:
colony identification PCR reaction parameters were as follows: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, annealing at 55℃for 15sec, extension at 72℃for 1min for 30sec, and then return to the annealing step for a total of 30 cycles, thorough extension at 72℃for 5min.
The PCR products were identified by 1% agarose gel electrophoresis, and as seen in lanes 5-7 of FIG. 1, corresponding bands appeared between 2500bp, and the brightness was better, and it could be preliminarily judged that the insertion of SPegtB-SPegtD was successful, then the PCR products were sequenced to a 2262bp complete reading frame by Beijing engine technology Co., ltd, and colonies with correct sequencing results were preserved for standby, so that recombinant expression plasmid PBBR1MCS-2-SPegtB-SPegtD was constructed (FIG. 2).
2.4 transformation and identification of Sphingomonas
HBJ-193 was introduced after vector construction by means of electrotransformation, which was described in Manjusha et al (Manjusha et al 2020) with major modifications, and the specific steps were: (1) Single colony of Sphingomonas HBJ-193 is picked up to LB liquid culture medium 37 ℃ and shake cultured for 12 hours to OD under the dark condition at 200r/min 600 =0.6 to 0.8; (2) Subpackaging the bacterial liquid in a 50ml sterile centrifuge tube, carrying out ice bath for 30min, centrifuging at 4 ℃ for 20min at 8000r/min, and discarding the supernatant; (3) Adding 30mL of ice-bath sterile water, fully and re-suspending, centrifuging, discarding supernatant, and respectively adding 20mL of ice-bath sterile water and 10mL of ice-bath sterile water for washing and collecting thalli; (4) Adding 2mL of ice-bath sterile water for resuspension, and sub-packaging 100 mu L/tube into a sterile 1.5mL centrifuge tube for ice-bath use; (5) Flushing the electric shock cup with sterile water for 3 times, then flushing with absolute ethyl alcohol for 3 times, and airing on ice for standby; (6) Gently mixing about 100ng recombinant expression plasmid into HBJ-193 competent cells, ice-bathing for 20min, shocking with an electrotransport device with parameters of 1.8kV and 25 mu F, pulse time interval after shocking being 4.5-5.5ms, rapidly adding 900 mu L of LB liquid culture medium, gently mixing, sucking into a 1.5mL sterile centrifuge tube, resuscitating at 37 ℃ for 50min at 200 r/min; (7) Concentrating the bacterial liquid to 100 mu L, coating the bacterial liquid on an LB solid plate containing 50 mu g/ml kanamycin, and culturing at 30 ℃; (8) After single colonies were grown, the colonies were identified using DNA polymerase 2X Rapid Taq Master Mix, and the reaction system and reaction parameters were as follows:
colony identification reaction system:
colony identification PCR reaction parameters were as follows: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, annealing at 55℃for 15sec, extension at 72℃for 1min for 30sec, and then return to the annealing step for a total of 30 cycles, thorough extension at 72℃for 5min.
After the PCR products are identified by 1% agarose gel electrophoresis, colonies with correct band sizes are preserved for standby.
Thus, the construction of the SpegtB and SPegtD overexpression strains of SpegtD and HBJ-193-BD is completed.
2.5 fermentation of recombinant bacteria
The PBBR1MCS-2 carrier has kanamycin resistance, so HBJ-193-BD can normally grow in the presence of kanamycin, and the loss of recombinant plasmids can be prevented by adding kanamycin into a culture medium, so the fermentation adopts the following method: (1) HBJ-193-BD single colony was picked up to 25ml LB liquid medium and 6.25. Mu.l kanamycin mother liquid was added, and cultured at 28℃for 16 hours at 150rpm as seed liquid; (2) The seed solution was aspirated at 2% inoculum size and added to 25ml fermentation medium, and 6.25. Mu.l kanamycin mother solution was added thereto, and the mixture was incubated at 28℃at 150rpm in the dark for 72 hours; (3) Adding the kanamycin mother solution equivalent to the step (2), continuously culturing at 28 ℃ and 150rpm in dark for 72 hours, and ending fermentation.
2.6 ergothioneine product detection
Taking 2ml of culture solution obtained after HBJ-193-BD fermentation for 144 hours, adding 4 times of ethanol extract, standing at 4 ℃ for 12 hours to obtain crude extract, taking 1ml of crude extract to a 1.5ml centrifuge tube, centrifuging at normal temperature of 12000r/min for 10 minutes, taking supernatant, using an organic phase filter head of 0.22 mu m for impurity removal and filtration, and then carrying out HPLC analysis.
