CN118360229B - Recombinant bacillus subtilis, construction method thereof and production method of ergothioneine - Google Patents
Recombinant bacillus subtilis, construction method thereof and production method of ergothioneine Download PDFInfo
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Abstract
The invention belongs to the technical field of ergothioneine production, and relates to recombinant bacillus subtilis, a construction method thereof and a production method of ergothioneine. The bacillus subtilis WB800N is taken as a starting bacterium, and the UPP gene is replaced by a genome containing EgtABCDE gene clusters, so that EgtABCDE gene clusters are overexpressed; the pdxST gene, ncEgt gene and CmEgt gene were overexpressed by transferring pdxST gene, ncEgt gene and CmEgt gene. According to the invention, through integrating the expression gene cluster EgtABCDE, the driven coenzyme PLP synthesis and the expression of Egt1 and Egt2, the synthesis way of ergothioneine is comprehensively enhanced, and the recombinant bacillus subtilis is constructed, so that the recombinant strain can obviously improve the fermentation yield of ergothioneine.
Description
Technical Field
The invention belongs to the technical field of ergothioneine production, and relates to recombinant bacillus subtilis, a construction method thereof and a production method of ergothioneine.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Ergothioneine (Ergothioneine, EGT) is a natural thiol-containing small molecular compound derived from histidine, cysteine and glutamic acid, is a rare natural chiral amino acid, and has strong antioxidant activity. At the earliest, it was isolated from ergot (CLAVICEPS PURPUREA), a fungus parasitic on rye. The antioxidant effect of the ergothioneine is mainly reflected in removing active free radicals in cells, chelating toxic metal ions, regulating oxidation-reduction balance in organisms, participating in cell injury repair and the like, and besides, the ergothioneine also has the function of resisting radiation, is an important additive component of cosmetics, so the ergothioneine has wide application in the fields of foods, medicines, cosmetics and the like.
At present, the synthesis method of ergothioneine is mainly a chemical method, the chemical synthesis steps are complicated, part of the steps involve expensive reagents, the safety of chemical reaction is difficult to ensure, the later separation and purification are difficult, and under the condition of a plurality of adverse factors, the chemical regulation modeling synthesis of ergothioneine still has certain challenges. In recent years, due to the continuous iterative maturation of biotechnology such as gene editing, molecular cloning, genetic engineering, metabolic engineering and the like, the development of synthetic biology has been promoted, and many chassis microorganisms have been developed, and the genetic background thereof has been gradually mined and updated. Some ergothioneine recombinant engineering bacteria have been developed, wherein both of patent publication No. CN113234652A, CN112251392A uses escherichia coli as chassis microorganisms, the fermentation time is 105h, the ergothioneine yield is 1.1g/L, and the fermentation time is 52h, and the ergothioneine yield is 2.9g/L. While the yield of the ergothioneine detected in fermentation broth of the microorganism with Saccharomyces cerevisiae as the chassis is only 6.6mg/L, the fermentation time of the microorganism with Corynebacterium glutamicum as the chassis is 56h, and the yield of the ergothioneine is 5.47g/L in the fermentation broth of the microorganism with Saccharomyces cerevisiae as the chassis in the patent publication No. CN 110607286A. Although the recombinant engineering bacteria can be used for fermentation to provide the yield of the ergothioneine, new recombinant engineering bacteria are still needed to be provided so as to further improve the yield of the ergothioneine prepared by fermentation.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the recombinant bacillus subtilis, the construction method thereof and the production method of the ergothioneine, and the recombinant bacillus subtilis is constructed by integrating the expression gene cluster EgtABCDE, the driven coenzyme PLP synthesis and the expression of Egt1 and Egt2 to comprehensively enhance the synthesis way of the ergothioneine, and the recombinant strain can obviously improve the fermentation yield of the ergothioneine.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
In the first aspect, a recombinant bacillus subtilis is prepared by taking bacillus subtilis WB800N as a starting strain, and replacing a UPP gene with a genome containing EgtABCDE gene clusters to enable EgtABCDE gene clusters to be over-expressed; the pdxST gene, ncEgt gene and CmEgt gene were overexpressed by transferring pdxST gene, ncEgt gene and CmEgt gene.
The genome containing EgtABCDE gene clusters of the present invention may be a genome containing at least two EgtABCDE gene clusters, or a genome containing a strong promoter and EgtABCDE gene clusters, and in some embodiments, the genome containing EgtABCDE gene clusters is a genome containing a strong promoter and EgtABCDE gene clusters. Specifically, the strong promoter in the genome containing EgtABCDE gene cluster is P43.
In some embodiments, the base sequence of the UPP gene is shown in SEQ ID NO.20, the base sequence of the EgtABCDE gene cluster is shown in SEQ ID NO.21, the base sequence of the NCBI accession number KR821087 of the pdxST gene, the base sequence of the NcEgt1 gene is shown in SEQ ID NO.22, and the base sequence of the CmEgt2 gene is shown in SEQ ID NO. 23.
In a second aspect, a method for constructing the recombinant bacillus subtilis includes the following steps:
Deleting and replacing UPP genes in a bacillus subtilis WB800N genome to a genome containing EgtABCDE gene clusters to obtain a recombinant integrated strain;
And transferring pdxST genes, ncEgt1 genes and CmEgt genes into the recombinant integration strain to obtain the recombinant bacillus subtilis.
In some embodiments, a recombinant vector comprising a homologous arm upstream and downstream of the UPP and a genome comprising the EgtABCDE gene cluster is transformed into bacillus subtilis WB800N by means of electrotransformation to obtain a recombinant integrated strain. The recombinant vector containing UPP upstream and downstream homology arms and genome containing EgtABCDE gene cluster is transferred into bacillus subtilis WB800N by an electrotransformation method, so that the UPP gene in the bacillus subtilis WB800N genome is deleted and replaced by the genome containing EgtABCDE gene cluster.
In one or more embodiments, the construction process of the recombinant vector comprising the upstream and downstream homology arms of the UPP and the genome comprising the EgtABCDE gene cluster comprises the steps of:
a. carrying out PCR amplification by using a vector PHT194ts as a template and adopting BEGT-ABCDE-F/BEGT-ABCDE-R primer pairs;
b. Performing PCR amplification by using a bacillus subtilis 168 genome as a template and respectively adopting a UPP-UP-F/UPP-UP-R primer pair and a UPP-DW-F/UPP-DW-R primer pair;
c. PCR amplification is carried out by taking a carrier PWB980 as a template and adopting a P43-F/P43-R primer pair;
d. Performing PCR amplification by using a Mycobacterium smegmatis genome as a template and adopting EgtABCDE-F/EgtABCDE-R primer pairs;
e. Carrying out overlap extension PCR on the amplification product obtained in the step b, the amplification product obtained in the step c and the amplification product obtained in the step d;
f. And d, connecting the amplification product obtained in the step a and the product obtained in the step e overlapping extension PCR to obtain a recombinant vector containing UPP upstream and downstream homology arms and a genome containing EgtABCDE gene clusters.
