CN117987338A - Recombinant corynebacterium glutamicum producing ergothioneine, and construction method and application thereof - Google Patents
Recombinant corynebacterium glutamicum producing ergothioneine, and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant corynebacterium glutamicum, which takes corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 (DE 3) as chassis fungus, and introduces a protein coding gene egtB, egtD, egtE related to ergothioneine synthesis into the chassis fungus in a gene cluster form through a vector for heterologous expression, so that the recombinant corynebacterium glutamicum has the capacity of producing ergothioneine, and the corynebacterium glutamicum can not secrete endotoxin in the process of producing the ergothioneine, can be applied in place of escherichia coli, has high biological safety, and greatly simplifies the post-extraction process. On the basis, the synthesis method optimizes the synthesis paths of three precursor amino acids required by ergothioneine synthesis through a synthesis biological means, thereby further improving the yield of the ergothioneine and having higher production and application potential of the ergothioneine.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant corynebacterium glutamicum, a construction method thereof and application of the recombinant corynebacterium glutamicum in preparation of ergothioneine.
Background
Ergothioneine (EGT) is a natural amino acid derivative derived from histidine, is very soluble in water, and is the only natural 2-thioimidazole amino acid currently found. The ergothioneine has a special thioketone structure and a higher reduction potential (-60 mV), and compared with other sulfur-containing antioxidants (such as glutathione and cysteine), the ergothioneine has a higher reduction potential (-200 mV to-320 mV), so that the ergothioneine has better oxidation resistance, can stably exist under physiological pH conditions and in strong alkali solutions, and is not easy to undergo autoxidation. Ergothioneine has the functions of anti-inflammatory, redox balance maintenance, detoxification, radiation resistance, whitening, aging resistance and the like, is a multifunctional cell physiological protective agent, and has wide application prospect.
Currently, the methods for preparing ergothioneine industrially include natural biological extraction, chemical synthesis and microbiological synthesis. The natural biological extraction method mainly extracts from natural mushrooms, but the prepared ergothioneine has higher cost and is difficult to separate and purify due to the extremely low content of the ergothioneine. The chemical synthesis method for synthesizing ergothioneine has the advantages of long synthetic route and low yield, and the raw material 2-mercaptoimidazole is difficult to prepare, thus being extremely easy to cause resource waste. Traditional microbial synthesis methods often use microorganisms that can synthesize ergothioneine themselves, obtained by solid state fermentation, but the methods are long and have low yields. Therefore, in order to increase the yield of ergothioneine production, a strategy for efficiently producing ergothioneine is urgently needed.
The strain and the production method for producing the ergothioneine which are developed at present restrict the industrial production and the large-scale application of the ergothioneine. The host strain for producing ergothioneine is mainly Escherichia coli; however, escherichia coli can secrete endotoxin in the growth process, and the separation process for removing the endotoxin is complicated, which severely limits the application of ergothioneine. In addition, the existing corynebacterium glutamicum for producing ergothioneine still has certain defects, for example MINHYE KIM et al construct a recombinant corynebacterium glutamicum capable of producing ergothioneine, ferment the recombinant corynebacterium glutamicum for 36 hours, only can make the content of ergothioneine in fermentation liquor reach 264.4mg/L, and the yield is too low; TAKASHI HIRASAWA et al introduced different synthetic pathways for ergothioneine in Corynebacterium glutamicum, which resulted in a strain with a yield of ergothioneine of 100mg/L in shake flasks for two weeks, which was still low enough for large-scale production.
Disclosure of Invention
Aiming at the problems of difficult endotoxin separation, low yield, long time consumption and the like in the process of producing ergothioneine by microbial fermentation in the prior art, the invention provides a recombinant corynebacterium glutamicum which has no endotoxin secretion in the process of producing ergothioneine, and optimizes three precursor amino acid synthesis paths required by ergothioneine synthesis by a synthesis biological means, thereby further improving the yield of ergothioneine and having higher production and application potential of ergothioneine.
