Method for enhancing expression of corynebacterium glutamicum transcriptional regulation factor Cgl1314 to improve yield of ergothioneine

Abstract

The invention discloses a method for increasing the yield of ergothioneine in corynebacterium glutamicum. Expression of a transcriptional regulation factor Cgl1314 is enhanced, and enhancement modes include 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. The nucleotide sequence of the transcription regulation factor Cgl1314 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. Cgl1314 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 to increase the production of ergothioneine. Cgl1314 to increase the production of ergothioneine.

[0006] In a specific embodiment, the enhancement of the expression of the transcriptional regulatory factor in Corynebacterium glutamicum Cgl1314 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: The present invention first provides the use of a transcriptional regulatory factor Cgl1314 in increasing the production of ergothioneine in Corynebacterium glutamicum.

[0008] Specifically, it is to enhance the expression of the transcriptional regulatory factor in Corynebacterium glutamicum Cgl1314 to achieve an increase in the production of ergothioneine.

[0009] The present invention also provides a method for increasing the production of ergothioneine by Corynebacterium glutamicum, which enhances the expression of a transcriptional regulatory factor in Corynebacterium glutamicum capable of synthesizing ergothioneine Cgl1314 to obtain a recombinant Corynebacterium glutamicum with increased ergothioneine production.

[0010] Specifically, the enhancement of the expression of the transcriptional regulatory factor Cgl1314 is achieved by plasmid overexpression, increasing the genomic expression copy number, and replacing the enhancer element.

[0011] Preferably, the amino acid sequence of the transcriptional regulatory factor Cgl1314 is as shown in SEQ ID NO.1.

[0012] More preferably, the coding nucleotide sequence of the transcriptional regulatory factor Cgl1314 is as shown in SEQ ID NO.2.

[0013] In a specific embodiment, plasmid overexpression is achieved by constructing an expression vector with the coding nucleotide sequence of the transcriptional regulatory factor Cgl1314 transforming Corynebacterium glutamicum capable of synthesizing ergothioneine to obtain a recombinant bacterium, and culturing the recombinant bacterium to produce ergothioneine.

[0014] Optionally, it further includes the step of isolating the produced ergothioneine.

[0015] Among them, the Corynebacterium glutamicum capable of synthesizing ergothioneine is obtained by introducing the key enzyme L-histidine-N-α-trimethyltransferase / heptylcysteine sulfoxide-S-oxidase gene in the ergothioneine synthesis pathway into the Corynebacterium glutamicum starting strain (such as egt1 gene) and heptylcysteine sulfoxide - S - oxidase lyase gene (such as those from fungi egt2 genes; or those from bacteria egtE genes).

[0016] Preferably, the key enzymes L - histidine - N - α - trimethyltransferase / heptylcysteine sulfoxide - S - oxygenase gene and heptylcysteine sulfoxide - S - oxidase lyase gene in the synthetic ergothioneine pathway are introduced. Preferably, the gene sequences are codon - optimized according to Corynebacterium glutamicum.

[0017] The advantage of the present invention is that the enhanced expression of the transcriptional regulator Cgl1314 in Corynebacterium glutamicum increases the production of ergothioneine in the engineered strain GW8 of Corynebacterium glutamicum, and it is verified in experiments that the production can be increased by up to 3.0 times at most. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 2 is the map of the over - expression of pEC - Cgl1314.

[0020] Figure 3 is the schematic diagram of the fermentation of strain GWJ3 under microplate conditions.

[0021] Figure 4 is the schematic diagram of the fermentation of strain GWJ3 under shake - flask conditions.

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

[0023] Figure 6 is the result peak map of the total amount of ergothioneine after sampling and treatment for the shake - flask fermentation of GWJ1 for 60 h.

[0024] Figure 7 is the result peak map of the total amount of ergothioneine after sampling and treatment for the shake - flask fermentation of GWJ3 for 60 h. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following further illustrates the present invention with reference to the 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.

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

[0027] In this example, the recombinant plasmid expression plasmids pXMJ19 and pECXK99E were purchased from BioVector NTCC company (http: / / www.biovector.net / ).

[0028] The ergothioneine standard was purchased from sigma company (http : / / www.sigmaaldrich.com / sigmaaldrich).

[0029] 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 / ).

[0030] 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.

[0031] 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.

[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 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.

[0033] 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.

[0034] Example 1: Construction of Corynebacterium glutamicum GW8 The wild-type Corynebacterium glutamicum itself does not contain the key enzyme L-histidine-N-α-trimethyltransferase / heptylcysteine sulfoxide-S-oxidase (Egt1, from e gt1gene encoding) and heptylcysteine sulfoxide-S-oxidative lyase (Egt2, from a fungal source egt2 gene; or EgtE, from a bacterial source egtE gene encoding), it is necessary to introduce exogenous genes to synthesize ergothioneine. The present invention constructs a plasmid basic vector pXMJ19-Egt1-EgtE capable of synthesizing ergothioneine. Based on the amino acid sequence (SEQ ID No. 3) of Egt1 (NCBI-ProteinID: XP_956324) reported in Neurospora crassa ( Neurospora crassa ), the coding 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 (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. Then, based on the amino acid sequence (SEQ ID No. 5) of EgtE (NCBI-ProteinID: ABK70212) reported in Mycobacterium smegmatis ( Mycobacterium smegmatis ), the coding 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 (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 plasmid map of pXMJ19-Egt1-EgtE is as shown in Figure 1 .