Specifically, the template in the step a is a vector PHT194ts. More specifically, the construction process of the vector PHT194ts is as follows: performing PCR amplification by adopting BEGT-ABCDE-F/BEGT-ABCDE-R primer pairs; performing PCR amplification by adopting BEGT ts-F/BEGT 194ts-R primer pairs; the two PCR amplified products were ligated to make vector PHT194ts.
Specifically, the template of step b is the bacillus subtilis 168 genome.
Specifically, the template of step c is a carrier PWB980.
In particular, the template of step d is the Mycobacterium smegmatis genome.
In some embodiments, pdxST genes, ncEgt1 genes, and CmEgt2 genes are transferred to the recombinant integrative strain by electrotransformation.
In a third aspect, a method for producing ergothioneine, comprising fermenting with the recombinant Bacillus subtilis.
In some embodiments, the fermentation is a fed-batch fermentation. Specifically, the initial concentration of glucose is 19-21 g/L; in the fermentation process, the concentration of glucose is controlled to be 9-11 g/L.
In one or more embodiments, the fermentation medium comprises: 19-21 g/L glucose, 11-13 g/L yeast powder, 5.5-6.5 g/L peptone, 5.5-6.5 g/L (NH 4)2SO4, 2.5-3.5 g/L histidine (His), 2.5-3.5 g/L cysteine (L-Cys), 0.8-1.2 g/L glutamic acid (Glu), 11.5-13.5 g/L K 2HPO4•3H2O、2.3~2.7 g/L KH2PO4 and 9-10 mL/L trace metal solution, the trace metal solution contains ：3.5~4.5 g/L FeSO4•7H2O、3.5~4.5 g/L CaCl2、0.8~1.2 g/LMnSO4•5H2O、0.35~0.45 g/L CoCl2•6H2O、0.18~0.22 g/L NaMoO4•2H2O、0.18~0.22 g/L ZnSO4•7H2O、0.09~0.11 g/L AlCl3•6H2O、0.09~0.11 g/L CuCl2•H2O、 0.045~0.055 g/L H3BO4.
In one or more embodiments, the feed medium contains: 700-800 g/L glucose solution, 9-11 g/L His, 9-11 g/L L-Cys, 2.8-3.2 g/L Glu, 250-350 g/L yeast powder and 100-200 g/L peptone.
In some embodiments, the temperature is 36-38 ℃ and the acid-base property of the culture medium is neutral and the concentration of dissolved oxygen is 20-30% in the fermentation process.
In some embodiments, isopropyl- β -D-thiogalactoside (IPTG) is added to induce when the cell concentration reaches an OD600 of 30-40.
The invention also provides a detection method for detecting ergothioneine, which adopts liquid chromatography for detection; in the liquid chromatography, the stationary phase of the chromatographic column is octadecylsilane chemically bonded silica, the mobile phase is water with the pH value of 4.9-5.1 (one or more of acetic acid, sodium acetate, boric acid, formic acid, butyric acid, oxalic acid and pyruvic acid are adopted for adjustment), the detection wavelength is 250-260 nm, preferably 254-nm, the column temperature is 15-40 ℃, preferably 20-30 ℃ and most preferably 25 ℃; the flow rate of the mobile phase is 0.7-1.0 mL/min, and most preferably 0.7 mL/min; the recording time of the chromatogram is within 30 min, preferably within 25 min, more preferably 20 min; the peak-out time of ergothioneine is 3-10 min, preferably 4-8 min, and most preferably 4-6 min.
The beneficial effects of the invention are as follows:
According to the invention, the safe strain bacillus subtilis is adopted as chassis microorganism, ergothioneine is synthesized by metabolic engineering means, and compared with the escherichia coli chassis microorganism, the bacillus subtilis used in the invention has no endotoxin and exotoxin, and is safer to apply in food and medicine fermentation. Compared with Saccharomyces cerevisiae and corynebacterium glutamicum, the recombinant bacillus subtilis disclosed by the invention is subjected to fed-batch fermentation in a 10L fermentation tank for 66 hours, the yield of ergothioneine reaches 9.26g/L, and the yield is higher than that of the recombinant engineering bacteria reported in the prior art.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic representation of the pathway of the biosynthesis of ergothioneine by Bacillus subtilis in the present invention;
FIG. 2 is a diagram of an integrative vector PHT194ts- (UPP-UP) -P43-EgtABCDE- (UPP-DW) constructed in the example of the present invention, wherein pE194ts ori is a replication initiation site, promoter is a promoter, terminator is a terminator, RBS is a ribosome binding site, cm is a chloramphenicol resistance gene, UPP-UP is an upstream integrative homology arm, UPP-DW is a downstream integrative homology arm, and P43 promoter is a P43 promoter (Bacillus subtilis constitutive promoter);
FIG. 3 is a schematic diagram showing the construction of a recombinant integration strain of Bacillus subtilis (BsWB N-EgtABCDE) in which pE194ts ori is the replication initiation site of Bacillus subtilis, promoter is the promoter, terminator is the terminator, RBS is the ribosome binding site, cm is the chloramphenicol resistance gene, UPP-UP is the upstream integration homology arm, UPP-DW is the downstream integration homology arm, P43 promoter is the P43 promoter (constitutive promoter of Bacillus subtilis), and AMP is the ampicillin resistance gene according to the embodiment of the present invention;
FIG. 4 is a map of PHT101-pNcEgt1-pCmEgt2-RBS-pdxST vector in the example of the present invention, wherein ColE1 Ori is the E.coli replication initiation site, cm is the chloramphenicol resistance gene, promoter is the promoter, terminator is the terminator, RBS is the ribosome binding site, pgrac promoter is the Pgrac promoter (Bacillus subtilis inducible promoter), repA is the Bacillus subtilis replication initiation site, and AMP is the ampicillin resistance gene;
FIG. 5 is a liquid chromatogram of ergothioneine produced by shake flask fermentation in an embodiment of the invention;
FIG. 6 is a graph showing results of ergothioneine production in a 10L fermenter fermentation test in an example of the present invention.
Detailed Description
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Examples
Materials: bacillus subtilis WB800N (product number YB00590, product company: shanghai Yubo biotechnology Co., ltd.); bacillus subtilis vector: pE194ts (cat# 37128, ex. ATCC), PHT101 (cat# Vt2073, ex. Excellent organism), PWB980 (cat# HG-VKH1349, ex. Orno).
The biosynthetic ergothioneine pathway is shown in FIG. 1.
