Application of transcriptional regulatory factor Cgl2988 in improving β-alanine production in Corynebacterium glutamicum

Abstract

The present invention belongs to the field of bioengineering technology and applications, and discloses the use of the transcriptional regulatory factor Cgl2988 in increasing β-alanine production in Corynebacterium glutamicum. By enhancing the expression of the transcriptional regulatory factor Cgl2988 in a strain capable of synthesizing β-alanine, β-alanine production in an engineered strain of Corynebacterium glutamicum can be significantly increased. In one experiment, enhanced expression of the transcriptional regulatory factor Cgl2988 in Corynebacterium glutamicum resulted in increased β-alanine production in the engineered strain CG19 of Corynebacterium glutamicum, with experimental verification demonstrating an increase of up to 211%, demonstrating its application value.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering technology and application, and particularly relates to the application of a transcriptional regulatory factor Cgl2988 in improving the beta-alanine production of Corynebacterium anguinale. Background Art

[0002] β-alanine is the only naturally occurring β-amino acid. It is a raw material for the synthesis of pharmaceuticals such as vitamin B5, guanidinopropionic acid, balsalazide, and carnosine. It can also be used as a food additive to improve food flavor, and has a wide range of applications in the pharmaceutical, chemical, and food industries. Currently, the main production methods for β-alanine are chemical synthesis methods, including the acrylate method, acrylic acid method, and iminodipropylene method. These reactions rely on extreme environments such as high temperature and 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 relies primarily on chemical synthesis, enzymatic methods, and microbial fermentation. Chemical synthesis is difficult to achieve on a large scale due to expensive raw materials and safety concerns. Enzymatic methods ultimately require conversion to a fumaric acid intermediate, and large-scale production increases the risk of non-renewable resource depletion and places a strain burden on the environment. In recent years, microbial fermentation, based on metabolic engineering and synthetic biology techniques, has become a research hotspot. This approach, which involves constructing engineered strains that produce high β-alanine yields, offers advantages such as low cost, readily available raw materials, and environmental friendliness. Existing research has primarily focused on genetic engineering of enzymes involved in the β-alanine biosynthesis pathway and central carbon metabolism, while the role of transcriptional regulators has been underexplored. Transcriptional regulators play a central role in cellular metabolic networks, orchestrating the expression of multiple genes to optimize metabolic flux distribution and resource allocation, thereby significantly improving the efficiency of target product synthesis. Therefore, in-depth research into the impact of transcriptional regulators on β-alanine biosynthesis will not only help reveal its metabolic regulatory mechanisms but also potentially identify new, efficient targets for engineering, providing new approaches for the industrial production of β-alanine.

[0004] Corynebacterium glutamicum ( Corynebacterium glutamicum As an important industrial microorganism, Corynebacterium glutamicum is widely used in the production of chemicals and biomaterials such as amino acids and organic acids. Its food-safe properties make it an ideal strain for production in the food and cosmetics sectors. It also serves as a promising platform for the biosynthesis of β-alanine. Due to its mature genetic manipulation system and high safety profile, Corynebacterium glutamicum has strong potential for industrial production of β-alanine. 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. Cgl2988A method for increasing β-alanine production by expressing

[0006] In a specific embodiment, the enhanced transcriptional regulatory factor in Corynebacterium glutamicum Cgl2988 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:

[0008] The present invention first provides a transcriptional regulatory factor Cgl2988 Application in improving β-alanine production in Corynebacterium glutamicum.

[0009] Specifically, it is to enhance the transcriptional regulatory factor in Corynebacterium glutamicum Cgl2988 expression to increase β-alanine production.

[0010] The present invention also provides a method for increasing the yield of β-alanine produced by Corynebacterium glutamicum, which is achieved by strengthening the transcriptional regulatory factor in Corynebacterium glutamicum capable of synthesizing β-alanine. Cgl2988 to obtain recombinant Corynebacterium glutamicum with improved β-alanine production.