In order to achieve the above object, the present invention provides a recombinant corynebacterium glutamicum, which comprises the following steps: the corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 (DE 3) was used as the Chassis bacteria, and the egtD gene and the egtE gene derived from Mycolicibacterium smegmatis and the egtB gene derived from Methylobacteriumpseudosasicola were expressed in a heterologous manner, and/or thrB gene in the genome of the Chaetoceros bacteria was knocked out.
The invention also provides a construction method of the recombinant corynebacterium glutamicum, which comprises the following steps: the corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 (DE 3) was used as the Chassis bacteria, and the egtD gene and the egtE gene derived from Mycolicibacteriumsmegmatis and the egtB gene derived from Methylobacteriumpseudosasicola were expressed in a heterologous manner, and/or thrB gene in the genome of the Chaetoceros bacteria was knocked out. As food-safe microorganisms, corynebacterium glutamicum is widely used in industrialized amino acid production, such as glutamic acid, lysine. Compared with the escherichia coli, the corynebacterium glutamicum can not secrete endotoxin, and products produced by using the corynebacterium glutamicum have wider application markets. In order to enhance the expression of the ergothioneine by the corynebacterium glutamicum, the invention introduces a protein coding gene egtB, egtD, egtE related to the synthesis of the ergothioneine into corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 (DE 3) in the form of a gene cluster through a vector for heterologous expression. Wherein, the egtD and egtE genes from Mycolicibacterium smegmatis respectively code for synthesizing SAM-dependent histidine methyltransferase and PLP-combined C-S lyase, and the egtB gene from Methylobacteriumpseudosasicola codes for synthesizing mononuclear non-heme iron enzyme. The egtB, egtD, egtE genes are chemically synthesized to form the ergothioneine synthesis gene cluster egtDBE, and codon optimization is performed for the Corynebacterium glutamicum expression system. The gene cluster egtDBE with the optimized base sequence shown as SEQ ID NO.1 is introduced into chassis bacteria through a vector for overexpression. Further, in order to enhance the accumulation of S-adenosylmethionine, a thrB gene in the genome of Chaetoceros is knocked out, and the base sequence of the thrB gene is preferably shown as SEQ ID NO. 6.
Preferably, the construction method further comprises the following steps: enhancing the expression of a precursor of ergothioneine in the Chassis fungus, wherein the precursor of ergothioneine is at least one selected from cysteine, S-adenosylmethionine and histidine. Three main precursors are cysteine, S-adenosylmethionine and histidine during the synthesis of ergothioneine. Thus, in order to enhance the accumulation of three amino acid precursors, the present invention optimizes the metabolic pathway of Corynebacterium glutamicum. For histidine metabolism, the invention selects the mutant hisG Y56M-T235P and/or hisE gene of the hisG gene from corynebacterium glutamicum to be over-expressed. The present invention also selected overexpression of metK gene derived from Corynebacterium glutamicum for S-adenosylmethionine metabolism. For cysteine metabolism, the invention selects the mutant gene cysE M201R from colibacillus or cysE gene from corynebacterium glutamicum and truncated serA gene serA △197 to be over-expressed. The metabolic flow of the precursor of the ergothioneine is increased by modifying the metabolic pathways of the three precursors, and the yield of the ergothioneine is further improved.
Specifically, the method for enhancing the expression of cysteine in the chassis bacteria comprises the following steps: at least one of cysE M201R gene, serA △197 gene, cysE gene is overexpressed in Chassis bacteria. Wherein the cysE M201R gene is derived from ESCHERICHIA COLI, and the base sequence of the cysE M201R gene is preferably shown as SEQ ID NO. 2; the serA △197 gene is derived from Corynebacterium glutamicum, and the base sequence of the serA △197 gene is preferably shown as SEQ ID NO. 3; the cysE gene is derived from Corynebacterium glutamicum, and the base sequence of the cysE gene is preferably shown in SEQ ID NO. 4.
