Method for enhancing expression of corynebacterium glutamicum transcriptional regulation factor Cgl1371 to improve yield of ergothioneine

Abstract

The invention discloses a method for increasing the yield of ergothioneine in corynebacterium glutamicum. Expression of a transcriptional regulatory factor Cgl1371 is enhanced, and enhancement modes include plasmid overexpression, increase of genome expression copy number, replacement of an emphasis regulatory element and the like, so that the yield of ergothioneine is improved. The nucleotide sequence of the transcription regulation factor Cgl1371 is as shown in SEQ ID NO. 2. The method for improving the target product ergothioneine by changing the expression level of transcriptional regulatory factors has guiding significance on 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. Cgl1371 A method for increasing ergothioneine production by expressing Background Art

[0002] Ergothioneine was first isolated 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 absorbed by the body through the production of a gene (a product of the mitochondrial gene). Ergothioneine is a potent scavenger of reactive oxygen species (ROS) and an inhibitor of lipid peroxidation. Due to its non-toxic and safe properties as a natural antioxidant, it is widely used in skincare, food, biology, and medicine.

[0003] Currently, the production of ergothioneine mainly relies on natural biological extraction, chemical synthesis, and biosynthesis. Natural extraction suffers from problems such as low yield and high impurities, limiting its industrial application. Chemical synthesis, however, is difficult to achieve large-scale production due to expensive raw materials and unsafe conditions. In recent years, microbial fermentation based on metabolic engineering and synthetic biology has become a research hotspot. This method, which involves constructing engineered strains that produce high ergothioneine yields, offers advantages such as low cost, readily available raw materials, and environmental friendliness. Existing research has primarily focused on genetic modification of key enzymes in the ergothioneine synthesis pathway and precursor supply pathway, while the role of transcriptional regulatory factors has not been thoroughly explored. Transcriptional regulatory factors play a central role in cellular metabolic networks, significantly improving the synthesis efficiency of target products by coordinating the expression of multiple genes and optimizing metabolic flux distribution and resource allocation. Therefore, in-depth research into the impact of transcriptional regulatory factors on ergothioneine biosynthesis will not only help reveal its metabolic regulation mechanisms but also potentially identify new, efficient modification targets, providing new ideas for the industrial production of ergothioneine.

[0004] Corynebacterium glutamicum ( Corynebacterium glutamicumAs an important industrial microorganism, Corynebacterium glutamicum is widely used in the production of chemicals such as amino acids and organic acids, as well as biomaterials. Its food-safe properties make it an ideal production strain for food, cosmetics, and other sectors. It also serves as a promising platform for the biosynthesis of ergothioneine. Due to its mature genetic manipulation system and high safety profile, Corynebacterium glutamicum has strong potential for industrial production of ergothioneine. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for enhancing the transcriptional regulatory factor in Corynebacterium glutamicum. Cgl1371 A method for increasing ergothioneine production by expressing

[0006] In a specific embodiment, the enhanced transcriptional regulatory factor in Corynebacterium glutamicum Cgl1371 The expression of the gene is achieved through plasmid overexpression, increasing the genomic expression copy number, replacing the regulatory elements, etc.

[0007] The technical solution of the present invention is summarized as follows: The present invention first provides a transcriptional regulatory factor Cgl1371 Application in improving ergothioneine production in Corynebacterium glutamicum.

[0008] Specifically, it is to enhance the transcriptional regulatory factor in Corynebacterium glutamicum Cgl1371 expression to increase ergothioneine production.

[0009] The present invention also provides a method for increasing the yield of ergothioneine produced by Corynebacterium glutamicum, which is achieved by strengthening the transcriptional regulatory factor in Corynebacterium glutamicum capable of synthesizing ergothioneine. Cgl1371 to obtain a recombinant Corynebacterium glutamicum with improved ergothioneine production.

[0010] Specifically, the enhanced transcriptional regulatory factor Cgl1371 The expression of is achieved through plasmid overexpression, increasing the genomic expression copy number, and replacing the regulatory elements.

[0011] Preferably, the transcriptional regulatory factor Cgl1371 The amino acid sequence is shown in SEQ ID NO.1.

[0012] More preferably, the transcriptional regulatory factor Cgl1371 The encoding nucleotide sequence is shown in SEQ ID NO.2.