[0011] Specifically, the enhanced transcriptional regulatory factor Cgl2988 The expression of is achieved through plasmid overexpression, increasing the genomic expression copy number, and replacing the regulatory elements.

[0012] Preferably, the transcriptional regulatory factor Cgl2988 The amino acid sequence is shown in SEQ ID NO.1.

[0013] More preferably, the transcriptional regulatory factor Cgl2988 The encoding nucleotide sequence is shown in SEQ ID NO.2.

[0014] In a specific embodiment, plasmid overexpression is achieved by inserting a transcriptional regulatory factor Cgl2988 An expression vector is constructed by using the coding nucleotide sequence of the present invention, and the recombinant bacteria are transformed into Corynebacterium glutamicum capable of synthesizing β-alanine to obtain the recombinant bacteria, and the recombinant bacteria are cultured to produce β-alanine.

[0015] Optionally, the method further comprises the step of isolating the produced β-alanine.

[0016] Wherein, the Corynebacterium glutamicum capable of synthesizing β-alanine is obtained by introducing the key enzyme aspartate-α-decarboxylase gene (such as the gene derived from Bacillus subtilis) in the β-alanine synthesis pathway into the Corynebacterium glutamicum starting bacteria. panD bs Gene).

[0017] Preferably, the aspartate-α-decarboxylase gene, a key enzyme in the β-alanine synthesis pathway, is introduced, and preferably, the gene sequence is codon-optimized according to Corynebacterium glutamicum.

[0018] The advantage of the present invention is that the transcriptional regulatory factor in Corynebacterium glutamicum Cgl2988 The enhanced expression of β-alanine increased the production of β-alanine in the engineered strain CG19 of Corynebacterium glutamicum, and experiments showed that the increase could be as high as 211%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 2 is a map of the pXMJ19-panDbs expression vector.

[0020] Figure 2 This is a map of pEC-Cgl2988 overexpression.

[0021] Figure 3 Schematic diagram of the fermentation of strain CGTF2988 under microplate conditions.

[0022] Figure 4 Schematic diagram of the fermentation of strain CGTF2988 under shake flask conditions.

[0023] Figure 5 This is the peak diagram of β-alanine standard.

[0024] Figure 6 This is the peak diagram of the total amount of β-alanine after sampling and processing of CG19WT after 60h of shake flask fermentation.

[0025] Figure 7 This is the peak diagram of the total amount of β-alanine after sampling and processing of CGTF2988 after 60 hours of shake flask fermentation. DETAILED DESCRIPTION

[0026] 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.

[0027] The original strain used in the present invention Corynebacterium glutamicum ATCC 13032 was obtained from the laboratory.

[0028] The recombinant expression plasmids pXMJ19 and pECXK99E involved in this example were purchased from BioVector NTCC (http: / / www.biovector.net / ).

[0029] β-Alanine standards were purchased from Sigma (http: / / www.sigmaaldrich.com / sigmaaldrich).

[0030] 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 / ).

[0031] 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.

[0032] 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.

[0033] BHI medium: Weigh 37 g of bovine brain heart extract powder, 10 g of (NH4)2SO4, 0.2 g of K2HPO4, 0.5 g of MgSO4·7H2O, and 0.3 g of NaH2PO4, dilute to 1 L with distilled water, adjust the pH to 7.2, and sterilize at 121°C for 20 min.

[0034] 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. Add distilled water to 1 L. Add 2% agar powder to the BHI solid medium and sterilize by high-pressure steam at 121°C for 20 min.

[0035] CGXII medium: Weigh 10 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. Bring the volume to 1 L with distilled water. Adjust the pH to 7.0 and sterilize at 121°C for 20 min. Before use, add biotin stock solution and trace element stock solution at a 1 / 1000 volume ratio.

