Application of transcriptional regulation factor Cgl2415 in improvement of yield of ergothioneine in corynebacterium glutamicum and method for preparing ergothioneine

Abstract

The invention belongs to the field of bioengineering technology and application, and discloses application of a transcriptional regulatory factor Cgl2415 in improving the yield of ergothioneine in corynebacterium glutamicum and a method for preparing the ergothioneine. According to the invention, the expression of a transcriptional regulation factor Cgl2415 is enhanced, and the enhancement modes comprise plasmid overexpression, increase of genome expression copy number, replacement of an emphasis regulation element and the like, so that the yield of ergothioneine is improved. 1, and the nucleotide sequence of the transcription regulation factor Cgl2415 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. Cgl2415 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 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. Cgl2415 A method for increasing ergothioneine production by expressing

[0006] In a specific embodiment, the enhanced transcriptional regulatory factor in Corynebacterium glutamicum Cgl2415 The expression of the gene is achieved by overexpression of plasmids, increasing the copy number of genomic expression, replacing the strong regulatory elements, etc.

[0007] The technical solution of the present invention is summarized as follows: The present invention first provides a transcriptional regulatory factor Cgl2415 Application in improving ergothioneine production in Corynebacterium glutamicum.

[0008] Specifically, it is to enhance the transcriptional regulatory factor in Corynebacterium glutamicum Cgl2415 expression to achieve increased 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. Cgl2415 to obtain a recombinant Corynebacterium glutamicum with improved ergothioneine production.

[0010] Specifically, the enhanced transcriptional regulatory factor Cgl2415 The expression is achieved by overexpression of plasmids, increasing the copy number of genomic expression, and replacing stress regulation elements.

[0011] Preferably, the transcriptional regulator Cgl2415 The amino acid sequence is shown in SEQ ID NO.1.

[0012] More preferably, the transcriptional regulatory factor Cgl2415 The encoding nucleotide sequence is shown in SEQ ID NO.2.

[0013] In a specific embodiment, plasmid overexpression is achieved by inserting a transcriptional regulator Cgl2415 An expression vector is constructed using the coding nucleotide sequence, and the recombinant bacteria are transformed into Corynebacterium glutamicum capable of synthesizing ergothioneine to obtain the recombinant bacteria, and the recombinant bacteria are cultured to produce ergothioneine.

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

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

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

[0017] The advantage of the present invention is that the transcriptional regulatory factor in Corynebacterium glutamicum Cgl2415 The enhanced expression of increased ergothioneine production in the engineered strain GW8 of Corynebacterium glutamicum was verified in experiments to be increased by 2.9 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- Cgl2415 Overexpression profile.

[0020] Figure 3 Schematic diagram of the fermentation of strain GWJ5 under microplate conditions.

[0021] Figure 4 Schematic diagram of the fermentation of strain GWJ5 under shake flask conditions.

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

[0023] Figure 6 This is the peak diagram of the total amount of ergothioneine after sampling and processing for GWJ1 after shaking flask fermentation for 60 hours.

[0024] Figure 7 This is the peak diagram of the total amount of ergothioneine after sampling and fermentation of GWJ5 for 60 hours. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with examples. The following examples are intended to enable those skilled in the art to better understand the present invention, but do not impose any limitation on the present invention.

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

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

[0028] Ergothioneine standard was 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 Company (http: / / www.thermoscientificbio.com / fermentas), and other biochemical reagents used were purchased from Sangon Biotechnology (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

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

[0031] BHIS medium: weigh 18.5 g of bovine brain heart extract powder, 91 g of sorbitol, dilute to 1 L with distilled water, and 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 bovine brain heart extract powder, 91 g of sorbitol, dilute to 1 L with distilled water, add 2% agar powder to the BHI solid medium, and sterilize at 121 °C with high pressure steam 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, 21 g MOPS, adjust pH to 7.0, distilled water to 1 L, sterilize at 121℃ for 20 min. Before use, add biotin stock solution and trace element stock solution at 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 gt1gene encoding) and heptylcysteine ​​sulfoxide-S-oxidase (Egt2, from fungi egt2 gene; or EgtE, from bacterial origin E 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 amino acid sequence of Egt1 (NCBI-ProteinID: XP_956324) (SEQ ID No. 3) was optimized according to the codon preference of Escherichia coli to obtain the optimized egt1 gene (SEQ ID No. 4) and sent it to Beijing Genewise Biotech Co., Ltd. for synthesis and ligation 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) was optimized according to the codon preference of Escherichia coli to obtain the optimized E The gene (SEQ ID No. 6) was sent to Beijing Genewise Biotech 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 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 E 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 of Corynebacterium glutamicum based on reports iCW773 (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 / ), and predicted the target sites of regulatory factors for ergothioneine biosynthesis. A total of 47 regulatory target genes that may promote ergothioneine synthesis were screened, among which the prediction results showed that the up-regulated transcriptional regulatory factors Cgl2415 The expression level of ergothioneine may be increased.