[0013] In a specific embodiment, plasmid overexpression is achieved by inserting a transcriptional regulatory factor Cgl1371 An expression vector is constructed using the coding nucleotide sequence of the present invention, which is transformed into Corynebacterium glutamicum capable of synthesizing ergothioneine to obtain a recombinant bacterium, and the recombinant bacterium is cultured to produce ergothioneine.

[0014] Optionally, the method further comprises the step of isolating the produced ergothioneine.

[0015] Wherein, the Corynebacterium glutamicum capable of synthesizing ergothioneine is a key enzyme L-histidine-N-α-trimethyltransferase / heptylcysteine ​​sulfoxide-S-oxidation synthase gene (such as e) introduced into the Corynebacterium glutamicum starting bacterium in the synthesis of ergothioneine pathway. gt1 genes) and heptylcysteine ​​sulfoxide-S-oxidase genes (e.g., fungal egt2 Gene; or bacterial origin egE Gene).

[0016] Preferably, the key enzyme L-histidine-N-α-trimethyltransferase / heptylcysteine ​​sulfoxide-S-oxidation synthase gene and heptylcysteine ​​sulfoxide-S-oxidation lyase gene in the synthetic ergothioneine pathway are imported, and preferably, the gene sequence is codon optimized according to Corynebacterium glutamicum.

[0017] The advantage of the present invention is that the transcriptional regulatory factor in Corynebacterium glutamicum Cgl1371 The enhanced expression of ergothioneine increased the production of ergothioneine in the engineered strain GW8 of Corynebacterium glutamicum, and experiments showed that it could be increased by 3.4 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a map of the pXMJ19-Egt1-EgtE expression vector.

[0019] Figure 2 For pEC- Cgl1371 Overexpression map.

[0020] Figure 3 Schematic diagram of the fermentation of strain GWJ2 under microplate conditions.

[0021] Figure 4 Schematic diagram of the fermentation of strain GWJ2 under shake flask conditions.

[0022] Figure 5 This is the standard peak of ergothioneine.

[0023] Figure 6 This is the peak diagram of the total amount of thioneine after GWJ1 was sampled and fermented in a shake flask for 60 h.

[0024] Figure 7 This is the peak diagram of the total amount of thioneine after shake flask fermentation for 60 h for GWJ2. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the following examples. The following examples are provided to enable those skilled in the art to better understand the present invention, but are not intended to limit the present invention in any way.

[0026] The original strain used in the present invention Corynebacterium glutamicum ATCC 13032 was obtained from the laboratory.

[0027] The recombinant expression plasmids pXMJ19 and pECXK99E involved in this example were purchased from BioVector NTCC (http: / / www.biovector.net / ).

[0028] Ergothioneine standards were purchased from Sigma (http: / / www.sigmaaldrich.com / sigmaaldrich).

[0029] The restriction endonucleases, dephosphorylases, DNA ligases, and other molecular biological reagents used were purchased from Thermo Scientific (http: / / www.thermoscientificbio.com / fermentas), and other biochemical reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0030] LB medium: Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, and dilute to 1 L with distilled water. Add 2% agar powder to the LB solid medium and sterilize by autoclaving at 121°C for 20 min.

[0031] BHIS medium: Weigh 18.5 g of bovine brain heart extract powder, 91 g of sorbitol, and dilute to 1 L with distilled water. Sterilize with high-pressure steam at 121°C for 20 min.

[0032] LBHIS medium: Weigh 5 g of tryptone, 10 g of NaCl, 2.5 g of yeast extract, 18.5 g of calf brain extract powder, and 91 g of sorbitol. Dose to 1 L with distilled water. Add 2% agar powder to the BHI solid medium and sterilize by high-pressure steam at 121°C for 20 min.

[0033] CGXⅡA medium: Weigh 5 g yeast extract, 20 g (NH₄)₂SO₄, 5 g Urea, 1 g KH₂PO₄, 1 g K₂HPO₄, 0.25 g MgSO₄·7H₂O, 0.01 g CaCl₂, and 21 g MOPS. Adjust the pH to 7.0, dilute to 1 L with distilled water, and sterilize at 121°C for 20 min. Before use, add biotin stock solution and trace element stock solution at a 1 / 1000 volume.

