Application of transcriptional regulator Cgl2415 in improving ergothioneine production in Corynebacterium glutamicum and method for preparing ergothioneine

Abstract

The present invention belongs to the field of biotechnology and its applications, and discloses an application of a transcriptional regulator Cgl2415 in improving the production of ergothioneine in Corynebacterium glutamicum and a method for preparing ergothioneine. The present invention enhances the expression of the transcriptional regulator Cgl2415 , and the enhancement methods include plasmid overexpression, increasing the genomic expression copy number, replacing the strong regulatory element, etc., so as to obtain an increase in the production of ergothioneine. The nucleotide sequence of the transcriptional regulator Cgl2415 is as shown in SEQ ID NO. 2. The method for improving the target product ergothioneine by changing the expression level of the transcriptional regulator of the present invention has guiding significance for the metabolic engineering synthesis and transformation of industrial strains.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and its applications, and particularly relates to a method for enhancing the production of ergothioneine by enhancing the expression of a transcriptional regulator in Corynebacterium glutamicum Cgl2415 Background Art Background Technology

[0002] Ergothioneine was first isolated and extracted by French pharmacist Tanret from ergot fungi ( Claviceps purpurea ) in 1909, and was subsequently found in certain tissues and organs of mammals, cereal plants, and some fungi and bacteria. Subsequent studies have shown that only some bacterial genera such as various fungi, Methylobacterium, Actinomycetes, Mycobacterium, and strains such as cyanobacteria have the ability to synthesize ergothioneine, while mammals do not have the ability to synthesize it themselves and need to utilize the ergothioneine transporter OCTN1 (organic cation transporter N1, SLC22A4 the product of the gene) present in different tissues to absorb ergothioneine. Ergothioneine is a powerful scavenger of reactive oxygen species (ROS) and also an inhibitor of lipid peroxidation. Due to its non-toxic and safe natural antioxidant properties, it is widely used in the fields of skin care products, food, and biology and medicine.

[0003] Currently, the production of ergothioneine mainly relies on natural biological extraction methods, chemical synthesis methods, and biosynthesis methods. Among them, the natural extraction method has problems such as low yield and many impurities, which limit its industrial application; the chemical synthesis method is difficult to achieve large-scale production due to expensive raw materials and difficult-to-guarantee safety. In recent years, the microbial fermentation method based on metabolic engineering and synthetic biology technologies has become a research hotspot. This method has the advantages of low cost, easy availability of raw materials, and environmental friendliness by constructing engineering strains with high ergothioneine production. Existing research mainly focuses on the modification of key enzyme genes in the ergothioneine synthesis pathway and the precursor supply pathway, and the role of transcriptional regulators has not been deeply explored. Transcriptional regulators play a core role in the cell metabolic network and can optimize the distribution of metabolic flux and resource allocation by coordinating the expression of multiple genes, thereby significantly improving the synthesis efficiency of target products. Therefore, in-depth study of the impact of transcriptional regulators on ergothioneine biosynthesis not only helps to reveal its metabolic regulation mechanism, but also may discover new efficient modification targets, providing new ideas for the industrial production of ergothioneine.

[0004] Corynebacterium glutamicum ( Corynebacterium glutamicum ) is an important industrial microorganism and is widely used in the production of chemicals such as amino acids and organic acids and biological materials. Its food safety grade characteristics make it an ideal production strain in the fields of food, cosmetics, etc., and it is also a potential high-quality chassis cell for ergothioneine biosynthesis. Based on its mature genetic operation system and high safety, Corynebacterium glutamicum has strong application potential in the industrial production of ergothioneine. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a method for enhancing the expression of a transcriptional regulatory factor in Corynebacterium glutamicum to increase the production of ergothioneine. Cgl2415 to increase the production of ergothioneine.

[0006] In the specific embodiments, the enhancement of the expression of the transcriptional regulatory factor in Corynebacterium glutamicum Cgl2415 is achieved by plasmid overexpression, increasing the genomic expression copy number, replacing the enhancer element, etc.

[0007] The technical solution of the present invention is outlined as follows:

[0008] The present invention first provides the use of a transcriptional regulatory factor Cgl2415 in increasing the production of ergothioneine in Corynebacterium glutamicum.

[0009] Specifically, it is to enhance the expression of the transcriptional regulatory factor in Corynebacterium glutamicum Cgl2415 to achieve an increase in the production of ergothioneine.

[0010] The present invention also provides a method for increasing the production of ergothioneine by Corynebacterium glutamicum, which enhances the expression of the transcriptional regulatory factor Cgl2415 in Corynebacterium glutamicum capable of synthesizing ergothioneine to obtain a recombinant Corynebacterium glutamicum with increased ergothioneine production.