High performance liquid chromatography detection of ergothioneine was performed using an Shimadzu LC 2030 CN instrument. The chromatographic column model is Welch Ultimate HILIC Amphion II (5 mu m 4.6X1250 mm), the detection wavelength of the ultraviolet detector is 257nm, the detection mobile phase is acetonitrile/water=80:20, the flow rate is 1mL/min, the sample injection amount is 20 mu L, and the column temperature is 30 ℃. Ergothioneine standard is dissolved in dd H 2 In O, the concentrations of ergothioneine standard substances are 5, 10, 20, 40 and 80mg/L respectively, and HPLC detection is carried out under the same conditions. After HPLC detection, peak areas corresponding to standard solutions with different concentrations are recorded, the peak areas are taken as an ordinate, the standard solution concentration (mg/L) is taken as an abscissa, and a linear regression equation is fitted.
The standard linear equation of ergothioneine is y= 74940x-29220, r 2 =1, linear range 5-80 mg/L. HPLC analysis of HBJ-193-BD broth is shown in FIG. 5, and according to the prepared ergothioneine standard curve, the final concentration of ergothioneine in HBJ-193-BD broth is 73.46mg/L, and the yield of ergothioneine is increased by 44.63% compared with that of HBJ-193 original strain (FIG. 4).
Example 3: construction and transformation of PBBR1MCS-2-SPegtD-SPegtE expression plasmid
The method for constructing the vector is a homologous recombination method, a gene fragment with a homology arm is connected into an expression vector PBBR1MCS-2 by utilizing homologous recombinase, and then the recombinant bacterium is constructed by introducing Sphingomonas through dielectric transformation.
3.1 addition of homology arms to Gene fragments
Using SpegtD (GE 002086) and SpegtE (GE 001348) sequences in SpegtD HNJ-193 genome of SpegtD as template&SPegtD-R、D-E&E-p, using DNA polymeraseMax Super-Fidelity DNA Polymerase amplified target genes SPegtD and SPegtE with homology arms, the reaction parameters were as follows:
adding a homology arm PCR reaction system:
PCR reaction parameters:
pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, SPegtD 60℃annealing for 15sec, extension at 72℃for 45sec, then back to the annealing step for a total of 35 cycles, and thorough extension at 72℃for 5min; pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, SPegtE60℃annealing for 15sec, extension at 72℃for 1min, followed by a total of 35 cycles of annealing and thorough extension at 72℃for 5min. .
After the PCR product is identified by gel electrophoresis through 1% agarose, the nucleic acid is recovered by adopting a HiPure Gel Pure DNA Mini Kit kit, and the detailed steps are shown in the specification.
3.2 double cleavage of the vector, see example 2 for double cleavage of the 2.2 vector.
3.3 construction of expression plasmids
The vector is constructed by ClonExpress Ultra One Step Cloning Kit homologous recombination enzyme, and a plurality of gene fragments with homology arms and a linearized vector fragment are subjected to homologous recombination to construct a plasmid with a circular structure. The specific reaction system and parameters are as follows:
homologous recombination reaction system:
homologous recombination reaction parameters: incubate at 50℃for 30min and immediately cool on ice. The recombinant product may be stored at-20 ℃.
Conversion into 0.1M CaCl 2 Construction of 2.3 expression vector of reference example 2 for colony identification of competent cells of E.coli DH 5. Alpha. Prepared.
The PCR products were identified by 1% agarose gel electrophoresis, and it was seen from lanes 8-10 of FIG. 1 that corresponding bands appeared between 2500bp, and the brightness was better, and it could be preliminarily judged that the insertion of SPegtD-SPegtE was successful, then the PCR products were sequenced to a 2238bp complete reading frame by Beijing engine technology Co., ltd, and colonies with correct sequencing results were preserved for standby, so that recombinant expression plasmid PBBR1MCS-2-SPegtD-SPegtE was constructed (FIG. 3).
3.4 transformation and identification of Sphingomonas, see transformation and identification of 2.4 Sphingomonas in example 2.
Thus, the construction of the SpegtD and SPegtE overexpression strains of Sphingomonas HBJ-193-DE is completed.
3.5 fermentation of recombinant bacteria, see fermentation of 2.5 recombinant bacteria of example 2.
3.6 ergothioneine product detection, reference example 2, 2.6 ergothioneine product detection.
HPLC analysis of HBJ-193-DE fermentation broth is shown in FIG. 6, and according to the prepared ergothioneine standard curve, the final concentration of ergothioneine in HBJ-193-DE fermentation broth is 55.18mg/L, and the yield of ergothioneine is increased by 26.30% compared with that of HBJ-193 original strain (FIG. 4).