[0035] 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.

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

[0037] Example 2: Obtaining the regulatory factor modification target Based on the reported high-quality genome-scale metabolic network model of Corynebacterium glutamicum iCW773 (PMID: 28680478), after adding the heterologous reaction information of 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. The prediction results showed that upregulating the expression level of the transcriptional regulatory factor Cgl1314 might increase the synthesis of ergothioneine. Subsequently, this was verified.

[0038] Example 3: Overexpression of the transcriptional regulatory factor Cgl1314 Obtaining of plasmid Using plasmid pECXK99E as the basic vector to construct the plasmid pEC-Cgl1314 for overexpressing the transcriptional regulatory factor Cgl1314 . Using the primers Cgl1314-F (SEQ ID NO.7) / Cgl1314-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 to amplify the Cgl1314 fragment and the pEC fragment, and then recombining the two fragments by homologous recombination to obtain the plasmid pEC-Cgl1314, whose map is shown in Figure 2 .

[0039] Table 1

[0040] .

[0041] Example 4: Construction of ergothioneine-producing strain and its flask fermentation (1) Construction of ergothioneine-producing strain The plasmid pEC-Cgl1314 with correct sequencing results was electrotransformed into Corynebacterium glutamicum GW8 and evenly spread on the LBHIS solid plate with Cm and Kan resistance. Single colonies were picked respectively and verified by PCR with the following primers test-F (SEQ ID NO.11: AGCTGTGGTATGGCTGTG) / test-R (SEQ ID NO.12: GAGAGTAGGGAACTGCCA).

[0042] The one with correct sequencing is the ergothioneine-producing strain GWJ3 into which the plasmid pEC-Cgl1314 was inserted. GWJ3 is an ergothioneine-producing strain based on GW8 overexpressing the transcriptional regulatory factor Cgl1314 in Corynebacterium glutamicum. GWJ1 is a control strain based on GW8 into which the empty plasmid pECXK99E without regulatory factors was inserted.

[0043] (2) Flask fermentation of the production strain Ferment strains GWJ1 and GWJ3 in microplates and shake flasks.

[0044] Inoculation method: First, streak GWJ1 and GWJ3 on BHIS solid medium and incubate 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. Incubate 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 incubate 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 rpm and shake and incubate 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 yield of ergothioneine of strain GWJ3 overexpressing the transcriptional regulatory gene Cgl1314 was 3.0 - fold higher than that of the control strain GWJ1 (27 mg / L), reaching 107.6 mg / L. Subsequently, a comparison was carried out under further shake flask conditions. Samples were taken every 12 h to measure the ergothioneine shake flask yield (see Figure 4 ), and the ergothioneine peak map (see Figure 5 , 6 and 7). The results showed that the yield of strain GWJ3 was further improved, and 204.6 mg / L of ergothioneine could be accumulated in 60 h.

[0045] The strain codes in the present invention, such as GWJ1 and GWJ3, are for convenience of description, but should not be construed as a limitation to the present invention.

[0046] For the construction of the strains of the present invention, the order of the steps is not limited. Those skilled in the art who achieve the purpose of the present invention according to the content disclosed in the present invention fall within the protection scope of the present invention.

Claims

1. Application of a transcriptional regulator Cgl1314 in increasing the production of ergothioneine in Corynebacterium glutamicum.

2. The application according to claim 1, characterized in that, It is to enhance the expression of transcriptional regulatory factors in Corynebacterium glutamicum Cgl1314 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 Cgl1314 to obtain recombinant Corynebacterium glutamicum with increased ergothioneine production.

4. The method according to claim 3, characterized in that, The enhanced transcriptional regulator Cgl1314 is expressed by plasmid overexpression, increasing the genomic expression copy number, or replacing the enhanced regulatory elements such as promoters.

5. The method according to claim 3, wherein Transcription regulatory factor Cgl1314 has the amino acid sequence shown in SEQ ID NO.

1.

6. The method according to claim 3, characterized in that Transcription regulatory factor Cgl1314 The coding nucleotide sequence thereof 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 Cgl1314 and transforming Corynebacterium glutamicum capable of synthesizing ergothioneine to obtain a recombinant strain, and culturing the recombinant strain 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 gt1 gene) and the heptylcysteine sulfoxide-S-oxygenolytic cleavage enzyme gene (such as the egt2 gene of fungal origin; or the egtE gene of bacterial origin) 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-oxygen cleavage enzyme gene in the ergothioneine synthesis pathway. Preferably, the gene sequences are codon-optimized according to Corynebacterium glutamicum.

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