Specifically, the method for enhancing the expression of S-adenosylmethionine in chassis bacteria comprises the following steps: the metK gene is overexpressed in Chaetomium. Wherein, the metK gene is derived from Corynebacterium glutamicum, and the base sequence is preferably shown in SEQ ID NO. 5.
Specifically, the method for enhancing histidine expression in chassis bacteria comprises the following steps: the hisG Y56M-T235P gene, and/or the hisE gene are overexpressed in Chaetomium. Wherein, the hisG Y56M-T235P gene and the hisE gene are derived from Corynebacterium glutamicum, and the base sequences of the hisG Y56M-T235P gene and the hisE gene are respectively preferably shown as SEQ ID NO.7 and SEQ ID NO. 8.
Preferably, the construction method further comprises the steps of: the egtD gene, egtE gene, egtB gene were ligated into the same over-expression vector. In order to further optimize the expression of the ergothioneine by the corynebacterium glutamicum, the invention forms an ergothioneine synthesis gene cluster egtDBE by a chemical synthesis mode through a protein coding gene egtB, egtD, egtE related to the synthesis of the ergothioneine, and carries out codon optimization on an expression system of the corynebacterium glutamicum. The gene cluster egtDBE with the optimized base sequence shown as SEQ ID NO.1 is introduced into chassis bacteria through a vector for overexpression.
The invention also provides application of the recombinant corynebacterium glutamicum or the recombinant corynebacterium glutamicum constructed by the method in preparation of ergothioneine, products containing the ergothioneine and ergothioneine derivatives. Preferably, the application includes: taking a raw material containing fermentable sugar as a substrate, and carrying out fermentation culture on the recombinant corynebacterium glutamicum to prepare ergothioneine; wherein, the fermentation culture is preferably carried out under the conditions of 25-30 ℃ and pH of 6.8-7.0,0.5-1 mM IPTG. The inducer IPTG is preferably added during fermentation for 5-8 h. In order to further increase the yield of ergothioneine, the recombinant corynebacterium glutamicum is preferably subjected to fed-batch fermentation, i.e., a carbon source and a nitrogen source are fed in the fermentation process. Finally, under certain culture conditions, the yield of the ergothioneine of the recombinant corynebacterium glutamicum after fermentation can reach 355.4mg/L.
Preferably, the fermentable sugar is at least one selected from molasses, glucose, corn steep liquor, mannose and glycerol. The recombinant corynebacterium glutamicum designed by the invention can utilize cheap raw materials such as glucose, molasses, corn steep liquor and the like as substrates, and can continuously ferment and produce ergothioneine under the condition of low salt, thereby greatly reducing the cost of the raw materials, simplifying the fermentation process and being beneficial to the industrial production and large-scale application of the ergothioneine.
The term "enhancement" in the present invention means that the activity of an enzyme encoded by a corresponding polynucleotide is increased by over-expression of a gene or substitution of an expression regulatory sequence (promoter substitution, etc.) of the gene on the genome.
The vector used in the present invention may not be particularly limited as long as the vector is replicable in a host, and any vector known in the art may be used.
The invention has the beneficial effects that: according to the invention, the protein coding gene egtB, egtD, egtE related to ergothioneine synthesis is introduced into the corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 (DE 3) of the chassis fungus through a vector in the form of gene cluster for heterologous expression, so that the corynebacterium glutamicum has the capacity of producing ergothioneine, and the corynebacterium glutamicum can not secrete endotoxin in the process of producing the ergothioneine, can replace escherichia coli for application, has high biological safety, and greatly simplifies the post-extraction process. In order to further increase the yield of ergothioneine, the invention also modifies the metabolism of histidine, S-adenosylmethionine and cysteine in chassis bacteria, and increases the metabolic flow of ergothioneine precursors. Wherein, by strengthening the expression of S-adenosylmethionine in chassis bacteria, the yield of the recombinant corynebacterium glutamicum ergothioneine is obviously improved and 355.4mg/L is reached. In addition, the recombinant corynebacterium glutamicum designed by the invention can utilize cheap raw materials such as molasses, glucose, corn steep liquor and the like to ferment, so that the cost of the raw materials is reduced, the fermentation process is simplified, and the industrial production and large-scale application of ergothioneine are facilitated.