1. Construction of bacillus subtilis integration vector:
1. PCR amplification of integrated vector material:
The vector pE194ts is used as a template, and the primer pair EGT194 ts-F/EGT 194ts-R is subjected to PCR amplification to obtain a 1.35Kb DNA fragment, wherein the fragment comprises a bacillus subtilis temperature-sensitive replication initiation site, plasmid stable replication inheritance is carried out at 37 ℃, and plasmid replication is stopped and gradually lost in offspring at 42 ℃. The polymerase used for PCR amplification is KOD-Plus-Neo, and the PCR procedure is as follows: pre-denaturation at 95℃for 1min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 3min, final extension at 68℃for 5min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 1min, 25 cycles were performed; the PCR product is purified and recovered by a purification kit for standby.
Carrying out PCR amplification by using a carrier PHT101 as a template and a primer pair BEGT ts-F/BEGT 194ts-R, wherein polymerase used for the PCR amplification is KOD-Plus-Neo, the PCR procedure is that the PCR is carried out at 95 ℃ for 3min, at 95 ℃ for 10s, at 58 ℃ for 10s, at 68 ℃ for 3min, at 68 ℃ for 5min, at 95 ℃ for 10s, at 58 ℃ for 10s and at 68 ℃ for 1min, and 25 cycles are executed; the PCR product is purified and recovered by a purification kit for standby.
The primer sequences used are shown in Table 1.
TABLE 1 primer sequences and templates used in step1
2. Preparation of vector PHT194 ts:
And (3) connecting the PCR products in the step (1) by adopting a seamless cloning kit, wherein the seamless cloning kit is purchased from Shandong Cisco, the product number AK0601-B is converted into DH5a competence by the connection product, and the final positive vector is obtained by sequencing and named PHT194ts.
3. Construction of the integration vector PHT194ts- (UPP-UP) -P43-EgtABCDE- (UPP-DW):
the vector PHT194ts is used as a template, and a primer pair BEGT-ABCDE-F/BEGT-ABCDE-R is used for PCR amplification, wherein polymerase used for the PCR amplification is KOD-Plus-Neo, and the PCR procedure is as follows: pre-denaturation at 95℃for 3min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 3min, final extension at 68℃for 5min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 3min, 25 cycles were performed; the PCR product is purified and recovered by a purification and recovery kit for standby.
The bacillus subtilis 168 genome is used as a template, and primer pairs UPP-UP-F/UPP-R, UPP-DW-F/UPP-DW-R are respectively used for PCR amplification, wherein polymerase used for the PCR amplification is KOD-Plus-Neo, and the PCR program is as follows: pre-denaturation at 95℃for 3min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 1min, final extension at 68℃for 5min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 1min, 25 cycles were performed; the PCR product is purified and recovered by a purification and recovery kit for standby.
The carrier PWB980 is used as a template, and the primer pair P43-F/P43-R is subjected to PCR amplification, wherein polymerase used for the PCR amplification is KOD-Plus-Neo, and the PCR procedure is as follows: pre-denaturation at 95℃for 3min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 1min, final extension at 68℃for 5min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 1min, 25 cycles were performed; the PCR product is purified and recovered by a purification and recovery kit for standby.
The genome of the mycobacterium smegmatis is used as a template, and primer pairs EgtABCDE-F/EgtABCDE-R are respectively used for PCR amplification, wherein polymerase used for the PCR amplification is KOD-Plus-Neo, and the PCR procedure is as follows: pre-denaturation at 95℃for 3min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 3min, final extension at 68℃for 5min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 3min, 25 cycles were performed; the PCR product is purified and recovered by a purification and recovery kit for standby.
The sequences and templates used in the above procedure in step 3 are shown in Table 2.
TABLE 2 sequences and templates employed in step 3
Overlapping extension PCR was performed with the primer pair UPP-UP-F/UPP-UP-R, P-F/P43-R, egtABCDE-F/EgtABCDE-R, UPP-DW-F/UPP-DW-R amplification recovery product. The procedure was to dilute 0.2ng of each PCR amplification recovery product to 50. Mu.L with water. The mixture of the PCR amplification recovery products is used as a template, and a primer pair UPP-UP-F/UPP-DW-R is used for PCR amplification, wherein polymerase used for the PCR amplification is KOD-Plus-Neo, and the PCR procedure is as follows: pre-denaturation at 95℃for 3min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 3min, final extension at 68℃for 5min, denaturation at 95℃for 10s, annealing at 58℃for 10s, extension at 68℃for 3min, 25 cycles were performed; the PCR product is purified and recovered by a gel cutting purification recovery kit for standby. The overlapping extension PCR product is 6.4Kb, and comprises a homologous arm integrated at the upstream of about 300bp UPP, a P43 constitutive promoter original of about 300bp, a 5.3Kb ergothioneine synthetic gene cluster and a homologous arm integrated at the downstream of about 300 bp.
The PCR amplified product of the primer pair BEGT-ABCDE-F/BEGT-ABCDE-R and the overlapping extension PCR recovery product are subjected to seamless cloning connection, a seamless cloning kit is purchased from Shandong Cisco, the product is AK0601-B, DH5a is converted into a competent product, and the final positive vector is obtained through sequencing, wherein the integrated vector is named PHT194ts- (UPP-UP) -P43-EgtABCDE- (UPP-DW) in the invention, and the vector map is shown in figure 2.
2. Constructing bacillus subtilis recombinant integration strain:
And (3) transforming the integrated vector constructed in the step (I) into bacillus subtilis WB800N by an electrotransformation method to obtain a recombinant integrated strain, and naming the recombinant strain as BsWB800N-EgtABCDE. The vector PHT194ts- (UPP-UP) -P43-EgtABCDE- (UPP-DW) is transformed into bacillus subtilis WB800N, and through the homologous sequence carried on the vector, ergothioneine synthesis EgtABCDE driven by the promoter P43 is integrated into a bacillus subtilis WB800N genome UPP gene locus, the UPP gene is substituted and deleted, the deletion of the UPP gene does not cause the death of the bacillus subtilis, and for pyrimidine anabolism, some microorganisms such as the bacillus subtilis can still be supplemented back through other ways, so that the substitution and deletion of the UPP gene does not cause great influence on the growth of the bacillus subtilis. The integration principle is shown in fig. 3.
Construction of recombinant Bacillus strain BsWB N-EgtABCDE detailed procedure:
1. Reagent configuration:
2YT Medium (1L): 16g peptone, 10g yeast powder, 5g NaCl, pH=7.2.
Electrotransport buffer (1L): 18g sorbitol, 18.5g mannitol, 20 mL g glycerol.
2. Preparation of bacillus electrotransformation competence:
1) Inoculating bacillus WB800N strain into 2YT culture medium, and culturing at 37deg.C and 180r overnight.
2) According to 1:100 inoculum size is transferred into a proper amount of 2YT culture medium, and the OD600 of the culture medium reaches 0.2-0.4 through 180r shaking culture at 37 ℃.
3) Then 1% (w/v) DL-threonine, 2% (w/v) DL-glycine, 0.1% (w/v) DL-tryptophan, 0.03% (w/v) Tween 80 were added to the medium treated in step 2), and the culture was shake-cultured at 37℃for 1 hour at 180 r.