[0038] Example 3: Overexpression of transcriptional regulators Cgl2415 Plasmid acquisition Using plasmid pECXK99E as the basic vector to construct overexpression transcriptional regulatory factors Cgl2415 The plasmid pEC-Cgl2415 was obtained by amplifying the plasmid using the primers Cgl2415-F (SEQ ID NO.7) / Cgl2415-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. Cgl2415 The fragment and the pEC fragment were then recombined by homologous recombination to obtain the plasmid pEC-Cgl2415, the map of which is shown in Figure 2 .

[0039] Table 1. Primers used

[0040] Example 4: Construction of ergothioneine production strain and shake flask fermentation (1) Construction of ergothioneine production strain The plasmid pEC-Cgl2415 with the correct sequencing results was introduced into Corynebacterium glutamicum GW8 by electrotransfection and evenly spread on the Cm and Kan resistant LBHIS solid plate. Single colonies were picked and PCR verified with the following primers test-F (SEQ ID NO.11) / test-R (SEQ ID NO.12). The one with correct sequencing was the ergothioneine production strain GWJ5 inserted with plasmid pEC-Cgl2415. GWJ5 is based on GW8 overexpressing transcriptional regulatory factors in Corynebacterium glutamicum Cgl2415 GWJ1 is a control strain based on GW8 with the pECXK99E empty plasmid without regulatory factors inserted.

[0041] (2) Shake flask fermentation of production strains The strains GWJ1 and GWJ5 were fermented in microtiter plates and shake flasks.

[0042] Inoculation method: First, streak GWJ1 and GWJ5 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 220rpm 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 induction agent (IPTG) was about 0.8, the ergothioneine production was measured after 48 h of culture (see Figure 3 ), overexpression of transcriptional regulatory genes Cgl1371 The strain GWJ5 increased 2.0 times compared with the control strain GWJ1 (27 mg / L) to 80.9 mg / L. Subsequently, further comparison was made under shake flask conditions, and samples were taken every 12 hours to determine the shake flask yield of ergothioneine (see Figure 4 ), ergothioneine peak diagram (see Figure 5 , 6 and 7). The results showed that the yield of strain GWJ5 was further improved, and it could accumulate 145.1 mg / L of ergothioneine in 60 h, which was still 128.6% higher than the control strain GWJ1 (63.5 mg / L). Cgl2415 There are few studies on transcriptional regulatory genes, and no reports have shown a direct association between them and ergothioneine or its precursors (histidine, cysteine, and methionine). Cgl2415 Gene overexpression has a promoting effect on ergothioneine biosynthesis.

[0043] The strain codes in the present invention, such as CWJ1, GWJ5, etc., are for the convenience of description, but should not be construed as limitations of the present invention.

[0044] The construction of the strain of the present invention has no limitation on the order of the steps. Any steps achieved by a person skilled in the art according to the contents disclosed in the present invention to achieve the purpose of the present invention shall fall within the protection scope of the present invention.

Claims

1. Transcriptional regulatory factors Cgl2415 Application in improving ergothioneine production in Corynebacterium glutamicum.

2. The use according to claim 1, characterized in that It is a transcriptional regulator that enhances Cgl2415 expression to achieve increased ergothioneine production.

3. A method for improving the production of ergothioneine by Corynebacterium glutamicum, characterized in that: By enhancing transcriptional regulatory factors in Corynebacterium glutamicum capable of synthesizing ergothioneine Cgl2415 to obtain a recombinant Corynebacterium glutamicum with improved ergothioneine production.

4. The method according to claim 3, characterized in that The enhanced transcriptional regulator Cgl2415 The expression is achieved by overexpression of plasmids, increasing the copy number of genomic expression, and replacing stress regulation elements.

5. The method according to claim 3, characterized in that Transcription regulator Cgl2415 The amino acid sequence is shown in SEQ ID NO.

1.

6. The method according to claim 3, characterized in that Transcription regulator Cgl2415 The encoding nucleotide sequence is shown in SEQ ID NO.

2.

7. The method according to claim 4, characterized in that Plasmid overexpression is the process of Cgl2415 An expression vector is constructed using the coding nucleotide sequence, and the recombinant bacteria are transformed into Corynebacterium glutamicum capable of synthesizing ergothioneine to obtain the recombinant bacteria, and the recombinant bacteria are cultured to produce ergothioneine.

8. The method according to claim 7, characterized in that Also included is the step of isolating the produced ergothioneine.

9. The method according to claim 7 or 8, characterized in that The Corynebacterium glutamicum capable of synthesizing ergothioneine is prepared by introducing the key enzyme L-histidine-N-α-trimethyltransferase / heptylcysteine ​​sulfoxide-S-oxidation synthase gene and the heptylcysteine ​​sulfoxide-S-oxidation lyase gene in the ergothioneine synthesis pathway into the Corynebacterium glutamicum starting bacteria.

10. The method according to claim 9, characterized in that The key enzymes L-histidine-N-α-trimethyltransferase / heptylcysteine ​​sulfoxide-S-oxidation synthase gene and heptylcysteine ​​sulfoxide-S-oxidation lyase gene in the ergothioneine synthesis pathway were introduced and codon-optimized according to Corynebacterium glutamicum.

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