[0034] Example 1: Construction of Corynebacterium glutamicum GW8 Corynebacterium glutamicum itself does not contain the key enzyme L-histidine-N-α-trimethyltransferase / heptylcysteine ​​sulfoxide-S-oxidase (Egt1, represented by e gt1 gene encoding) and heptylcysteine ​​sulfoxide-S-oxidase (Egt2, derived from fungi egt2 Gene; or EgtE, of bacterial origin egE Gene encoding), it is necessary to introduce exogenous genes to synthesize ergothioneine. The present invention constructs a plasmid basic vector pXMJ19-Egt1-EgtE that can synthesize ergothioneine. Based on Neurospora crassa ( Neurospora crassa ) reported by the Egt1 (NCBI-ProteinID: XP_956324) amino acid sequence (SEQ ID No. 3), the coding gene of Egt1 was optimized according to the codon preference of Escherichia coli to obtain the optimized egt1 The gene (SEQ ID No. 4) was sent to Beijing Jinweizhi Biotechnology Co., Ltd. for synthesis and ligated with the 5' flanking sequence "GAAAGGAGGCCCTTCAG" between the restriction sites PstI and XbaI of the pXMJ19 vector to construct the pX-Egt1 plasmid. Then, based on Mycobacterium smegmatis ( Mycobacterium smegmatis ) reported by the EgtE (NCBI-ProteinID: ABK70212) amino acid sequence (SEQ ID No. 5), the EgtE coding gene was optimized according to the codon preference of Escherichia coli to obtain the optimized egE The gene (SEQ ID No. 6) was sent to GENEWIZ Biotech (Beijing) Co., Ltd. for synthesis and ligated with the 5' flanking sequence "AAAGGAGGACAACC" between the restriction sites XmaI and SacI of the pXMJ19-Egt1 plasmid to construct the pXMJ19-Egt1-EgtE plasmid. The final pXMJ19-Egt1-EgtE plasmid map is shown in the figure. Figure 1 shown.

[0035] The structure of the recombinant expression vector pXMJ19-Egt1-EgtE is described as follows: egt1 The gene (SEQ ID No. 4) was inserted between the restriction sites PstI and XbaI of the pXMJ19 vector, and the optimized egE The recombinant vector was obtained by inserting the gene (SEQ ID No. 6) between the restriction sites XmaI and SacI.

[0036] pXMJ19-Egt1-EgtE was transformed into Corynebacterium glutamicum ATCC 13032 strain by electroporation to obtain recombinant strain GW8.

[0037] Example 2: Acquisition of regulatory factor modification targets A high-quality genome-scale metabolic network model based on the reported Corynebacterium glutamicum i CW773 (PMID: 28680478) added heterologous reaction information of ergothioneine synthesis and integrated the regulatory information of Corynebacterium glutamicum in the CoryneRegNet database (https: / / exbio.wzw.tum.de / coryneregnet / ) to predict the target sites of regulatory factors for ergothioneine biosynthesis. The prediction results showed that the transcriptional regulatory factors were upregulated. Cgl1371 The expression level of ergothioneine may be increased. This was subsequently verified.

[0038] Example 3: Overexpression of transcriptional regulatory factors Cgl1371 Plasmid acquisition Plasmid pECXK99E was used as the basic vector to construct overexpression transcriptional regulatory factors Cgl1371 The plasmid pEC-Cgl1371 was amplified using the primers Cgl1371-F (SEQ ID NO.7) / Cgl1371-R (SEQ ID NO.8) and pEC-F (SEQ ID NO.9) / pEC-R (SEQ ID NO.10) shown in Table 1 as upstream and downstream primers. Cgl1371 The fragment and the pEC fragment were then recombined by homologous recombination to obtain the plasmid pEC-Cgl1371, the map of which is shown in Figure 2 .

[0039] Table 1

[0040] Example 4: Construction of thioneine-producing strain and shake flask fermentation thereof (1) Construction of ergothioneine-producing strain Plasmid pEC-Cgl1371, which showed a positive sequencing result, was electroporated into Corynebacterium glutamicum GW8 and evenly plated onto Cm- and Kan-resistant LBHIS plates. Single colonies were selected for PCR verification using the following primers: test-F (SEQ ID NO. 11: AGCTGTGGTATGGCTGTG) and test-R (SEQ ID NO. 12: GAGAGTAGGGAACTGCCA).