Comparative example 1: construction and transformation of PBBR1MCS-2-SPegtB expression plasmid
Referring to the experimental methods of example 2 or 3, homology arms were first added to the desired gene fragment SPegtB, then the SPegtB fragment with homology arms was ligated to a double digested plasmid vector by homologous recombination method, construction of recombinant bacteria was completed by electrotransformation, and then the production of ergothioneine by the recombinant bacteria broth was examined by HPLC. The difference is that:
addition of homology arms to gene fragments: the primer of the PCR reaction system added by the homologous arm of SPegtB (GE 002087) is p-B & B-p.
Reaction parameters: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, annealing at 55℃for 15sec, extension at 72℃for 1min, followed by a total of 35 cycles of annealing and thorough extension at 72℃for 5min.
Construction of expression plasmids: the amount of SPegtB having a homology arm in the homologous recombination reaction system was 54ng.
Homologous recombination reaction parameters: incubate at 50℃for 15min and immediately cool on ice. The recombinant product may be stored at-20 ℃.
The recombinant plasmid PBBR1MCS-2-SPegtB (FIG. 7) was obtained after the correct identification by electrophoresis, and recombinant bacteria HBJ-193-B were obtained by dielectric transformation into HBJ-193.
HPLC analysis of HBJ-193-B broth is shown in FIG. 11, and according to the prepared ergothioneine standard curve, the final concentration of ergothioneine in HBJ-193-B broth is 17.83mg/L, and the yield of ergothioneine is 56.15% lower than that of HBJ-193 original strain (FIG. 4).
Comparative example 2: construction and transformation of PBBR1MCS-2-SPegtD expression plasmid
Referring to the experimental methods of example 2 or 3, homology arms were first added to the desired gene SPegtD, then SPegtD fragments with homology arms were ligated to a double digested plasmid vector by homologous recombination method, construction of recombinant bacteria was completed by electrotransformation, and then the production of ergothioneine by recombinant bacteria fermentation broth was examined by HPLC. The difference is that:
addition of homology arms to gene fragments: the primer of the PCR reaction system added by the homologous arm of SPegtD (GE 002086) is p-D & D-p.
Reaction parameters: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, annealing at 60℃for 15sec, extension at 72℃for 45sec, and then back to the annealing step for a total of 35 cycles, and thorough extension at 72℃for 5min.
Construction of expression plasmids: the amount of SPegtD having homology arms in the homologous recombination reaction system was 42ng.
Homologous recombination reaction parameters: incubate at 50℃for 15min and immediately cool on ice. The recombinant product may be stored at-20 ℃.
The recombinant plasmid PBBR1MCS-2-SPegtD (FIG. 8) was obtained after the correct electrophoretic identification, and recombinant bacteria HBJ-193-D were obtained by dielectric transformation into HBJ-193.
HPLC analysis of HBJ-193-D fermentation broth is shown in FIG. 12, and according to the prepared ergothioneine standard curve, the final concentration of ergothioneine in HBJ-193-D fermentation broth is 14.23mg/L, and the yield of ergothioneine is 65.01% lower than that of HBJ-193 original strain (FIG. 4).
Comparative example 3: construction and transformation of PBBR1MCS-2-SPegtE expression plasmid
Referring to the experimental methods of example 2 or 3, a homology arm was first added to the target gene SPegtE, then the target gene SPegtE with the homology arm was ligated to a double digested plasmid vector by a homologous recombination method, construction of recombinant bacteria was completed by electrotransformation, and then the ergothioneine yield of the recombinant bacteria broth was examined by HPLC. The difference is that:
addition of homology arms to gene fragments: the primer of the PCR reaction system added by the homologous arm of the SPegtE (GE 001348) is p-E & E-p.
Reaction parameters: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, annealing at 60℃for 15sec, extension at 72℃for 1min, followed by a total of 35 cycles of annealing and thorough extension at 72℃for 5min.
Construction of expression plasmids: the amount of SPegtE having homology arms in the homologous recombination reaction system was 53ng.
Homologous recombination reaction parameters: incubate at 50℃for 15min and immediately cool on ice. The recombinant product may be stored at-20 ℃.
The recombinant plasmid PBBR1MCS-2-SPegtE (FIG. 9) was obtained after the correct electrophoretic identification, and recombinant bacteria HBJ-193-E were obtained by dielectric transformation into HBJ-193.
HPLC analysis of HBJ-193-E fermentation broth is shown in FIG. 13, and according to the prepared ergothioneine standard curve, the final concentration of ergothioneine in HBJ-193-E fermentation broth is 14.68mg/L, and the yield of ergothioneine is 63.90% lower than that of HBJ-193 original strain (FIG. 4).