Drawings
FIG. 1 is a map of recombinant expression plasmid pDXW-egtDBE-gene of example 1 of the present invention.
FIG. 2 is a graph showing the content of ergothioneine in fermentation broths obtained by fermentation of different recombinant Corynebacterium glutamicum under shake flask conditions in examples 1 to 3 of the present invention.
FIG. 3 is a diagram showing the growth of fed-batch fermentation cells in a 3L fermenter of recombinant Corynebacterium glutamicum Egt1, egt4, egt11 according to example 5 of the present invention.
FIG. 4 is a graph showing the yield of ergothioneine from a fed-batch fermentation in a 3L fermenter of recombinant Corynebacterium glutamicum Egt1, egt4, egt11 according to example 5 of the present invention.
FIG. 5 shows the results of liquid phase detection of ergothioneine.
FIG. 6 is a full scale of strains corresponding to recombinant Corynebacterium glutamicum.
Detailed Description
The following specific examples are presented to illustrate the present invention, and those skilled in the art will readily appreciate the additional advantages and capabilities of the present invention as disclosed herein. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be noted that the following embodiments and features in the embodiments may be combined with each other without conflict. The methods used in the examples of the present invention are conventional methods, and the reagents used are commercially available.
Bacterial strain and plasmid
Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 (DE 3) referred to in the examples below was deposited by the laboratory; coli (ESCHERICHIA COLI) DH 5. Alpha. Was purchased from the company Shanghai Co., ltd; pJYS3 plasmid was purchased from Bao-organism; the pDXW-T7-recT cgl plasmid was given by university of Jiangnan; the artificially synthesized egtDE, egtB genes from ergothioneine original production strains Mycobacterium smegmatis, methylobacteriumpseudosasicola, respectively, were purchased from the biotechnology company, san francisco.
(II) Medium
LB liquid medium: 10g/L NaCl, 10g/L peptone, 5g/L corn steep liquor.
LB liquid medium: 10g/L NaCl, 10g/L peptone, 5g/L corn steep liquor and 18g/L agar powder.
Shake flask medium: glucose 60g/L, corn steep liquor 15g/L, yeast powder 10g/L,(NH4)2SO430 g/L,KH2PO46g/L,Na2HPO43 g/L,NaCl 1g/L,Na2S2O35 g/L,0.1mM CaCl2.
3L fermenter Medium: glucose 100g/L, corn steep liquor 15g/L, yeast powder 10g/L,(NH4)2SO430 g/L,KH2PO46 g/L,Na2HPO43 g/L,NaCl 1g/L,Na2S2O35 g/L,1mL trace elements.
(III) detection method
High Performance Liquid Chromatography (HPLC) detection method: agilent 1200, uv detector, NH2 column (250×4.6mm,5 μm), mobile phase ratio: water acetonitrile=7:3, flow rate 1mL/min, column temperature 30 ℃, uv absorption: 254nm, sample injection volume of 5 μl, detection duration: 20min. The results of the liquid phase detection of ergothioneine are shown in FIG. 4.