[0011] Specifically, the enhancement of the expression of the transcriptional regulatory factor Cgl2415 is achieved by plasmid overexpression, increasing the genomic expression copy number, and replacing the enhancer element.

[0012] Preferably, the amino acid sequence of the transcriptional regulatory factor Cgl2415 is as shown in SEQ ID NO.1.

[0013] More preferably, the coding nucleotide sequence of the transcriptional regulatory factor Cgl2415 is as shown in SEQ ID NO.2.

[0014] In the specific embodiments, plasmid overexpression is achieved by constructing an expression vector with the coding nucleotide sequence of the transcriptional regulatory factor Cgl2415 and transforming Corynebacterium glutamicum capable of synthesizing ergothioneine to obtain a recombinant bacterium, and culturing the recombinant bacterium to produce ergothioneine.

[0015] Optionally, it further includes the step of isolating the produced ergothioneine.

[0016] Among them, the Corynebacterium glutamicum capable of synthesizing ergothioneine is obtained by introducing the key enzymes L-histidine-N-α-trimethyltransferase / heptylcysteine sulfoxide-S-oxidase gene (such as e gt1 gene) and heptylcysteine sulfoxide-S-oxidative lyase gene (such as the egt2 gene derived from fungi; or the egtE gene derived from bacteria) into the starting strain of Corynebacterium glutamicum.

[0017] Preferably, the key enzymes L-histidine-N-α-trimethyltransferase / heptylcysteine sulfoxide-S-oxidase gene and heptylcysteine sulfoxide-S-oxidative lyase gene in the ergothioneine synthesis pathway are codon-optimized according to Corynebacterium glutamicum.

[0018] The advantage of the present invention is that the overexpression of the transcriptional regulator Cgl2415 in Corynebacterium glutamicum increases the yield of ergothioneine in the engineered strain GW8 of Corynebacterium glutamicum, and it is verified in the experiment that the yield can be increased by 2.9 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the map of the pXMJ19-Egt1-EgtE expression vector.

[0020] Figure 2 is the map of the pEC- Cgl2415 overexpression.

[0021] Figure 3 is the fermentation schematic diagram of strain GWJ5 under microplate conditions.

[0022] Figure 4 is the fermentation schematic diagram of strain GWJ5 under shake flask conditions.

[0023] Figure 5 is the peak map of the ergothioneine standard.

[0024] Figure 6 is the result peak map of the total amount of ergothioneine after sampling and treatment during the 60-hour shake flask fermentation of GWJ1.

[0025] Figure 7 is the result peak map of the total amount of ergothioneine after sampling and treatment during the 60-hour shake flask fermentation of GWJ5. DETAILED DESCRIPTION OF THE INVENTION

[0026] The following examples are used to further illustrate the present invention. The following examples are intended to enable those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

[0027] The original strain used in the present invention Corynebacterium glutamicumATCC 13032 is from the laboratory.

[0028] In this example, the recombinant plasmid expression plasmids pXMJ19 and pECXK99E were purchased from BioVector NTCC Company (http: / / www.biovector.net / ).

[0029] The ergothioneine standard was purchased from sigma company (http : / / www.sigmaaldrich.com / sigmaaldrich).

[0030] Molecular biology reagents such as restriction endonucleases, dephosphorylases, and DNA ligases used were purchased from thermo company (http: / / www.thermoscientificbio.com / fermentas), and other biochemical reagents used were purchased from Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0031] LB medium: Weigh 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and make up the volume to 1 L with distilled water. 2% agar powder needs to be added to the LB solid medium, and autoclave at 121 °C for 20 min.

[0032] BHIS medium: Weigh 18.5 g of bovine brain heart infusion powder and 91 g of sorbitol, and make up the volume to 1 L with distilled water. Autoclave at 121 °C for 20 min.

[0033] LBHIS medium: Weigh 5 g of tryptone, 10 g of NaCl, 2.5 g of yeast extract, 18.5 g of bovine brain heart infusion powder, and 91 g of sorbitol, and make up the volume to 1 L with distilled water. 2% agar powder needs to be added to the BHI solid medium, and autoclave at 121 °C for 20 min.

[0034] CGXⅡA medium: Weigh 5 g of yeast extract, 20 g of (NH4)2SO4, 5 g of Urea, 1 g of KH2PO4, 1 g of K2HPO4, 0.25 g of MgSO4·7H2O, 0.01 g of CaCl2, 21 g of MOPS, adjust the pH to 7.0, and make up the volume to 1 L with distilled water. Autoclave at 121 °C for 20 min. Before use, add biotin stock solution and trace element stock solution according to 1 / 1000 volume.

