Application of transcriptional regulation factor Cgl2988 in improving yield of beta-alanine of corynebacterium glutamicum

Abstract

The invention belongs to the field of bioengineering technology and application, and discloses application of a transcriptional regulation factor Cgl2988 in improving the yield of beta-alanine of corynebacterium glutamicum. The yield of the beta-alanine in the corynebacterium glutamicum engineering strain can be greatly increased by performing enhanced expression on a transcriptional regulation factor Cgl2988 in the process of synthesizing the beta-alanine, and in an experiment, the yield of the beta-alanine in the corynebacterium glutamicum engineering strain CG19 is increased by performing enhanced expression on the transcriptional regulation factor Cgl2988 in the corynebacterium glutamicum. Experiments prove that the maximum value can be increased by 211%, and the method has application value.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology and applications, and specifically relates to the application of the transcriptional regulator Cgl2988 in improving the β-alanine production of Corynebacterium glutamicum. Background Art

[0002] β-alanine is the only naturally occurring β-type amino acid, and is a synthetic raw material for drugs such as vitamin B5, guanidinoacetic acid, balsalazide, and carnosine. It can also be used as a food additive to improve food flavor, and has wide applications in the fields of medicine, chemical industry, food, etc. Currently, the main production methods of β-alanine are chemical synthesis methods, including acrylonitrile method, acrylic acid method, iminodiacrylonitrile method, etc. The reaction process relies on extreme environments such as high temperature and high pressure, and some processes use nitrile-containing raw materials, which pose potential hazards to the environment and organisms.

[0003] Currently, the production of β-alanine mainly relies on chemical synthesis methods, biocatalytic methods, and microbial fermentation methods. Among them, the chemical synthesis method is difficult to achieve large-scale production due to expensive raw materials and difficult-to-guarantee safety; the biocatalytic method ultimately needs to be converted through fumaric acid intermediates, and its large-scale production exacerbates the risk of depletion of non-renewable resources and leads to the burden on the ecological environment. 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 β-alanine production. Existing research mainly focuses on the genetic modification of enzyme-catalyzing genes in the β-alanine synthesis pathway and the central carbon metabolism 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 influence of transcriptional regulators on β-alanine biosynthesis not only helps to reveal its metabolic regulation mechanism, but may also discover new efficient modification targets, providing new ideas for the industrial production of β-alanine.

[0004] Corynebacterium glutamicum ( Corynebacterium glutamicum ) as an important industrial microorganism 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 β-alanine biosynthesis. Based on its mature genetic operation system and high safety, Corynebacterium glutamicum has strong application potential in the industrial production of β-alanine. 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 strengthening the transcriptional regulator in Corynebacterium glutamicum Cgl2988Method for increasing β-alanine production by expression

[0006] In a specific embodiment, the expression of the transcriptional regulator in Corynebacterium glutamicum Cgl2988 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 application of the transcriptional regulator Cgl2988 in increasing the production of β-alanine in Corynebacterium glutamicum.

[0008] Specifically, it is to enhance the expression of the transcriptional regulator in Corynebacterium glutamicum Cgl2988 to achieve an increase in the production of β-alanine.

[0009] The present invention also provides a method for increasing the production of β-alanine by Corynebacterium glutamicum, which is to enhance the expression of the transcriptional regulator in Corynebacterium glutamicum capable of synthesizing β-alanine Cgl2988 to obtain a recombinant Corynebacterium glutamicum with increased β-alanine production.

[0010] Specifically, the enhancement of the expression of the transcriptional regulator Cgl2988 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 regulator Cgl2988 is as shown in SEQ ID NO.1.

[0012] More preferably, the coding nucleotide sequence of the transcriptional regulator Cgl2988 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 regulator Cgl2988 and transforming Corynebacterium glutamicum capable of synthesizing β-alanine to obtain a recombinant bacterium, and culturing the recombinant bacterium to produce β-alanine.

[0014] Optionally, it further includes the step of separating the produced β-alanine.

[0015] Among them, the Corynebacterium glutamicum capable of synthesizing β-alanine is obtained by introducing the key enzyme gene of the β-alanine synthesis pathway, aspartate-α-decarboxylase gene (such as the panD bs gene) from Bacillus subtilis into the starting strain of Corynebacterium glutamicum.

[0016] Preferably, the gene of aspartate-α-decarboxylase, a key enzyme in the synthetic β-alanine pathway, is introduced, and preferably, the gene sequence is codon-optimized according to Corynebacterium glutamicum.

[0017] The advantage of the present invention is that the enhanced expression of the transcriptional regulatory factor Cgl2988 in Corynebacterium glutamicum increases the β-alanine production in the engineered strain CG19 of Corynebacterium glutamicum, and the verification in the experiment shows that it can be increased by up to 211% at most. Description of the Drawings

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

[0019] Figure 2 is the map of the overexpression of pEC-Cgl2988.