4) Precooling the culture medium treated in the step 3) on ice for 20min, and then centrifuging at 4000r at 4 ℃ to collect thalli.
5) And (3) sequentially re-suspending and centrifuging the thalli collected in the step (4), and washing the thalli with an electrotransport buffer solution for 2 times.
6) And finally, adding a proper amount of electrotransport buffer solution into the thalli obtained in the step 5), and sub-packaging competent cells for standby.
3. Electric conversion:
Taking a prepared electrotransformation competent cell, adding a 50 ng DNA carrier, uniformly mixing with the competent cell, pre-cooling on ice for 5: 5min, transferring to a pre-cooled 2. 2mm electric shock cup, setting electric shock parameters to be 2.5 KV in voltage, 200 omega in resistance and 25 [ mu ] F in capacitance, adding 1 mL 2YT medium containing mannitol of 0.5M sorbitol and 0.38M immediately after electric shock, and carrying out shaking table resuscitation for 3 hours at 37 ℃ and 180: 180 r. Then a chloramphenicol resistant plate was applied with a final chloramphenicol concentration of 5 μg/mL.
4. Positive clone screening:
In this example, the integration vector PHT194ts- (UPP-UP) -P43-EgtABCDE- (UPP-DW) was transformed into Bacillus subtilis WB800N, and the ergothioneine synthesis EgtABCDE driven by the promoter P43 was integrated into the Bacillus subtilis WB800N genome UPP gene locus by the homologous sequence carried on the vector, and the UPP gene was deleted by substitution. The UPP gene of bacillus subtilis encodes uracil phosphoribosyl transferase (UPRTase), which catalyzes uracil to produce uridine monophosphate, allowing the cell to utilize extracellular uracil. 5-fluorouracil is a pyrimidine analog, and can be catalyzed by UPRTase to generate a strong inhibitor 5-F-dUMP of bacillus subtilis thymidylate synthase, and the existence of the strong inhibitor can lead to death of bacillus subtilis cells.
Firstly, picking the transformed monoclonal into a 2YT culture medium at 37 ℃, shaking the culture in a 180 r shaking table overnight, and then according to 1:100 is transferred into a proper amount of 2YT culture medium with 10 mu M/L of 5-fluorouracil, the mixture is recovered by a 180 r shaking table at 37 ℃ for 4-6 hours, and then the culture solution is diluted for plating.
PCR colony validation:
the diluted bacterial solution was spread on a 2YT solid plate containing 10. Mu.M/L of 5-fluorouracil, incubated overnight at 37℃and then picked up for colony PCR verification. Colony PCR-verified primers are shown in Table 3.
TABLE 3 primer sequences for colony PCR verification
Positive clones were identified, primers VerdeUPP-up-F and VerdeUPP-dw-R were designed on the Bacillus subtilis genome, primers VerdeUPP-up-R and VerdeUPP-dw-F were designed on the integrating vector sequence outside the homology arm upstream and downstream of the integrating vector, and thus colony PCR was performed on the same single clone with primer pair VerdeUPP-up-F/VerdeUPP-up-R and primer pair VerdeUPP-dw-F/VerdeUPP-dw-R, respectively, and both pairs of primers amplified a fragment of about 1.5Kb on the same clone at the same time, which was regarded as double crossover integration of the strain, further sequencing confirmation of the PCR product, and finally the recombinant strain was designated BsWB N-EgtABCDE.
3. Constructing a gene overexpression vector:
Since part of the genes are proteins with PLP as cofactor in the pathway of synthesis of ergothioneine, efficient supply of PLP as cofactor will contribute to the synthesis of L-ergothioneine. Here we constructed overexpression vectors of pdxST, ncEgt1 and CmEgt, on the one hand enhancing the supply of cofactor PLP and, on the other hand, enhancing the synthesis of L-ergothioneine in Bacillus subtilis from another route. Genes pdxST, ncEgt1 and CmEgt have the codon preference of the bacillus subtilis of Shanghai engineering bioengineering, codon optimization is carried out, then a plasmid vector map provided by the embodiment is synthesized and constructed on a vector PHT101 as shown in FIG. 4, and the constructed vector is named PHT101-pNcEgt1-pCmEgt2-RBS-pdxST.
4. Construction of recombinant Bacillus subtilis BsWB N-EGT:
And (3) transforming the vector PHT101-pNcEgt1-pCmEgt2-RBS-pdxST obtained in the step three into the recombinant strain BsWB N-EgtABCDE obtained in the step two by an electrotransformation method to obtain recombinant bacillus subtilis, and further naming the strain as BsWB N-EGT. The mixture is placed in glycerol for preservation at the temperature of-80 ℃.
5. Shake flask fermentation test:
1. The shake flask fermentation medium comprises: 60 g/L glucose, 12 g/L yeast powder, 6 g/L peptone 、6 g/L(NH4)2SO4、3 g/L L-His、3 g/L L-Cys、1 g/L L-Glu 、12.5 g/L K2HPO4•3H2O、2.5 g/L KH2PO4 and 10 mL/L trace metal solution. The trace metal solution contains ：4.0 g/L FeSO4•7H2O、4.0 g/L CaCl2、1.0 g/L MnSO4•5H2O、0.4 g/L CoCl2•6H2O、0.2 g/L NaMoO4•2H2O、0.2 g/L ZnSO4•7H2O、0.1 g/L AlCl3•6H2O、0.1 g/L CuCl2•H2O、 0.05 g/L H3BO4. amino acids in the culture medium which are sterilized by filtration.
2. Shake flask fermentation culture:
Collecting glycerol-deposited bacteria at-80deg.C, inoculating loop, picking the deposited bacteria in 100mL small shake flask with 20mL 2YT medium, and shake culturing at 37deg.C and 180r overnight. Transferring seed liquid with an inoculum size of 5% into a 500mL large shake flask with 100mL fermentation medium, culturing at 37 ℃ under the condition of rotating speed of 200r, adding IPTG (concentration of 0.5 mM) for induction when the concentration of the thallus reaches OD600 to 6-8, sampling and centrifugally detecting the ergothioneine content of the culture liquid, purifying the thallus by using water for two times, adding an equal aqueous solution before centrifugation for resuspension and crushing, centrifugally taking the supernatant, detecting the intracellular ergothioneine content by using liquid chromatography, and finally, displaying that the shake flask is fermented for 48 hours, the concentration of the thallus is about 37, the total intracellular and extracellular contents of the ergothioneine is 1.63g/L, wherein the existence of 0.34g/L of the fermentation liquid indicates that the extracellular ratio is 21.4%, and the part of ergothioneine is discharged outside in the fermentation process.