Comparative example 4: construction and transformation of PBBR1MCS-2-SPegtB-SPegtD-SPegtE expression plasmid
Referring to the experimental methods of comparative examples 1, 2, and 3, a homology arm was added to the target gene SPegtB, SPegtD, SPegtE first, then the target gene SPegtB, SPegtD, SPegtE with the homology arm was linked to a double digested plasmid vector by a homologous recombination method, construction of recombinant bacteria was completed by electrotransformation, and then the ergothioneine yield of the recombinant bacteria broth was detected by HPLC. The difference is that:
the primer of the PCR reaction system added by the homologous arm of the SPegtB (GE 002087) is p-B & SPegtB-R.
Reaction parameters: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, annealing at 55℃for 15sec, extension at 72℃for 1min, followed by a total of 35 cycles of annealing and thorough extension at 72℃for 5min.
The primer of the PCR reaction system added by the homologous arm of the SPegtD (GE 002086) is B-D & SPegtD-R.
Reaction parameters: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, annealing at 60℃for 15sec, extension at 72℃for 45sec, and then back to the annealing step for a total of 35 cycles, and thorough extension at 72℃for 5min.
The primer of the PCR reaction system added by the homologous arm of the SPegtE (GE 001348) is D-E & E-p.
Reaction parameters: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15sec, annealing at 60℃for 15sec, extension at 72℃for 1min, followed by a total of 35 cycles of annealing and thorough extension at 72℃for 5min.
Construction of expression plasmids: the amount of SPegtB having homology arms in the homologous recombination reaction system was 26ng, the amount of SPegtD having homology arms was 20ng, and the amount of SPegtE having homology arms was 26ng.
Homologous recombination reaction parameters: incubate at 50℃for 30min and immediately cool on ice. The recombinant product may be stored at-20 ℃.
The recombinant plasmid PBBR1MCS-2-SPegtB-SPegtD-SPegtE (FIG. 10) was obtained after the correct electrophoretic identification, and recombinant bacteria HBJ-193-BDE were obtained by dielectric transformation into HBJ-193.
HPLC analysis of HBJ-193-BDE fermentation broth is shown in FIG. 14, and according to the prepared ergothioneine standard curve, the final concentration of ergothioneine in HBJ-193-BDE fermentation broth is 25.38mg/L, and the yield of ergothioneine is 37.60% lower than that of HBJ-193 original strain (FIG. 4).
The embodiments described above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made in the equivalent manner, and are included in the scope of the present invention.
Claims (10)
1. A sphingomonas ergothioneine synthesis gene or combination of genes, characterized by: is any one or combination of the following genes: (1) SPegtB; (2) SPegtD; (3) SPegtE; (4) SPegtB+SPegtD; (5) SPegtD+SPegtE; (6) SPegtB+SPegtD+SPegtE; wherein the nucleotide sequence of the SPegtB is shown as SEQ ID NO. 1, the nucleotide sequence of the SPegtD is shown as SEQ ID NO. 3, and the nucleotide sequence of the SPegtE is shown as SEQ ID NO. 5.
2. The biological material related to the sphingomonas ergothioneine synthesis gene or gene combination as claimed in claim 1, characterized in that: is any one of the following biological materials:
1) The sphingomonas ergothioneine synthesis gene or the protein encoded by the gene combination;
2) An expression cassette containing said sphingomonas ergothioneine synthesis gene or combination of genes;
3) A recombinant vector comprising said sphingomonas ergothioneine synthetic gene or gene combination, or a recombinant vector comprising said expression cassette of 2);
4) Recombinant microorganisms comprising said sphingomonas ergothioneine synthesis gene or gene combination, or recombinant microorganisms comprising said expression cassette of 2), or recombinant microorganisms comprising said recombinant vector of 3).
3. The biological material related to the sphingomonas ergothioneine synthesis gene or gene combination according to claim 2, characterized in that:
1) The protein is any one or combination of the following proteins: (1) SPegtB; (2) SPegtD; (3) SPegtE; (4) SPegtB+SPegtD; (5) SPegtD+SPegtE; (6) SPegtB+SPegtD+SPegtE; wherein the amino acid sequence of SPegtB is shown as SEQ ID NO.2, the amino acid sequence of SPegtD is shown as SEQ ID NO. 4, and the amino acid sequence of SPegtE is shown as SEQ ID NO. 6;
3) The recombinant vector is obtained by constructing the gene or the gene combination into a PBBR1MCS-2 plasmid;
4) The recombinant microorganism is obtained by transforming the recombinant vector described in 3) into Sphingomonas.
