Application of transcriptional regulator Cgl0020 in improving ergothioneine production in Corynebacterium glutamicum and method for preparing ergothioneine

Abstract

The present invention belongs to the field of bioengineering technology and applications, and discloses an application of a transcriptional regulator Cgl0020 in improving the production of ergothioneine in Corynebacterium glutamicum and a method for preparing ergothioneine. By strengthening the expression of the transcriptional regulator Cgl0020 , the strengthening 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 Cgl0020 is shown in SEQ ID NO. 2. The method for improving the target product ergothioneine by changing the expression level of the transcriptional regulator 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 bioengineering technology and application, and specifically relates to a method for enhancing the transcriptional regulatory factor of Corynebacterium glutamicum. Cgl0020 A method for increasing ergothioneine production by expressing Background Art

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

[0003] At present, the production of ergothioneine mainly relies on natural biological extraction, chemical synthesis and biosynthesis. Among them, the natural extraction method has problems such as low yield and many impurities, which limits its industrial application; the chemical synthesis method is difficult to achieve large-scale production due to expensive raw materials and difficult to ensure safety. In recent years, microbial fermentation based on metabolic engineering and synthetic biology technology has become a research hotspot. This method has the advantages of low cost, easy availability of raw materials and environmental friendliness by constructing an engineering strain with high ergothioneine production. Existing research mainly focuses on the genetic modification of key enzymes in the ergothioneine synthesis pathway and precursor supply pathway, while the role of transcriptional regulatory factors has not been deeply explored. Transcriptional regulatory factors play a core role in the cell metabolic network, and can coordinate the expression of multiple genes, optimize metabolic flow distribution and resource allocation, thereby significantly improving the synthesis efficiency of the target product. Therefore, in-depth research on the effect of transcriptional regulatory factors on the biosynthesis of ergothioneine will not only help to reveal its metabolic regulation mechanism, but also may discover new efficient transformation 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 and biomaterials such as amino acids and organic acids. Its food safety grade characteristics make it an ideal production strain in the fields of food and cosmetics, and it is also a potential high-quality chassis cell for the biosynthesis of ergothioneine. 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 Cgl0020 to increase the production of ergothioneine.

[0006] In the specific embodiments, the enhancement of the expression of the transcriptional regulatory factor in Corynebacterium glutamicum Cgl0020 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 Cgl0020 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 Cgl0020 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 is achieved by enhancing the expression of a transcriptional regulatory factor in Corynebacterium glutamicum capable of synthesizing ergothioneine Cgl0020 to obtain a recombinant Corynebacterium glutamicum with increased ergothioneine production.

[0011] Specifically, the enhancement of the expression of the transcriptional regulatory factor Cgl0020 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 Cgl0020 is as shown in SEQ ID NO.1.

[0013] More preferably, the coding nucleotide sequence of the transcriptional regulatory factor Cgl0020 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 Cgl0020 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 enzyme L-histidine-N-α-trimethyltransferase / heptylcysteine sulfoxide-S-oxygenase gene (such as the gt1 gene) and the heptylcysteine sulfoxide-S-oxygen cleavage enzyme gene (such as the gene derived from fungi egt2 ; or the gene derived from bacteria egtE gene) into the starting strain of Corynebacterium glutamicum.

[0017] Preferably, the key enzyme L-histidine-N-α-trimethyltransferase / heptylcysteine sulfoxide-S-oxygenase gene and the heptylcysteine sulfoxide-S-oxygen cleavage enzyme gene introduced into 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 Cgl0020 in Corynebacterium glutamicum increases the production of ergothioneine in the engineered strain GW8 of Corynebacterium glutamicum, and it is verified in the experiment that the production can be increased by 2.0 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- Cgl0020 overexpression.

[0021] Figure 3 is the schematic diagram of the fermentation of strain GWJ6 under microplate conditions.

[0022] Figure 4 is the schematic diagram of the fermentation of strain GWJ6 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 of GWJ1 after 60 h of shake flask fermentation.