[0036] Example 1: Construction of Corynebacterium glutamicum CG19

[0037] Although wild-type Corynebacterium glutamicum has a potential metabolic pathway for synthesizing β-alanine, it encodes aspartate-α-decarboxylase (ADC) in its natural state. panD The gene expression level of the gene encoding the gene is low and the enzyme activity is insufficient, resulting in the inability to effectively accumulate β-alanine through this pathway. panDGenes are used to enhance the expression of key enzymes to achieve the accumulation of β-alanine. The present invention constructs a plasmid basic vector pXMJ19-panDbs that can synthesize β-alanine. Based on Bacillus subtilis ( Bacillus subtilis ) reported ADC (NCBI-ProteinID: UUV45455.1) amino acid sequence (SEQ ID No. 3), the ADC coding gene was optimized according to the codon preference of E. coli, and the optimized panD bs The gene (SEQ ID No. 4) was sent to GENEWIZ Biotech (Beijing) Co., Ltd. for synthesis and ligated with the 5' flanking sequence "AAAGGAGGACAACC" between the restriction sites PstI and XbaI of the pXMJ19 vector to construct the pXMJ19-panDbs plasmid. The final pXMJ19-panDbs plasmid map is shown in the figure. Figure 1 shown.

[0038] The structure of the recombinant expression vector pXMJ19-panDbs is described as follows: panD bs The recombinant vector was obtained by inserting the gene (SEQ ID No. 4) between the restriction sites PstI and XbaI of the pXMJ19 vector.

[0039] pXMJ19-panDbs was transformed into Corynebacterium glutamicum ATCC 13032 strain by electroporation to obtain recombinant strain CG19.

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

[0041] A high-quality genome-scale metabolic network model based on the reported Corynebacterium glutamicum i CW773 (PMID: 28680478) added heterologous reaction information for β-alanine 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 β-alanine biosynthesis. The prediction results showed that the up-regulation of transcriptional regulatory factors Cgl2988 The expression level of β-alanine may be increased. Cgl2988 It belongs to the MarR family of regulatory factors. The physiological and biochemical functions of this family of regulatory factors mainly involve redox sensing based on cysteine ​​oxidation and regulation of substrate metabolism. Literature research shows that Cgl2988 Involved in stress-responsive cell envelope remodeling in Corynebacterium glutamicum (PMID: 31164873) and acts as a MalR transcriptional regulator binding to the malic enzyme gene malEThe promoter region of Cgl2988 Will reduce malE The expression level of Cgl2988 Then malE The expression level of malE However, there is currently no direct evidence that this regulatory factor and the genes it regulates have a repressive effect. malE The specific function of the gene in the process of β-alanine synthesis and its potential role still need to be further studied. Cgl2988 A wet experiment was conducted to verify whether β-alanine synthesis could be improved.

[0042] Example 3: Overexpression of transcriptional regulatory factors Cgl2988 Plasmid acquisition

[0043] Plasmid pECXK99E was used as the basic vector to construct overexpression transcriptional regulatory factors Cgl2988 The plasmid pEC-Cgl2988 was amplified using the primers Cgl2988-F (SEQ ID NO.5) / Cgl2988-R (SEQ ID NO.6) and pEC-F (SEQ ID NO.7) / pEC-R (SEQ ID NO.8) shown in Table 1 as upstream and downstream primers. Cgl2988 The fragment and the pEC fragment were then recombined by homologous recombination to obtain the plasmid pEC-Cgl2988, the map of which is shown in Figure 2 .

[0044] Table 1

[0045]

[0046] Example 4: Construction of β-alanine production strain and shake flask fermentation

[0047] (1) Construction of β-alanine-producing strain

[0048] Plasmid pEC-Cgl2988, which showed a positive sequencing result, was electroporated into Corynebacterium glutamicum CG19 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. 9: AGCTGTGGTATGGCTGTG) / test-R (SEQ ID NO. 10: tggcagttccctactctc).

[0049] The strain with the correct sequencing result is the β-alanine production strain CGTF2988 inserted into the plasmid pEC-Cgl2988. CGTF2988 is based on CG19 overexpressing the transcriptional regulatory factor in Corynebacterium glutamicum. Cgl2988 β-alanine-producing strain. CG19WT is a control strain based on CG19 inserted into the pECXK99E empty plasmid without regulatory factors.