Example 1: construction of recombinant Corynebacterium glutamicum Egt1
Chemically synthesizing (Jin Weizhi company) ergothioneine synthesis gene cluster egtDBE, and carrying out PCR (polymerase chain reaction) amplification on gene egtDBE by taking egtDBE-F, egtDBE-R as a primer to obtain an amplified fragment of egtDBE; performing inverse PCR amplification on plasmid PDXW-T7-recTcgl through a primer pDXW-F, pDXW-R to obtain a vector linear fragment, connecting the amplified fragment with the linear vector by adopting a one-step cloning method, and converting the amplified fragment into DH5 alpha competence of escherichia coli; coating the transformation product on LB solid medium containing 50 mug.mL -1 kanamycin, and inversely culturing for 12 hours in a constant temperature incubator at 37 ℃ to obtain a transformant; and selecting the transformant for colony PCR verification, inoculating the transformant with correct verification into LB liquid culture medium, culturing overnight, extracting plasmid for further verification after sequencing, and obtaining recombinant plasmid pDXW-egtDBE after correct verification.
The recombinant plasmid pDXW-egtDBE was electrotransformed into the competence of Corynebacterium glutamicum ATCC13032 (DE 3), the transformed product was spread on BHI solid medium containing 50. Mu.g/mL kanamycin, and cultured upside down in a 30℃incubator for 24 hours to obtain a transformant, namely Corynebacterium glutamicum Egt1.
TABLE 1 example 1 primer Table
Modification of the ergothioneine precursor synthesis pathway
Example 2: construction of the over-expression plasmid
The following gene fragments were subjected to de-amplification with the corresponding primers (Table 2), inverse PCR was performed on the plasmid pDXW-egtDBE prepared in example 1 using the primers to obtain vector linear fragments, one or more of the amplified fragments metK (base sequence shown as SEQ ID NO. 5), hisG Y56M-T235P (base sequence shown as SEQ ID NO. 7), serA △197 (base sequence shown as SEQ ID NO. 3), cysE M201R (base sequence shown as SEQ ID NO. 2), hisE (base sequence shown as SEQ ID NO. 8), cysE (base sequence shown as SEQ ID NO. 4) were ligated with the resulting linearized pDXW-egtDBE vector to obtain recombinant plasmids, respectively, and the recombinant plasmids were transformed into E.coli DH 5. Alpha. Competent cells, respectively; coating the transformation product on LB solid medium containing 50 mug/mL streptomycin, and inversely culturing for 12 hours in a constant temperature incubator at 37 ℃ to obtain a transformant; and selecting the transformant for colony PCR verification, inoculating the transformant with correct verification into LB liquid culture medium, culturing overnight, extracting plasmid for further verification after sequencing, and obtaining relevant recombinant plasmid after correct verification.
The obtained recombinant plasmids pDXW-egtDBE-cysEM201R、pDXW-egtDBE-serA△197、pDXW-egtDBE-metK、pDXW-egtDBE-hisGY56M-T235P、pDXW-egtDBE-cysE、pDXW-egtDBE-hisE were transformed into the competence of Corynebacterium glutamicum ATCC13032 (DE 3), respectively, the transformed products were spread on a solid medium containing 50. Mu.g/mL kanamycin BHI, and were cultured upside down in a constant temperature incubator at 30℃for 24 hours to obtain transformants, namely, recombinant Corynebacterium glutamicum Egt2, egt3, egt4, egt5, egt6 and Egt7.
TABLE 2 example 2 primer tables
Example 3: construction of CRISPR/Cpf1 Gene knockout vector
The crRNA is designed by taking Corynebacterium glutamicum ATCC13032 as a template, and the specific operation method is that a target gene nucleotide sequence is input on a crRNA special design website (http:// crispor.tefor.net /), a Corynebacterium glutamicum genome is selected, 21p-TTTN-Cpf1 is selected as a PAM site for analysis, and a crRNA sequence with low off-target rate is selected. The crRNA is reversely amplified by designing corresponding primers (Table 3) and taking pJYS-delta crtYf plasmid as a template to replace the original crRNA sequence with the crRNA sequence of thrB gene (the base sequence is shown as SEQ ID NO. 6).
And querying the upstream and downstream gene sequences of the knocked-out genes through NCBI, and designing corresponding upstream and downstream homology arms. The upstream and downstream homology arm templates were obtained by designing primers for amplification using Corynebacterium glutamicum ATCC13032 genomic DNA as a template, and the primers used are shown in Table 3.