4. A sphingomonas ergothioneine synthesis gene or gene combination related biomaterial according to claim 3, characterised in that:
3) The recombinant vector is obtained by constructing SPegtB+SPegtD or SPegtD+SPegtE into a PBBR1MCS-2 plasmid;
4) The recombinant microorganism described in (3) is obtained by transforming the recombinant vector described in (2) into Sphingomonas sp.) HBJ-193.
5. Use of a sphingomonas ergothioneine synthesis gene or combination of genes as described in claim 1 or of a related biomaterial as described in any of claims 2-4 for the synthesis of ergothioneine.
6. The use according to claim 5, characterized in that:
the application is that the sphingomonas ergothioneine synthetic gene or gene combination is overexpressed in sphingomonas.
7. The use according to claim 6, characterized in that:
the application is to over-express the SPegtB+SPegtD or the SPegtD+SPegtE in the Sphingomonas body;
the Sphingomonas is Sphingomonas sp HBJ-193.
8. A method for the biosynthesis of ergothioneine, characterized by: the method comprises the following steps:
(1) construction of expression plasmids PBBR1MCS-2-SPegtB-SPegtD or PBBR1 MCS-2-SPegtD-SPegtE:
carrying out homologous recombination on the SPegtD gene fragment with the homology arm and the SPegtB or SPegtE gene fragment with the homology arm and the linearized PBBR1MCS-2 carrier fragment simultaneously by utilizing homologous recombination enzyme to obtain an expression plasmid PBBR1MCS-2-SPegtB-SPegtD or PBBR1MCS-2-SPegtD-SPegtE; wherein the nucleotide sequence of the SPegtB is shown as SEQ ID NO. 1, the nucleotide sequence of the SPegtD is shown as SEQ ID NO. 3, and the nucleotide sequence of the SPegtE is shown as SEQ ID NO. 5;
(2) construction of recombinant bacteria HBJ-193-BD or HBJ-193-DE:
introducing plasmid PBBR1MCS-2-SPegtB-SPegtD or PBBR1MCS-2-SPegtD-SPegtE into Sphingomonas HBJ-193 by adopting a dielectric transformation method to obtain recombinant bacteria HBJ-193-BD or HBJ-193-DE;
(3) fermenting recombinant bacteria:
selecting recombinant bacteria HBJ-193-BD or HBJ-193-DE single colony to LB liquid culture medium, adding kanamycin, and culturing to obtain seed liquid; adding seed solution into fermentation medium, adding kanamycin, and culturing in dark place; adding equal amount of kanamycin again, continuing to culture in dark place, and ending fermentation;
(4) ergothioneine isolation and purification:
adding ethanol extract into the fermented culture solution, standing to obtain crude extract, centrifuging the crude extract, collecting supernatant, removing impurities, and filtering to obtain ergothioneine.
9. The method for the biosynthesis of ergothioneine according to claim 8, characterized in that: the method comprises the following steps:
(1) the homologous recombination is realized by adopting a ClonExpress Ultra One Step Cloning Kit kit;
(1) the 10 mu L reaction system for homologous recombination comprises the following components:
(1) reaction parameters for homologous recombination described in (a): incubate at 50℃for 30min and immediately cool.
10. A method of ergothioneine biosynthesis according to claim 8 or 9, characterized in that:
(3) the inoculation amount of the seed liquid is 2%;
(3) the conditions of the light-shielding culture are 28 ℃,150rpm and 72 hours;
(3) the adding amount of kanamycin is 1mg according to the volume ratio of the kanamycin to the culture medium: a 40ml meter;
(4) the composition of the ethanol extract is as follows: 1.67g sodium dodecyl sulfate, 700mL absolute ethyl alcohol and the balance of water are contained in each 1L system;
(4) the adding amount of the ethanol extract is 4 times of the volume of the fermented culture solution;
(4) the standing condition is 4 ℃ and 12 hours;
(4) the centrifugation conditions are 12000r/min and 10min.
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| CN116970546A (en) * | 2023-09-22 | 2023-10-31 | 江苏省中国科学院植物研究所 | Engineering strain for synthesizing ergothioneine and application thereof |
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| CN116970546A (en) * | 2023-09-22 | 2023-10-31 | 江苏省中国科学院植物研究所 | Engineering strain for synthesizing ergothioneine and application thereof |
| CN116970546B (en) * | 2023-09-22 | 2023-12-26 | 江苏省中国科学院植物研究所 | Engineering strain for synthesizing ergothioneine and application thereof |
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