Liquid chromatography analysis:
Liquid chromatography detection: octadecylsilane chemically bonded silica (ODS-BP, 4.6X105 nm, 5 μm) as stationary phase, with purified water as mobile phase, then the mobile phase is adjusted to pH 5.0 with acetic acid, and the detection wavelength is 254nm; the column temperature is 25 ℃; the flow rate of the mobile phase is 0.7mL/min; the recording time of the chromatogram is 20min; the peak time of ergothioneine is 4.696 min, the relative retention time is 1min, the total of the peak areas of ergothioneine and the peak areas of ergothioneine is 73.685% in the effective time of 4-20 min, the theoretical plate number is 10048, the tailing factor is 1.015, and the separation degree is 10.08 which is in the normal range and is consistent with the peak time of the standard sample, as shown in figure 5.
6. Fermentation test in 10L fermenter:
1. Fermentation medium: 20 g/L glucose, 12 g/L yeast powder, 6 g/L peptone 、6 g/L(NH4)2SO4、3g/L L-His、3g/L L-Cys、1g/L L-Glu、12.5 g/L K2HPO4•3H2O、2.5 g/L KH2PO4 and 10 mL/L trace metal solution. The trace metal solution contains ：4.0 g/L FeSO4•7H2O、4.0 g/L CaCl2、 1.0 g/LMnSO4•5H2O、0.4 g/L CoCl2• 6H2O、0.2 g/L NaMoO4•2H2O、 0.2 g/LZnSO4•7H2O、0.1 g/L AlCl3•6H2O、0.1 g/L CuCl2•H2O、0.05 g/L H3BO4.
Feed medium: 750 g/L glucose solution, 10 g/L L-His, 10 g/L L-Cys, 3 g/L L-Glu,300 g/L yeast powder, 150 g/L peptone.
2. Fed-batch fermentation on a 10L fermenter:
Collecting glycerol-deposited bacteria at-80deg.C, inoculating loop, picking the deposited bacteria in 2YT medium with 20 mL in 100 mL hr shake flask, and shake culturing at 37deg.C with 180 r shaker overnight. Transferring seed liquid with an inoculum size of 5% into a 500mL large shake flask with a fermentation medium of 100 mL, culturing for 4h at a temperature of 37 ℃ and a rotating speed of 200r for seed liquid activation, transferring the activated seed liquid with an inoculum size of 3% into a 10L fermentation tank, culturing at a temperature of 37 ℃ and an initial stirring rotating speed of 200rpm, regulating pH to be maintained at 7.0 by ammonia water, controlling the aeration rate to be 1.5 VVM (aeration ratio or aeration rate, aeration ratio=aeration rate (m 3/min)/fermentation liquid volume (m 3)), detecting the concentration of dissolved oxygen by an oxygen dissolving electrode on line, and controlling the concentration of dissolved oxygen to be 25%. When the cell concentration reached an OD600 of 35, induction was performed by adding IPTG. The initial concentration of glucose in the fermentation medium is 20 g/L, the concentration of glucose is controlled to be 10 g/L by adding a feed medium in the fermentation process, sampling and detecting are carried out every 2 h, the total fermentation time is 20 h, the yield of ergothioneine is shown in figure 6, and when 66 h is fermented, the yield of ergothioneine can reach 9.26 g/L.
The base sequence of the promoter P43 is as follows:
gaattcgagctcagcattattgagtggatgattatattccttttgataggtggtatgttttcgcttgaacttttaaatacagccattgaacatacggttgatttaataactgacaaacatcaccctcttgctaaagcggccaaggacgctgccgccggggctgtttgcgtttttgccgtgatttcgtgtatcattggtttacttatttttttgccaaagctgtaatggctgaaaattcttacatttattttacatttttagaaatgggcgtgaaaaaaagcgcgcgattatgtaaaatataaagtgatagcggtaccaggagggctggaagaagcagaccgctaacacagtacataaaaaaggagacatgaac, As shown in SEQ ID NO. 19.
The UPP gene has the accession number CAB15706.1 in the GenBank database of NCBI, and the base sequence is:
atgggaaaggtttatgtatttgatcatcctttaattcagcacaagctgacatatatacggaatgaaaatacaggtacgaaggattttagagagttagtagatgaagtggctacactcatggcatttgaaattacccgcgatcttcctctggaagaagtggatatcaatacaccggttcaggctgcgaaatcgaaagtcatctcagggaaaaaactcggagtggttcctatcctcagagcaggattgggaatggttgacggcattttaaagctgattcctgcggcaaaagtgggacatgtcggcctttaccgtgatccagaaaccttaaaacccgtggaatactatgtcaagcttccttctgatgtggaagagcgtgaattcatcgtggttgacccgatgctcgctacaggcggttccgcagttgaagccattcacagccttaaaaaacgcggtgcgaaaaatatccgtttcatgtgtcttgtagcagcgccggagggtgtggaagaattgcagaagcatcattcggacgttgatatttacattgcggcgctagatgaaaaattaaatgaaaaaggatatattgttccaggtctcggagatgcgggtgaccgcatgtttggaacaaaataa, As shown in SEQ ID No. 20.