[0020] Figure 3 is the fermentation schematic diagram of the strain CGTF2988 under the microplate condition.

[0021] Figure 4 is the fermentation schematic diagram of the strain CGTF2988 under the shake flask condition.

[0022] Figure 5 is the peak map of the β-alanine standard.

[0023] Figure 6 is the result peak map of the total amount of β-alanine after sampling and treatment of CG19WT after 60 h of shake flask fermentation.

[0024] Figure 7 is the result peak map of the total amount of β-alanine after sampling and treatment of CGTF2988 after 60 h of shake flask fermentation. Detailed Embodiments

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

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

[0029] Molecular biology reagents such as restriction endonucleases, dephosphorylases, DNA ligases, etc. were purchased from Thermo (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, 10 g of NaCl, and make up 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 to 1 L with distilled water. Autoclave at 121 °C for 20 min.

[0032] BHI medium: Weigh 37 g of bovine brain heart infusion powder, 10 g of (NH4)2SO4, 0.2 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.3 g of NaH2PO4, make up to 1 L with distilled water, adjust the pH to 7.2, and 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 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] CGXⅡ medium: Weigh 10 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, make up to 1 L with distilled water, adjust the pH to 7.0, and 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 CG19 Although wild-type Corynebacterium glutamicum has a potential metabolic pathway for synthesizing β-alanine, under natural conditions, the gene encoding aspartate-α-decarboxylase (ADC, encoded by panD gene) has a low gene expression level and insufficient enzyme activity, resulting in the inability to effectively accumulate β-alanine through this pathway. It is necessary to introduce exogenous panDEnhancing the expression of key enzymes by genes to achieve the accumulation of β-alanine. In the present invention, the plasmid basic vector pXMJ19-panDbs capable of synthesizing β-alanine was constructed. Based on the reported ADC (NCBI-ProteinID: UUV45455.1) amino acid sequence (SEQ ID No.3) of Bacillus subtilis ( Bacillus subtilis ), the coding gene of ADC was optimized according to the codon preference of Escherichia coli, and the optimized panD bs gene (SEQ ID No.4) was obtained and sent to Genewiz Biotechnology (Beijing) Co., Ltd. for synthesis and ligated together with the 5'-terminal flanking sequence "AAAGGAGGACAACC" between the restriction enzyme sites PstI and XbaI of the pXMJ19 vector to construct the pXMJ19-panDbs plasmid. The final plasmid map of pXMJ19-panDbs is as shown in Figure 1 .

[0036] The structure of the recombinant expression vector pXMJ19-panDbs is described as: the recombinant vector obtained after inserting the optimized panD bs gene (SEQ ID No.4) between the restriction enzyme sites PstI and XbaI of the pXMJ19 vector.

[0037] The pXMJ19-panDbs was transferred into the Corynebacterium glutamicum ATCC 13032 strain by electrotransformation to obtain the recombinant strain CG19.

[0038] Example 2: Obtaining the regulatory factor modification target Based on the reported high-quality genome-scale metabolic network model of Corynebacterium glutamicum i CW773 (PMID: 28680478), adding the heterologous reaction information of β-alanine 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 β-alanine biosynthesis was carried out. The prediction results showed that upregulating the expression level of the transcriptional regulatory factor Cgl2988 might increase the synthesis of β-alanine. Cgl2988 Belongs to the MarR family of regulatory factors, and the physiological and biochemical functions of the regulatory factors in this family mainly involve redox sensing based on cysteine oxidation and the regulation of substrate metabolism. Literature research shows that Cgl2988 is involved in the stress-responsive remodeling of the cell envelope in Corynebacterium glutamicum (PMID: 31164873), and binds to the promoter region of the malic enzyme gene malE as the MalR transcriptional regulatory factor. OverexpressingCgl2988 will reduce the malE expression level, while knocking out Cgl2988 has a positive impact on the malE expression level, indicating that this regulatory factor has a repressive effect on the malE gene (PMID: 22261175). However, there is currently no direct evidence showing the specific functions of this regulatory factor and the malE gene it regulates in the process of β-alanine synthesis, and its potential role still needs to be further studied. Subsequently, wet experiments were conducted to verify whether overexpressing the transcriptional regulatory factor Cgl2988 can improve β-alanine synthesis.

[0039] Example 3: Obtaining the plasmid for overexpressing the transcriptional regulatory factor Cgl2988 The plasmid pEC-Cgl2988 for overexpressing the transcriptional regulatory factor was constructed using the plasmid pECXK99E as the basic vector. Using the primers Cgl2988-F (SEQ ID NO.5) / Cgl2988-R (SEQ ID NO.6), pEC-F (SEQ ID NO.7) / pEC-R (SEQ ID NO.8) shown in Table 1 as the upstream and downstream primers respectively, the Cgl2988 fragment and the pEC fragment were amplified, and then the two fragments were recombined by homologous recombination to obtain the plasmid pEC-Cgl2988, and its map is shown in Cgl2988 Figure 2 Figure 2 .