[0050] (2) Shake flask fermentation of production strains

[0051] The strains CG19WT and CGTF2988 were fermented in microtiter plates and shake flasks.

[0052] Inoculation method: First, streak CG19WT and CGTF2988 on BHIS solid culture medium and culture in a 30℃ incubator for about 18h. Pick a single colony on the plate and inoculate it into 1mL of BHI liquid culture medium. Culture it at 30℃ and 220rpm for about 12h. Take 0.1mL and transfer it to 10mL of BHI liquid culture medium and continue to culture it for 12h. Inoculate the bacterial liquid into the prepared CGXⅡ fermentation medium at an initial concentration of 30g / L glucose at 1%, and place it in a constant temperature shaker at 30℃ and 220rpm for 4-6h to OD 600 When the pH value is about 0.8, the inducer (IPTG) is added. After culturing for 36 hours, the β-alanine production is measured (see Figure 3 ), overexpression of transcriptional regulatory genes Cgl2988 The strain CGTF2988 increased its β-alanine production by 83% to 1.7 g / L compared to the control strain CG19WT (0.93 g / L). Subsequently, further comparison was made under shake flask conditions, and samples were taken every 12 hours to measure their β-alanine shake flask production (see Figure 4 ), β-alanine peak diagram (see Figure 5 、 6 The results showed that the production of strain CGTF2988 was further improved, and it could accumulate 3.45 g / L of β-alanine in 60 h, which was a 211% increase compared to the control strain CG19WT (1.11 g / L).

[0053] The strain codes in the present invention, such as CG19WT, CGTF2988, etc., are for the convenience of description and should not be construed as limiting the present invention.

[0054] 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. The application of the transcriptional regulatory factor Cgl2988 in increasing the β-alanine production of Corynebacterium glutamicum is characterized in that: The method is to enhance the expression of the transcriptional regulatory factor Cgl2988 in Corynebacterium glutamicum which can synthesize β-alanine so as to increase the production of β-alanine; The Corynebacterium glutamicum capable of synthesizing β-alanine is prepared by introducing the gene for aspartate-α-decarboxylase, a key enzyme in the β-alanine synthesis pathway, into the Corynebacterium glutamicum starting bacteria; The amino acid sequence of the transcriptional regulatory factor Cgl2988 is shown in SEQ ID NO.

1.

2. A method for increasing the yield of β-alanine produced by Corynebacterium glutamicum, characterized in that: By enhancing the expression of the transcriptional regulatory factor Cgl2988 in Corynebacterium glutamicum that can synthesize β-alanine, a recombinant Corynebacterium glutamicum with increased β-alanine production is obtained; The Corynebacterium glutamicum capable of synthesizing β-alanine is prepared by introducing the gene for aspartate-α-decarboxylase, a key enzyme in the β-alanine synthesis pathway, into the Corynebacterium glutamicum starting bacteria; The amino acid sequence of the transcriptional regulatory factor Cgl2988 is shown in SEQ ID NO.

1.

3. The method according to claim 2, wherein The expression of the enhanced transcriptional regulatory factor Cgl2988 is achieved by overexpression of a plasmid, increasing the number of genome expression copies, or replacing a strong regulatory element.

4. The method according to claim 2, wherein The nucleotide sequence encoding the transcriptional regulatory factor Cgl2988 is shown in SEQ ID NO.

2.

5. The method according to claim 3, wherein Plasmid overexpression is achieved by constructing an expression vector with the coding nucleotide sequence of the transcriptional regulatory factor Cgl2988, transforming Corynebacterium glutamicum capable of synthesizing β-alanine to obtain a recombinant bacterium, and culturing the recombinant bacterium to produce β-alanine.

6. The method according to claim 5, wherein The process also includes the step of isolating the produced β-alanine.

7. The method according to claim 2, wherein The aspartate-α-decarboxylase gene, a key enzyme in the β-alanine biosynthesis pathway, was codon-optimized based on that of Corynebacterium glutamicum.

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