The base sequence of EgtABCDE gene cluster is:
atggccttacccgccagaagtgattctggctgtgcggttccggtcgagttcaccagcgcggagcaggccgccgcccacatcggtgccaacagcctgcaggacggtccgatcggccgggtgggcctggagatcgaggcgcactgtttcgacctgagcaatccgacgcggcgaccgagctgggacgaactgtccgccgtgatcgcggacgtaccgccactgcccggtggcagccggatcaccgtggaacccggcggcgcggtcgaactgtccggtccgccgtacgacggtccgctcgccgcggtcgccgcgctgcaggccgaccgtgctgtgctgcgtgcggagttcgcccgcaggaatcttggcctggtgctgctcggcaccgatccgctgcggccgacccgccgggtcaacccgggtgcgcgctactcggccatggaacagttcttcaccgcgagcggtaccgccgaggccggcgccgcgatgatgacggcaaccgcgtcggtgcaggtcaacctggatgccggtccgcgcgacggctgggccgagcgggtccggttggcacatgcgctggggcccacgatgatcgcgatcaccgccaactcaccgatgctgggtggacagttcacgggctggtgttcgacgcggcaacgcgtgtgggggcagctcgactcggcgcggtgcgggccggtgctgggtgtggacggcgacgatcccgcctccgaatgggcgcgctacgcgctgcgcgcgccggtgatgctggtgaactctccggacgcggtgcccgtgaccaactgggtgccgttcgcggactgggccgacgggcgcgccgtgctgggtggacgcagaccgaccgaggccgacctggactatcacctgacgacgctgttcccgccggtgcggccgcgccgctggctggagatccgctatctcgacagcgtgcccgacgcgctgtggccggccgctgtgttcaccctcaccacattgctcgacgatcccgtcgcggccgaaagtgccgctgaggcaacacgtccagtcgccacagcctgggaccgcgccgcgcggatgggcctgacggaccgacatctgcacaccgcggccctgacgtgcgtgcgcctcgcggcggagcgggcaccggccgagctcgaggaatcgatgacgctgttgatgcgttcggtccagcagaggcgtagtcccgccgacgacttctcggaccgggtggtggcacgtggaatcgcagccgccgtccgggaactggcgaaaggtgagctttgatcgcacgcgagacactggccgacgagctggccctggcccgcgaacgcacgttgcggctcgtggagttcgacgacgcggaactgcatcgccagtacaacccgctgatgagcccgctcgtgtgggacctcgcgcacatcgggcagcaggaagaactgtggctgctgcgcgacggcaaccccgaccgccccggcatgctcgcacccgaggtggaccggctttacgacgcgttcgagcactcacgcgccagccgggtcaacctcccgttgctgccgccttcggatgcgcgcgcctactgcgcgacggtgcgggccaaggcgctcgacaccctcgacacgctgcccgaggacgatccgggcttccggttcgcgctggtgatcagccacgagaaccagcacgacgagaccatgctgcaggcactcaacctgcgcgagggcccacccctgctcgacaccggaattcccctgcccgcgggcaggccaggcgtggcaggcacgtcggtgctggtgccgggcggcccgttcgtgctcggggtcgacgcgctgaccgaaccgcactcactggacaacgaacggcccgcccacgtcgtggacatcccgtcgttccggatcggccgcgtgccggtcaccaacgccgaatggcgcgagttcatcgacgacggtggctacgaccaaccgcgctggtggtcgccacgcggctgggcgcaccgccaggaggcgggcctggtggccccgcagttctggaaccccgacggcacccgcacccggttcgggcacatcgaggagatcccgggtgacgaacccgtgcagcacgtgacgttcttcgaagccgaggcctacgcggcgtgggccggtgctcggttgcccaccgagatcgaatgggagaaggcctgcgcgtgggatccggtcgccggtgctcggcgccggttcccctggggctcagcacaacccagcgcggcgctggccaacctcggcggtgacgcacgccgcccggcgccggtcggggcctacccggcgggggcgtcggcctatggcgccgagcagatgctgggcgacgtgtgggagtggacctcctcgccgctgcggccgtggcccggtttcacgccgatgatctacgagcgctacagcacgccgttcttcgagggcaccacatccggtgactaccgcgtgctgcgcggcgggtcatgggccgttgcaccgggaatcctgcggcccagcttccgcaactgggaccacccgatccggcggcagatcttctcgggtgtccgcctggcctgggacgtctgatgtgccggcatgtggcgtggctgggcgcgccgcggtcgttggccgacctggtgctcgacccgccgcagggactgctggtgcagtcctacgcaccgcgacgacagaagcacggtctgatgaacgccgacggttggggcgcagggtttttcgacgacgagggagtggcccgccgctggcgcagcgacaaaccgctgtggggtgatgcgtcgttcgcgtcggtggcacccgcactacgcagtcgttgcgtgctggccgcggtgcgctcggccaccatcggcatgcccatcgaaccgtcggcgtcggcgccgttcagcgacgggcagtggctgctgtcgcacaacggcctggtcgaccgcggggtgctcccgttgaccggtgccgccgagtccacggtggacagcgcgatcgtcgcggcgctcatcttctcccgtggcctcgacgcgctcggcgccaccatcgccgaggtcggcgaactcgacccgaacgcgcggttgaacatcctggccgccaacggttcccggctgctcgccaccacctggggggacacgctgtcggtcctgcaccgccccgacggcgtcgtcctcgcgagcgaaccctacgacgacgatcccggctggtcggacatcccggaccggcacctcgtcgacgtccgcgacgcccacgtcgtcgtgacacccctgtgaaaggacgccccatgacgctctcactggccaactacctggcagccgactcggccgccgaagcactgcgccgtgacgtccgcgcgggcctcaccgcggcaccgaagagtctgccgcccaagtggttctacgacgccgtcggcagtgatctgttcgaccagatcacccggctccccgagtattaccccacccgcaccgaggcgcagatcctgcggacccggtcggcggagatcatcgcggccgcgggtgccgacaccctggtggaactgggcagtggtacgtcggagaaaacccgcatgctgctcgacgccatgcgcgacgccgagttgctgcgccgcttcatcccgttcgacgtcgacgcgggcgtgctgcgctcggccggggcggcaatcggcgcggagtaccccggtatcgagatcgacgcggtatgtggcgatttcgaggaacatctgggcaagatcccgcatgtcggacggcggctcgtggtgttcctggggtcgaccatcggcaacctgacacccgcgccccgcgcggagttcctcagtactctcgcggacacgctgcagccgggcgacagcctgctgctgggcaccgatctggtgaaggacaccggccggttggtgcgcgcgtacgacgacgcggccggcgtcaccgcggcgttcaaccgcaacgtgctggccgtggtgaaccgcgaactgtccgccgatttcgacctcgacgcgttcgagcatgtcgcgaagtggaactccgacgaggaacgcatcgagatgtggttgcgtgcccgcaccgcacagcatgtccgcgtcgcggcactggacctggaggtcgacttcgccgcgggtgaggagatgctcaccgaggtgtcctgcaagttccgtcccgagaacgtcgtcgccgagctggcggaagccggtctgcggcagacgcattggtggaccgatccggccggggatttcgggttgtcgctggcggtgcggtgatgctcgcgcagcagtggcgtgacgcccgtcccaaggttgccgggttgcacctggacagcggggcatgttcgcggcagagcttcgcggtgatcgacgcgaccaccgcacacgcacgccacgaggccgaggtgggtggttatgtggcggccgaggctgcgacgccggcgctcgacgccgggcgggccgcggtcgcgtcgctcatcggttttgcggcgtcggacgtggtgtacaccagcggatccaaccacgccatcgacctgttgctgtcgagctggccggggaagcgcacgctggcctgcctgcccggcgagtacgggccgaatctgtctgccatggcggccaacggtttccaggtgcgtgcgctaccggtcgacgacgacgggcgggtgctggtcgacgaggcgtcgcacgaactgtcggcccatcccgtcgcgctcgtacacctcaccgcattggcaagccatcgcgggatcgcgcaacccgcggcagaactcgtcgaggcctgccacaatgcggggatccccgtggtgatcgacgccgcgcaggcgctggggcatctggactgcaatgtcggggccgacgcggtgtactcatcgtcgcgcaagtggctcgccggcccgcgtggtgtcggggtgctcgcggtgcggcccgaactcgccgagcgtctgcaaccgcggatccccccgtccgactggccaattccgatgagcgtcttggagaagctcgaactaggtgagcacaacgcggcggcgcgtgtgggattctccgtcgcggttggtgagcatctcgcagcagggcccacggcggtgcgcgaacgactcgccgaggtggggcgtctctctcggcaggtgctggcagaggtcgacgggtggcgcgtcgtcgaacccgtcgaccaacccaccgcgatcaccacccttgagtccaccgatggtgccgatcccgcgtcggtgcgctcgtggctgatcgcggagcgtggcatcgtgaccaccgcgtgtgaactcgcgcgggcaccgttcgagatgcgcacgccggtgctgcgaatctcgccgcacgtcgacgtgacggtcgacgaactggagcagttcgccgcagcgttgcgtgaggcgccctga, As shown in SEQ ID NO. 21.