[0040] Table 1

[0041] Example 4: Construction of β-alanine producing strain and its flask fermentation (1) Construction of β-alanine producing strain The plasmid pEC-Cgl2988 with correct sequencing results was electrotransformed into Corynebacterium glutamicum CG19 and evenly spread on the LBHIS solid plate with Cm and Kan resistances. Single colonies were picked respectively and PCR verified with the following primers test-F (SEQ ID NO.9: AGCTGTGGTATGGCTGTG) / test-R (SEQ ID NO.10: tggcagttccctactctc).

[0042] The β-alanine producing strain CGTF2988 with the plasmid pEC-Cgl2988 inserted was the one with correct sequencing. CGTF2988 is a β-alanine producing strain based on CG19 overexpressing the transcriptional regulatory factor Cgl2988 in Corynebacterium glutamicum. CG19WT is a control strain based on CG19 inserted with the empty plasmid pECXK99E without the regulatory factor.

[0043] (2) Shake flask fermentation of the production strain Perform microplate and shake flask fermentation on strains CG19WT and CGTF2988.

[0044] Inoculation method: First, streak CG19WT and CGTF2988 on a BHIS solid medium and place it in an incubator at 30 °C for about 18 h. Pick a single colony from the plate and inoculate it into 1 mL of BHI liquid medium. Incubate at 30 °C and 220 rpm for about 12 h. Take 0.1 mL and transfer it to 10 mL of BHI liquid medium and continue to incubate for 12 h. Inoculate the bacterial solution into the prepared CGXII fermentation medium at an initial concentration of 30 g / L of glucose at 1% and place it in a constant temperature shaker at 30 °C and 220 rpm for oscillating culture for 4 - 6 h until the OD 600 is about 0.8, then add the inducer (IPTG). After culturing for 36 h, measure its β-alanine production (see Figure 3 ), and the strain CGTF2988 overexpressing the transcriptional regulatory gene Cgl2988 increased by 83% compared to the control strain CG19WT (0.93 g / L) and reached 1.7 g / L. Subsequently, a comparison was made under further shake flask conditions. Samples were taken every 12 h to measure the β-alanine shake flask production (see Figure 4 ), the β-alanine peak map (see Figure 5 , 6 and 7). The results showed that the production of strain CGTF2988 was further improved, and 3.45 g / L of β-alanine could be accumulated in 60 h, which was 211% higher than that of the control strain CG19WT (1.11 g / L).

[0045] The strain codes in the present invention, such as CG19WT, CGTF2988, etc., are for convenience of description, but should not be construed as a limitation of the present invention.

[0046] 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 fall within the protection scope of the present invention.

Claims

1. Use of transcriptional regulator Cgl2988 in increasing the β-alanine production of Corynebacterium glutamicum.

2. The application according to claim 1, characterized in that, It is to enhance the expression of transcriptional regulator Cgl2988 in Corynebacterium glutamicum to achieve an increase in β-alanine production.

3. A method for increasing the production yield of β-alanine by Corynebacterium glutamicum, characterized in that: By enhancing the expression of transcriptional regulator Cgl2988 in Corynebacterium glutamicum capable of synthesizing β-alanine, a recombinant Corynebacterium glutamicum with increased β-alanine production is obtained.

4. The method according to claim 3, wherein The enhancement of the expression of transcriptional regulator Cgl2988 is achieved by plasmid overexpression, increasing the genomic expression copy number, or replacing the enhancer element.

5. The method according to claim 3, characterized in that The amino acid sequence of transcriptional regulator Cgl2988 is as shown in SEQ ID NO.

1.

6. The method according to claim 3, wherein The coding nucleotide sequence of transcriptional regulator Cgl2988 is as 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 transcriptional regulator Cgl2988, transforming Corynebacterium glutamicum capable of synthesizing β-alanine to obtain a recombinant bacterium, and culturing the recombinant bacterium to produce β-alanine.

8. The method according to claim 7, wherein It also includes the step of isolating the produced β-alanine.

9. The method according to claim 7 or 8, characterized in that, The Corynebacterium glutamicum capable of synthesizing β-alanine is obtained by introducing the gene of the key enzyme aspartate-α-decarboxylase in the β-alanine synthesis pathway into the starting strain of Corynebacterium glutamicum.

10. The method according to claim 9, characterized in that, The introduction of the gene of the key enzyme aspartate-α-decarboxylase in the β-alanine synthesis pathway is codon-optimized according to Corynebacterium glutamicum.

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