[0035] Example 1: Construction of Corynebacterium glutamicum GW8

[0036] Corynebacterium glutamicum itself does not contain the key enzymes L-histidine-N-α-trimethyltransferase / heptylcysteine sulfoxide-S-oxidase (Egt1, encoded by the e gt1 gene) and heptylcysteine sulfoxide-S-oxidative lyase (Egt2, a gene from a fungal source egt2 ; or EgtE, encoded by a gene from a bacterial source egtE ), and exogenous genes need to be introduced to synthesize ergothioneine. The present invention constructs a plasmid basic vector pXMJ19-Egt1-EgtE capable of synthesizing ergothioneine. Based on the amino acid sequence of Egt1 (NCBI-ProteinID: XP_956324) (SEQ ID No. 3) reported in Neurospora crassa ( Neurospora crassa ), the encoding gene of Egt1 was optimized according to the codon preference of Escherichia coli to obtain the optimized egt1 gene (SEQ ID No. 4), which was sent to GenScript Biotech Corporation (Beijing) for synthesis and ligated with the 5'-terminal flanking sequence "GAAAGGAGGCCCTTCAG" between the restriction enzyme sites PstI and XbaI of the pXMJ19 vector to construct the pX-Egt1 plasmid. Then, based on the amino acid sequence of EgtE (NCBI-ProteinID: ABK70212) (SEQ ID No. 5) reported in Mycobacterium smegmatis ( Mycobacterium smegmatis ), the encoding gene of EgtE was optimized according to the codon preference of Escherichia coli to obtain the optimized egtE gene (SEQ ID No. 6), which was sent to GenScript Biotech Corporation (Beijing) for synthesis and ligated with the 5'-terminal flanking sequence "AAAGGAGGACAACC" between the restriction enzyme sites XmaI and SacI of the pXMJ19-Egt1 plasmid to construct the pXMJ19-Egt1-EgtE plasmid. The final map of the pXMJ19-Egt1-EgtE plasmid is as shown in Figure 1 .

[0037] The structure of the recombinant expression vector pXMJ19-Egt1-EgtE is described as: the optimized egt1 gene (SEQ ID No.4) was inserted between the restriction enzyme sites PstI and XbaI of the pXMJ19 vector, and at the same time the optimized egtE gene (SEQ ID No. 6) was inserted between the restriction enzyme sites XmaI and SacI to obtain the recombinant vector.

[0038] The pXMJ19-Egt1-EgtE was introduced into the Corynebacterium glutamicum ATCC 13032 strain by electroporation to obtain the recombinant strain GW8.

[0039] Example 2: Obtaining of regulatory factor modification targets

[0040] Based on the reported high-quality genome-scale metabolic network model of Corynebacterium glutamicum i CW773 (PMID: 28680478), after adding the heterologous reaction information for ergothioneine synthesis and integrating the regulatory information of Corynebacterium glutamicum in the CoryneRegNet database (https: / / exbio.wzw.tum.de / coryneregnet / ), target prediction of regulatory factors for ergothioneine biosynthesis was carried out, and a total of 47 regulatory target genes that may promote ergothioneine synthesis were screened out. Among them, the prediction results showed that upregulating the expression level of the transcriptional regulatory factor Cgl2415 might increase the synthesis of ergothioneine.

[0041] Example 3: Overexpression of transcriptional regulatory factor Cgl2415 Obtaining of plasmids

[0042] Using plasmid pECXK99E as the basic vector to construct the plasmid pEC-Cgl2415 for overexpressing the transcriptional regulatory factor Cgl2415 . Using the primers Cgl2415-F (SEQ ID NO.7) / Cgl2415-R (SEQ ID NO.8), pEC-F (SEQ ID NO.9) / pEC-R (SEQ ID NO.10) shown in Table 1 as the upstream and downstream primers respectively to amplify Cgl2415 the fragment and the pEC fragment, and then recombining the two fragments by homologous recombination to obtain the plasmid pEC-Cgl2415, whose map is shown in Figure 2 .

[0043] Table 1. Primers used

[0044]

[0045] Example 4: Construction of ergothioneine-producing strain and its shake-flask fermentation

[0046] (1) Construction of ergothioneine-producing strain

[0047] The plasmid pEC-Cgl2415 with correct sequencing results was electrotransformed into Corynebacterium glutamicum GW8 and evenly spread on the LBHIS solid plate with Cm and Kan resistances. Single colonies were picked respectively and verified by PCR with the following primers test-F (SEQ ID NO.11) / test-R (SEQ ID NO.12). The strain with correct sequencing was the ergothioneine-producing strain GWJ5 into which the plasmid pEC-Cgl2415 was inserted. GWJ5 is an ergothioneine-producing strain based on GW8 overexpressing the transcriptional regulatory factor in Corynebacterium glutamicum Cgl2415Ergothioneine-producing strain. GWJ1 is a control strain based on GW8 inserted with the empty plasmid pECXK99E without regulatory factors.