The NcEgt gene has the accession number XP_956324.3 in the GenBank database of NCBI, and the base sequence is:
ATGCCAAGTGCCGAATCAATGACACCTTCAAGTGCGTTAGGCCAATTAAAAGCCACTGGCCAACATGTTTTGTCGAAACTTCAACAGCAGACATCTAACGCCGACATTATAGACATCCGGCGCGTTGCCGTGGAAATCAACCTTAAAACAGAGATTACATCGATGTTTAGACCAAAAGATGGCCCGAGACAACTGCCGACGCTTTTGCTCTATAACGAGCGGGGCCTGCAGCTTTTCGAAAGAATCACATACCTTGAGGAGTATTATTTGACGAACGACGAAATTAAAATACTTACAAAACATGCAACAGAAATGGCCTCCTTTATTCCTAGCGGCGCCATGATTATTGAGCTCGGCTCCGGGAACCTCAGAAAGGTTAATCTGTTATTAGAGGCGTTAGACAACGCAGGTAAGGCGATCGACTATTATGCACTCGACCTCAGCCGAGAAGAATTGGAAAGAACATTGGCTCAGGTGCCATCATACAAGCACGTCAAATGCCACGGCCTCTTAGGTACATACGACGACGGGAGAGACTGGTTAAAAGCACCTGAAAATATCAATAAGCAGAAATGTATCTTACACCTTGGAAGCTCTATAGGCAACTTTAACCGCTCGGATGCAGCTACCTTTCTCAAGGGGTTTACGGACGTCCTTGGTCCTAACGATAAGATGTTAATCGGAGTCGACGCGTGCAATGACCCAGCTAGAGTTTATCATGCGTATAATGATAAAGTAGGCATCACGCATGAGTTTATTCTTAATGGATTAAGAAATGCGAACGAAATCATTGGGGAAACAGCCTTTATCGAAGGCGATTGGCGCGTTATCGGCGAATATGTCTATGATGAGGAAGGTGGACGGCATCAAGCGTTTTATGCCCCTACACGGGACACAATGGTTATGGGTGAATTAATTCGGTCTCATGACCGCATCCAAATCGAACAAAGCCTGAAATACTCGAAGGAGGAATCGGAACGGTTGTGGAGTACAGCTGGCTTGGAACAGGTATCCGAGTGGACATACGGAAATGAATACGGCTTGCATTTGTTGGCCAAAAGCCGCATGTCGTTTTCATTAATCCCGAGCGTATACGCGCGCTCGGCCCTCCCTACCTTAGATGACTGGGAAGCGTTATGGGCAACATGGGATGTGGTGACTCGCCAAATGCTGCCGCAAGAAGAATTGTTGGAAAAACCGATAAAATTGAGAAACGCATGCATTTTCTATTTGGGCCATATTCCGACGTTCCTTGATATCCAGTTGACGAAAACCACGAAACAAGCACCATCAGAACCTGCACATTTTTGCAAAATCTTTGAACGGGGTATTGACCCGGATGTTGACAACCCGGAATTATGCCATGCGCATTCAGAAATTCCAGATGAATGGCCACCGGTCGAGGAAATTTTAACGTACCAAGAGACAGTTCGGTCCAGACTTAGAGGCTTGTATGCACATGGCATTGCAAATATTCCACGTAATGTAGGCCGTGCTATCTGGGTGGGATTCGAACACGAACTCATGCATATTGAAACACTTCTCTACATGATGCTCCAATCAGATAAAACACTGATCCCAACACATATTCCTCGTCCGGACTTTGATAAACTGGCACGAAAAGCGGAGTCTGAAAGAGTCCCAAATCAATGGTTTAAAATCCCGGCACAAGAGATTACAATTGGACTGGACGACCCGGAAGACGGGTCTGATATAAACAAGCACTATGGATGGGATAATGAGAAACCTCCTAGAAGAGTCCAGGTAGCTGCTTTTCAAGCGCAGGGTAGACCTATCACTAATGAAGAATACGCCCAGTATCTGTTGGAAAAGAATATCGATAAACTGCCAGCTAGCTGGGCTCGCCTGGACAACGAAAATATTTCAAATGGTACAACCAATTCCGTATCAGGGCATCACTCAAATCGCACGTCTAAACAACAGTTGCCAAGTTCATTTCTTGAAAAAACGGCCGTTCGGACCGTTTACGGACTCGTGCCATTGAAACATGCTCTGGACTGGCCGGTTTTTGCATCCTACGATGAGTTGGCGGGGTGTGCGGCTTATATGGGTGGTAGAATCCCTACGTTTGAAGAGACCCGGTCAATCTATGCCTATGCCGATGCATTAAAAAAAAAAAAGGAAGCGGAACGTCAATTGGGAAGAACGGTGCCTGCAGTAAACGCTCACCTTACCAATAACGGTGTGGAAATCACCCCACCTAGCTCTCCAAGCTCGGAAACGCCAGCGGAATCAAGCTCACCGTCAGATAGCAATACAACATTGATTACGACTGAAGATCTGTTTTCCGATTTGGATGGGGCTAACGTTGGCTTCCATAATTGGCATCCGATGCCGATCACGTCAAAGGGGAATACATTGGTGGGTCAAGGAGAATTGGGAGGGGTCTGGGAATGGACATCCTCCGTGTTAAGAAAATGGGAAGGATTTGAGCCAATGGAATTGTATCCAGGGTATACAGCGGATTTCTTTGACGAAAAACACAACATAGTTCTTGGCGGCTCATGGGCCACGCACCCACGCATCGCAGGTAGAAAATCATTTGTGAACTGGTACCAACGGAATTACCCTTATGCCTGGGTGGGGGCTAGAGTAGTGCGGGATCTCTGA, As shown in SEQ ID No. 22.