[0041] The strain with the correct sequencing result is the ergothioneine production strain GWJ2 inserted into the plasmid pEC-Cgl1371. GWJ2 is based on GW8 overexpression of transcriptional regulatory factors in Corynebacterium glutamicum. Cgl1371 GWJ1 is a control strain based on GW8 with the pECXK99E empty plasmid containing no regulatory factors inserted.

[0042] (2) Shake flask fermentation of production strains The strains GWJ1 and GWJ2 were fermented in microtiter plates and shake flasks.

[0043] Inoculation method: First, streak GWJ1 and GWJ2 on BHIS solid culture medium and culture in a 30°C incubator for about 18 hours. Pick a single colony on the plate and inoculate it into 1 mL of LBHIS liquid culture medium. Culture at 30°C and 220 rpm for about 12 hours. Take 0.1 mL and transfer it to 10 mL of LBHIS liquid culture medium and continue to culture for 12 hours. With an initial concentration of 30 g / L glucose, inoculate the bacterial liquid into the prepared CGXⅡA fermentation medium at 1%, add 1 g / L of precursor amino acids (histidine, methionine, cysteine), add AFC (ammonium ferric 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 for shaking culture for 4-6 hours until OD 600 When the pH value was about 0.8, the inducer (IPTG) was added. After 48 hours of culture, the ergothioneine production was determined (see Figure 3 ), overexpression of transcriptional regulatory genes Cgl1371 The strain GWJ2 increased 3.4 times to 120.1 mg / L compared to the control strain GWJ1 (27 mg / L). Subsequently, further comparison was made under shake flask conditions, and samples were taken every 12 hours to determine the ergothioneine shake flask yield (see Figure 4 ), Ergothioneine Peak Diagram (see Figure 5 、 6 and 7). The results showed that the yield of strain GWJ2 was further improved, and it could accumulate 255.0 mg / L of ergothioneine in 60 h.

[0044] The strain codes in the present invention, such as GWJ1, GWJ2, etc., are for the convenience of description and should not be construed as limiting the present invention.

[0045] The construction of the strain of the present invention is not limited to the order of the steps. Any steps achieved by those skilled in the art according to the disclosure of the present invention fall within the scope of protection of the present invention.

Claims

1. Application of a transcriptional regulator Cgl1371 in increasing the production of ergothioneine in Corynebacterium glutamicum.

2. The application according to claim 1, wherein It is to enhance the expression of transcriptional regulatory factors in Corynebacterium glutamicum Cgl1371 to achieve an increase in the production of ergothioneine.

3. A method for improving the production yield of ergothioneine by Corynebacterium glutamicum, characterized in that: By enhancing the expression of the transcriptional regulator in Corynebacterium glutamicum capable of synthesizing ergothioneine Cgl1371 to obtain recombinant Corynebacterium glutamicum with increased ergothioneine production.

4. The method according to claim 3, wherein The expression of the enhanced transcriptional regulator Cgl1371 is achieved by plasmid overexpression, increasing the genomic expression copy number, or replacing the enhancer elements such as promoters.

5. The method according to claim 3, wherein Transcription regulatory factor Cgl1371 The amino acid sequence is shown in SEQ ID NO.

1.

6. The method according to claim 3, wherein Transcription regulatory factor Cgl1371 The encoded nucleotide sequence is shown in SEQ ID NO.

2.

7. 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 Cgl1371 and transforming Corynebacterium glutamicum capable of synthesizing ergothioneine to obtain a recombinant bacterium, and cultivating the recombinant bacterium to produce ergothioneine.

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

9. The method according to any one of claims 3 to 7, characterized in that 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 e gt1 gene) and heptylcysteine sulfoxide-S-oxygen cleavage enzyme gene (such as the egt2 gene derived from fungi; or the egtE gene derived from bacteria) into the starting strain of Corynebacterium glutamicum.

10. The method according to claim 9, wherein Import the key enzymes L-histidine-N-α-trimethyltransferase / heptylcysteine sulfoxide-S-oxygenase gene and heptylcysteine sulfoxide-S-oxygenolytic cleavage enzyme gene in the ergothioneine synthesis pathway. Preferably, the gene sequences are codon-optimized according to Corynebacterium glutamicum.

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