[0048] (2)Flask fermentation of the production strain

[0049] Perform microplate and flask fermentations on strains GWJ1 and GWJ5.

[0050] Inoculation method: First, streak GWJ1 and GWJ5 on BHIS solid medium and incubate in a 30°C incubator for about 18 h. Pick single colonies on the plate and inoculate them into 1 mL of LBHIS liquid medium, culture at 30°C and 220 rpm for about 12 h, then take 0.1 mL and transfer it to 10 mL of LBHIS liquid medium and continue to culture for 12 h. Inoculate the bacterial solution into the prepared CGXⅡA fermentation medium at an initial concentration of 30 g / L of glucose at 1%, add 1 g / L of precursor amino acids (histidine, methionine, cysteine), add AFC (ferric ammonium citrate) at 0.06 g / L, add 0.1 M of PLP (pyridoxal phosphate) at 1 / 100, place it in a constant temperature shaker at 30°C and 220 rmp and shake culture for 4 - 6 h until OD 600 is about 0.8 or so, then add the inducer (IPTG). After culturing for 48 h, measure its ergothioneine yield (see Figure 3 ), the strain GWJ5 overexpressing the transcriptional regulatory gene Cgl1371 increased by 2.0 times compared to the control strain GWJ1 (27 mg / L), reaching 80.9 mg / L. Subsequently, under further flask conditions for comparison, take samples every 12 h and measure its flask yield of ergothioneine (see Figure 4 ), the ergothioneine peak map (see Figure 5 , 6 and 7). The results show that the yield of strain GWJ5 is further improved, and it can accumulate 145.1 mg / L of ergothioneine in 60 h, still increasing by 128.6% compared to the control strain GWJ1 (63.5 mg / L). After literature and database retrieval, there are few studies on Cgl2415 transcriptional regulatory genes at present, and there is no report on its direct association with ergothioneine or its precursors (histidine, cysteine, methionine). Therefore, this study first confirmed that Cgl2415 gene overexpression has a promoting effect on ergothioneine biosynthesis.

[0051] The strain codes in the present invention such as CWJ1, GWJ5, etc. are for convenience of description, but should not be construed as a limitation of the present invention.

[0052] The construction of the strain of the present invention is not limited by the order of the steps, and those skilled in the art who achieve the object of the present invention according to the content disclosed in the present invention fall within the protection scope of the present invention.

Claims

1. Use of transcriptional regulator Cgl2415 in increasing the production of ergothioneine in Corynebacterium glutamicum, characterized in that, The Corynebacterium glutamicum is a Corynebacterium glutamicum capable of producing ergothioneine obtained by introducing the egt1 gene and the egt2 gene in the synthetic ergothioneine pathway, or by introducing the egt1 gene and the egtE gene in the synthetic ergothioneine pathway; The amino acid sequence of the transcriptional regulator Cgl2415 is shown in SEQ ID NO.

1.

2. The application according to claim 1, wherein It is to enhance the expression of the transcriptional regulator Cgl2415 in Corynebacterium glutamicum to achieve an increase in the yield of ergothioneine.

3. A method for increasing the yield of ergothioneine produced by Corynebacterium glutamicum, characterized in that: By enhancing the expression of the transcriptional regulator Cgl2415 in Corynebacterium glutamicum capable of synthesizing ergothioneine, a recombinant Corynebacterium glutamicum with increased ergothioneine yield is obtained. The Corynebacterium glutamicum capable of synthesizing ergothioneine is a Corynebacterium glutamicum capable of producing ergothioneine by introducing the egt1 gene and the egt2 gene in the synthetic ergothioneine pathway, or by introducing the egt1 gene and the egtE gene in the synthetic ergothioneine pathway; The amino acid sequence of the transcriptional regulator Cgl2415 is shown in SEQ ID NO.

1.

4. The method according to claim 3, wherein The enhancement of the expression of the transcriptional regulator Cgl2415 is achieved by plasmid overexpression, increasing the genomic expression copy number, and replacing the enhancer element.

5. The method according to claim 3, characterized in that, The coding nucleotide sequence of the transcriptional regulator Cgl2415 is shown in SEQ ID NO.

2.

6. The method according to claim 4, wherein Plasmid overexpression is achieved by constructing an expression vector with the coding nucleotide sequence of the transcriptional regulator Cgl2415, transforming Corynebacterium glutamicum capable of synthesizing ergothioneine to obtain a recombinant bacterium, and culturing the recombinant bacterium to produce ergothioneine.

7. The method according to claim 6, wherein It also includes the step of isolating the produced ergothioneine.

8. The method according to claim 3, wherein The egt1 gene and the egt2 gene in the synthetic ergothioneine pathway, or the egt1 gene and the egtE gene in the synthetic ergothioneine pathway are codon-optimized according to Corynebacterium glutamicum.

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