The CmEgt gene has the accession number XP_006666863.1 in the GenBank database of NCBI, and the base sequence is:
ATGACAGTGACAAATCAGACAGATTTACCTACACGCGGAAAATATTTAGAGTTCGGGGGCCAACTTAAAACATTATTTCCACAAGCCCAAGATTGGGTAAATCTCAATCATGGAAGCTATGGCACGATGCCGTTAGTGATTCGCGAAAAGTTTCGTGCATATCAGGACCTCGCCGAGGCAACTCCGGATAAATTCATTCGCTACGACCAAGGTAAACTTATAGATGAAAGCCGCGAGGCAGTGGCTAAATTGGTTAACGCGCCTACTGACACAGTGGTCTTTGTAACGAATGCAACAGAAGCTGTGAATACCGTTTTTCGCAACATGAAATGGAATGAAGACGGGAAGGATGTTATCCTTTTTTTTAGCACAATTTATCCTGCCTGCGCGAAAATTGCCGATTTTATGGTTGACTACTTTGGCAGCCATCGCGTGGGTATTCATGAGATTCCGCTTCATTATCCTCTGGAAGATGAAGATATTATTCAACTTTTCCGGGACGCAGTTGCAGCACTTGAAAAACAAGGCAAACGGGCCCGTATTTGTACATTCGATGTCGTCTCATCGAATCCTGGCCTTGTGTTCCCGTGGGAAGCACTTTGTAAAGCTTGCAAGGAGCTTGACGTCTTGAGCATGGTAGATGGTGCACAGGGAATTGGGATGGTAGAACTGGATCTGGCTGCTGCGGATCCTGACTTCTTCACTTCAAATTGTCACAAGTGGTTACACGTCCCGCGGGGATGCGCTATTTTGTACTGCCCTTTGCGCAATCAAGATATGATCGCAACTCCGCTCTCAACATCACATGGTTATGTGCCGAGAACAGCTGTTCGTCGCGACGTTTTGCCACCGAACACAAAGCCGCCTTTCGTCAATAGATTTGAGCTCGTTGCTACTAAAGATAGATCCCAAGACATCGTGACTAAAGACGCTATCGCATGGAGAAGAGATGTCTGCGGAGGGGAAGCACGCATAATGGCCTATCTTTGGGATCTTAATAAACGGGGCAGCCGACATGTCGCCGCTCGATTGCGTACGGAAGTGTTGGAAAACACGAAAGGCACATTAACGAATTGTGCTATGGCTAATATTGCGTTGCCGATTTGGTTACCAGGAGGTAAAGGCAAAGGGGCAGGAGAACATGAAGAGGACATGGTTGTTCCAGAACATGAGGCAGTACAGGTTCGTCTGTGGATGATGCAGACAATGAAGGATGAATACCACACACTGATGCCGCTGTTCTGGATGGATGATCGCCTGTGGGTACGAACCTCGGCACAAATTTATCTTGACATGAAGGATTATGAGTACGCAGCTAAGGTGTTAGAAGAATTGGCTGCAAGAGTAGCTAATGGAGAATACAAGATTTGA, As shown in SEQ ID NO. 23.
The pdxST gene has the accession number KR821087 in the GenBank database of NCBI.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A recombinant bacillus subtilis is characterized in that bacillus subtilis WB800N is taken as a starting strain, and UPP genes are replaced by genomes containing EgtABCDE gene clusters, so that EgtABCDE gene clusters are overexpressed; over-expression of pdxST gene, ncEgt gene and CmEgt gene is achieved by transferring pdxST gene, ncEgt gene and CmEgt gene;
The UPP gene sequence is shown as SEQ ID NO.20, the EgtABCDE gene cluster sequence is shown as SEQ ID NO.21, the GenBank accession number of pdxST gene is KR821087, the NcEgt1 gene sequence is shown as SEQ ID NO.22, and the CmEgt2 gene sequence is shown as SEQ ID NO. 23;
The genome containing EgtABCDE gene clusters is a genome containing a strong promoter and EgtABCDE gene clusters;
The strong promoter in the genome containing EgtABCDE gene cluster is P43, and the nucleotide sequence is shown as SEQ ID NO. 19.
2. A method of constructing a recombinant bacillus subtilis according to claim 1, comprising the steps of:
Deleting and replacing UPP genes in a bacillus subtilis WB800N genome to a genome containing EgtABCDE gene clusters to obtain a recombinant integrated strain;
And transferring pdxST genes, ncEgt1 genes and CmEgt genes into the recombinant integration strain to obtain the recombinant bacillus subtilis.
3. The method for constructing recombinant bacillus subtilis according to claim 2, wherein the recombinant vector containing upstream and downstream homology arms of UPP and genome containing EgtABCDE gene cluster is transferred into bacillus subtilis WB800N by an electrotransformation method to obtain recombinant integrated strain.
4. The method for constructing a recombinant bacillus subtilis according to claim 3, wherein the construction process of the recombinant vector comprising upstream and downstream homology arms of UPP and genome comprising EgtABCDE gene clusters comprises the following steps:
a. carrying out PCR amplification by using a vector PHT194ts as a template and adopting BEGT-ABCDE-F/BEGT-ABCDE-R primer pairs;
b. Performing PCR amplification by using a bacillus subtilis 168 genome as a template and respectively adopting a UPP-UP-F/UPP-UP-R primer pair and a UPP-DW-F/UPP-DW-R primer pair;
c. PCR amplification is carried out by taking a carrier PWB980 as a template and adopting a P43-F/P43-R primer pair;
d. Performing PCR amplification by using a Mycobacterium smegmatis genome as a template and adopting EgtABCDE-F/EgtABCDE-R primer pairs;
e. Carrying out overlap extension PCR on the amplification product obtained in the step b, the amplification product obtained in the step c and the amplification product obtained in the step d;
f. Ligating the amplified product obtained in the step a and the product obtained in the step e overlapped extension PCR to obtain a recombinant vector containing UPP upstream and downstream homology arms and a genome containing EgtABCDE gene clusters;
the carrier used in the step a is PHT194ts, and the construction steps are as follows:
i1. PCR amplification is carried out by taking a vector pE194ts as a template and adopting an EGT194 ts-F/EGT 194ts-R primer pair;
i2. Carrying out PCR amplification by using a vector PHT101 as a template and BEGT ts-F/BEGT 194ts-R primer pairs;
i3. Adopting a seamless cloning kit to connect PCR products in i1 and i2, converting DH5a competence by the connected products, and sequencing to obtain a final positive vector, which is named PHT194ts; the nucleotide sequences of the primer pair EGT194 ts-F/EGT 194ts-R, BEGT194 ts-F/BEGT ts-R are respectively shown as SEQ ID NO.1, 2, 3 and 4.
5. The method for constructing recombinant bacillus subtilis according to claim 2, wherein pdxST gene, ncEgt gene and CmEgt gene are transferred into the recombinant integrative strain by an electrotransformation method.
6. A method for producing ergothioneine, which is characterized by adopting the recombinant bacillus subtilis of claim 1 for fermentation.
7. The method for producing ergothioneine according to claim 6, wherein the fermentation is fed-batch fermentation, and the initial concentration of glucose is 19-21 g/L; in the fermentation process, the concentration of glucose is controlled to be 9-11 g/L.
8. The method for producing ergothioneine according to claim 6, wherein when the cell concentration reaches an OD600 of 30-40, isopropyl-beta-D-thiogalactoside is added for induction.
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