[0025] Figure 7 is the result peak map of the total amount of ergothioneine after sampling and treatment of GWJ6 after 60 h of shake flask fermentation. DETAILED DESCRIPTION OF THE INVENTION

[0026] The following further describes the present invention in conjunction with embodiments. The following embodiments 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 Inc. (http: / / www.biovector.net / ).

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

[0030] The molecular biology reagents such as restriction endonucleases, dephosphorylating enzymes, 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 the 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 in the ergothioneine synthesis pathway, namely L-histidine-N-α-trimethyltransferase / heptylcysteine sulfoxide-S-oxygenase (Egt1, encoded by the gt1 gene) and heptylcysteine sulfoxide-S-oxygenolytic enzyme (Egt2, a gene from fungal origin egt2 ; or EgtE, encoded by a gene from bacterial origin egtE ). Therefore, exogenous genes need to be introduced to synthesize ergothioneine. In the present invention, a plasmid basic vector pXMJ19-Egt1-EgtE capable of synthesizing ergothioneine was constructed. Based on the amino acid sequence of Egt1 (NCBI-ProteinID: XP_956324) (SEQ ID No. 3) reported in Neurospora crassa ( Neurospora crassa ), the coding gene of Egt1 was optimized according to the codon preference of Escherichia coli, and the optimized egt1 gene (SEQ ID No. 4) was obtained. It was sent to Genewiz Biotechnology (Beijing) Co., Ltd. for synthesis and ligated together 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.

[0037] Then, based on the amino acid sequence of EgtE (NCBI-ProteinID: AFP42516) (SEQ ID No. 5) reported in Mycobacterium smegmatis ( Mycobacterium smegmatis ), the coding gene of EgtE was optimized according to the codon preference of Escherichia coli, and the optimized egtE gene (SEQ ID No. 6) was obtained. It was sent to Genewiz Biotechnology (Beijing) Co., Ltd. for synthesis and ligated together 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 .

[0038] 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.

[0039] The pXMJ19-Egt1-EgtE was introduced into Corynebacterium glutamicum ATCC 13032 strain by electroporation to obtain the recombinant strain GW8.

[0040] Example 2: Obtaining of regulatory factor modification targets

[0041] Based on the reported high-quality genome-scale metabolic network model of Corynebacterium glutamicum i CW773 (PMID: 28680478), 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 / ), the target prediction of regulatory factors for ergothioneine biosynthesis was carried out. A total of 47 regulatory target genes that may promote ergothioneine synthesis were screened out. Among them, the prediction results showed that up-regulating the expression level of the transcriptional regulatory factor Cgl0020 might increase the synthesis of ergothioneine. Subsequent verification was carried out on this.

[0042] Example 3: Overexpression of transcriptional regulatory factor Cgl0020 Obtaining of plasmids

[0043] The plasmid pEC-Cgl0020 for overexpressing the transcriptional regulatory factor Cgl0020 was constructed using the plasmid pECXK99E as the basic vector. Using the primers Cgl0020-F (SEQ ID NO.7) / Cgl0020-R (SEQ ID NO.8) and pEC-F (SEQ ID NO.9) / pEC-R (SEQ ID NO.10) shown in Table 1 as the upstream and downstream primers respectively, the Cgl0020 fragment and the pEC fragment were amplified, and then the two fragments were recombined by homologous recombination to obtain the plasmid pEC-Cgl0020, and its map is shown in Figure 2 .

[0044] Primers used in Table 1

[0045]

[0046] Example 4: Construction of ergothioneine-producing strain and its shake-flask fermentation

[0047] (1) Construction of ergothioneine-producing strain

[0048] The plasmid pEC-Cgl0020 with correct sequencing results was introduced into Corynebacterium glutamicum GW8 by electroporation and evenly spread on the LBHIS solid plate with Cm and Kan resistance. Single colonies were separately picked and verified by PCR using 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 GWJ6 into which the plasmid pEC-Cgl0020 was inserted. GWJ6 is an ergothioneine-producing strain based on GW8 overexpressing the transcriptional regulator Cgl0020 in Corynebacterium glutamicum. GWJ1 is a control strain based on GW8 into which the empty plasmid pECXK99E without a regulatory factor was inserted.