Carrying out reverse amplification by taking pJYS-delta crtYf plasmid as a template to obtain a pJYS vector, connecting corresponding gene fragments to pJYS plasmid according to the sequence of thrB1-thrB2-crRNA by a one-step cloning method, and constructing to obtain a pJYS-delta thrB knockout vector.
The pJYS-DeltatherB knockout vector thus obtained was introduced into Corynebacterium glutamicum ATCC13032 (DE 3), competent cells were prepared after successful gene knockout, and the expression plasmids pDXW-egtDBE、pDXW-egtDBE-cysEM201R、pDXW-egtDBE-serA△197、pDXW-egtDBE-metK、pDXW-egtDBE-hisGY56M-T235P、pDXW-egtDBE-cysE and pDXW-egtDBE-hisE prepared in example 1 and example 2 were electrically transformed into competent cells to obtain transformants, namely, recombinant Corynebacterium glutamicum abbreviated as Egt8, egt9, egt10, egt11, egt12, egt13 and Egt14.
TABLE 3 example 3 primer tables
| crRNA-thrB-F | TCCACGACACCACTGCTCCACATTTAAATAAAACGAAAGGCTCAGTCGAAAG |
| crRNA-thrB-F | GTGGAGCAGTGGTGTCGTGGAATCTACAACAGTAGAAATTCGGATCCAT |
| ThrB-upstream-F | GAACAACTGTTCACCGGGCCGCAATTCCAGTGGTTGGCCC |
| ThrB-upstream-R | GTTGGGCAGTGTCAAAGCCAGGTCCGAGGTTTGCAG |
| ThrB-downstream-F | TGGCTTTGACACTGCCCAACAAGGAAGGCCCCC |
| ThrB-downstream-R | CTGAGCTAGCTGTCAATCTAGCGCCCCTACGTGGTCTATCG |
| pJYS3-F | CTAGATTGACAGCTAGCTCAGTCCTAG |
| pJYS3-R | GGCCCGGTGAACAGTTGTTCTAC |
Example 4: shake flask culture of recombinant corynebacterium glutamicum
All recombinant corynebacterium glutamicum obtained in examples 1 to 3 were subjected to shake flask culture, and the shake flask medium was: glucose 60g/L, corn steep liquor 15g/L, yeast powder 10g/L,(NH4)2SO430 g/L,KH2PO46g/L,Na2HPO43 g/L,NaCl 1g/L,Na2S2O35 g/L,0.1mM CaCl2. shake flask culture conditions are: the strain cultured for 18 hours was added to the fermentation system in an amount of 5mL/100mL of the medium, cultured at 30℃for 6 hours, and then subjected to induction culture by adding 0.5mM IPTG. After 72h of incubation, the intracellular and extracellular products were collected and tested for ergothioneine content, as shown in FIG. 2. As shown in FIG. 2, the expression of S-adenosylmethionine in Chassis bacteria can be further improved by over-expressing metK gene and knocking out delta thrB gene, and the synthesis capacity of recombinant Corynebacterium glutamicum on ergothioneine can be further improved, wherein the yield of the ergothioneine of recombinant Corynebacterium glutamicum Egt1, egt4 and Egt11 can reach 23.3mg/L, 34.7mg/L and 60.3mg/L respectively in the shake flask culture stage.
Example 5: 3L fermenter fed-batch fermentation culture of recombinant Corynebacterium glutamicum
The recombinant Corynebacterium glutamicum Egt1, egt4 and Egt11 constructed in examples 1 to 3 were subjected to fed-batch fermentation in a 3L fermenter.