[0049] (2)Flask fermentation of the production strain

[0050] The strains GWJ1 and GWJ6 were subjected to microplate and flask fermentation.

[0051] Inoculation method: First, streak GWJ1 and GWJ6 on the BHIS solid medium and place them in an incubator at 30 °C 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. 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 PLP (pyridoxal phosphate) at 1 / 100, and place it in a constant temperature shaker at 30 °C and 220 rpm and shake culture for 4 - 6 h until the OD 600 is about 0.8, then add the inducer (IPTG). After culturing for 48 h, measure its ergothioneine yield (see Figure 3 ). The strain GWJ6 overexpressing the transcriptional regulatory gene Cgl0020 was 2.0 times higher than the control strain GWJ1 (27 mg / L), reaching 81.0 mg / L.

[0052] Subsequently, a comparison was carried out under further flask conditions. Samples were taken every 12 h to measure its flask yield of ergothioneine (see Figure 4 ), and the ergothioneine peak map (see Figure 5 , 6 and 7). The results showed that the yield of the strain GWJ6 was further improved, and 100.2 mg / L of ergothioneine could be accumulated in 60 h, still increasing by 57.8% compared with the control strain GWJ1 (63.5 mg / L). After searching the literature and databases, currently regarding Cgl0020There has been little research on transcriptional regulatory genes, and there are no reports of their direct association with ergothioneine or its precursors (histidine, cysteine, methionine). Therefore, this study for the first time confirmed that the overexpression of the Cgl0020 gene promotes the biosynthesis of ergothioneine.

[0053] The strain codes in the present invention, such as GWJ1, GWJ6, etc., are for convenience of description, but should not be construed as a limitation to the present invention.

[0054] For the construction of the strains of the present invention, the order of the steps is not limited, and those skilled in the art who achieve the purpose of the present invention according to the content disclosed in the present invention all fall within the protection scope of the present invention.

Claims

1. Use of the transcriptional regulator Cgl0020 in increasing the production of ergothioneine in Corynebacterium glutamicum, characterized in that, It is used to increase the production of ergothioneine in Corynebacterium glutamicum capable of synthesizing ergothioneine, and the Corynebacterium glutamicum capable of synthesizing ergothioneine is obtained by introducing 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 into the parental strain of Corynebacterium glutamicum; the amino acid sequence of the transcriptional regulator Cgl0020 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 Cgl0020 in Corynebacterium glutamicum to increase the production 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 Cgl0020 in Corynebacterium glutamicum capable of synthesizing ergothioneine, a recombinant Corynebacterium glutamicum with increased ergothioneine production is obtained; The Corynebacterium glutamicum capable of synthesizing ergothioneine is obtained by introducing the key enzyme genes egt1 and egt2 in the synthetic ergothioneine pathway, or the egt1 gene and the egtE gene in the synthetic ergothioneine pathway into the parental strain of Corynebacterium glutamicum; The amino acid sequence of the transcriptional regulator Cgl0020 is shown in SEQ ID NO.

1.

4. The method according to claim 3, wherein The enhancement of the expression of the transcriptional regulator Cgl0020 is achieved by plasmid overexpression, increasing the genomic expression copy number, or replacing the enhancer element.

5. The method according to claim 3, wherein The coding nucleotide sequence of the transcriptional regulator Cgl0020 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 Cgl0020, transforming Corynebacterium glutamicum capable of synthesizing ergothioneine to obtain a recombinant strain, and culturing the recombinant strain 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 key enzyme genes egt1, egt2, and egtE in the synthetic ergothioneine pathway are codon-optimized according to Corynebacterium glutamicum.

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