The amount of liquid in the 3L fermenter was 1L, and recombinant Corynebacterium glutamicum Egt1, egt4, egt11, which had been cultured for 16 hours, was added to the fermentation system in an amount of 100mL/1000mL of the medium. 3L fermenter Medium: glucose 100g/L, corn steep liquor 15g/L, yeast powder 10g/L,(NH4)2SO430 g/L,KH2PO46 g/L,Na2HPO43 g/L,NaCl 1g/L,Na2S2O35g/L,1mL trace elements. The culture conditions are as follows: the culture was performed at 30℃with ventilation of 1-5vvm, 30% dissolved oxygen, pH 6.0-7.0 for 6 hours, and 0.5mM IPTG was added for induction culture. During the culture, ammonia water is used to regulate pH to 6.0-7.0, and during the culture, bacteria growth, glucose consumption and ergothioneine content are measured. In addition, when the glucose concentration during fermentation was lower than about 10g/L, glucose was fed so as to be maintained at about 10g/L, and at the same time 15g/L sodium thiosulfate and 100g/L ammonium sulfate were fed for 24 hours at 10 mL/h. The output diagram of the recombinant corynebacterium glutamicum Egt1, egt4 and Egt11 after 48h fermentation is shown in FIG. 4, and the highest output obtained in 36h is 230.9mg/L, 355.4mg/L and 85.7mg/L respectively. In addition, the 3L fermentation tank culture medium adopts cheap raw materials of glucose and corn steep liquor as carbon sources, the strain grows rapidly in 20h of culture, the OD600 reaches 200, and the ergothioneine is continuously fermented and produced under the condition of low salt, so that the raw material cost is greatly reduced, the fermentation process is simplified, and the industrial production and large-scale application of the ergothioneine are facilitated.
The above examples are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The recombinant corynebacterium glutamicum is characterized in that the construction method comprises the following steps: the corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 (DE 3) was used as the Chassis bacteria, and the egtD gene and the egtE gene derived from Mycolicibacterium smegmatis and the egtB gene derived from Methylobacterium pseudosasicola were expressed in a heterologous manner, and/or thrB gene in the genome of the Chaetoceros bacteria was knocked out.
2. The method for constructing recombinant corynebacterium glutamicum according to claim 1, comprising the steps of: the corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 (DE 3) was used as the Chassis bacteria, and the egtD gene and the egtE gene derived from Mycolicibacterium smegmatis and the egtB gene derived from Methylobacterium pseudosasicola were expressed in a heterologous manner, and/or thrB gene in the genome of the Chaetoceros bacteria was knocked out.
3. The method of claim 2, further comprising the step of: enhancing the expression of a precursor of ergothioneine synthesis in Chaetomium, said precursor of ergothioneine synthesis being selected from at least one of cysteine, S-adenosylmethionine, histidine.
4. The method of claim 3, wherein the method of enhancing cysteine expression in the chassis bacteria is: at least one of cysE M201R gene, serA Δ197 gene, cysE gene is overexpressed in Chassis bacteria.
5. The method of claim 3, wherein the method of enhancing expression of S-adenosylmethionine in a chassis bacterium is: the metK gene is overexpressed in Chaetomium.
6. The method of claim 3, wherein the method of enhancing histidine expression in the chassis fungus is: the hisG Y56M-T235P gene, and/or the hisE gene are overexpressed in Chaetomium.
7. The method of claim 2, wherein the construction method further comprises the steps of: the egtD gene, egtE gene, egtB gene were ligated into the same over-expression vector.
8. Use of a recombinant corynebacterium glutamicum according to claim 1 or constructed by a method according to any one of claims 2 to 7 for the preparation of ergothioneine, products containing ergothioneine, derivatives of ergothioneine.
9. The application of claim 8, wherein the application comprises: and (3) taking a raw material containing fermentable sugar as a substrate, and carrying out fermentation culture on the recombinant corynebacterium glutamicum to prepare the ergothioneine.
10. The use according to claim 9, wherein the fermentable sugar is selected from at least one of molasses, glucose, corn steep liquor, mannose, glycerol.
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