Bacillus amyloliquefaciens theophylline ribose switch regulation and control system and application thereof

Abstract

The invention discloses a bacillus amyloliquefaciens riboswitch regulation and control system and application thereof. The regulation and control system is composed of a P43 promoter, a theophylline aptamer TC, RBS, RepA-tag and a required regulation and control gene. A theophylline ribose switch regulation element is integrated on the upstream of a bacillus amyloliquefaciens regulation gene, and the expression quantity of the gene is precisely regulated through theophylline induced expression. Furthermore, a theophylline ribose switch regulation element is integrated to the upstream of the genes hemX and mtnN to obtain the dual-regulation engineering bacterium HZ-B. Under induction of 2mM theophylline, the heme yield and the 5-adenosylmethionine (SAM) yield of the dual-regulation engineering bacterium HZ-B are remarkably increased and are respectively increased by 33.1 times and 0.5 times compared with those of an original strain HZ-12.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering technology, specifically relating to a theophylline-ribose switch regulation system of Bacillus amyloliquefaciens and its application. Background Technology

[0002] Bacillus amyloliquefaciens is a widely used industrial microorganism. Due to its non-pathogenicity, strong secretory capacity, and lack of codon bias, it can be applied to food synthetic biology. With the development of biotechnology, building efficient cell factories has become a core goal, making the development of rapid and efficient synthetic biology tools a research hotspot in the field of Bacillus amyloliquefaciens. Gene regulation technologies such as CRISPRi, riboswitches, and synthetic promoters can precisely regulate gene expression and are widely used in Escherichia coli or yeast host systems. However, gene regulation technologies rely on the high transformation efficiency of the host bacteria; currently, the transformation efficiency of Bacillus amyloliquefaciens is generally low, limiting the application of gene regulation technologies in Bacillus amyloliquefaciens.

[0003] This invention, based on riboswitch gene regulatory elements, constructs a theophylline riboswitch regulatory system in Bacillus amyloliquefaciens. This system precisely regulates gene expression levels by adding different concentrations of theophylline, thereby further regulating gene expression. hemX and mtnN Amyloliquefaciens strain that yields increased production of both heme and 5-adenosylmethionine (SAM). Summary of the Invention

[0004] This invention protects a theophylline riboside switch regulatory system of Bacillus amyloliquefaciens, the regulatory system comprising a theophylline riboside switch regulatory element (named P43TCR, sequence shown in SEQ ID NO.1) composed of a P43 promoter, theophylline aptamer TC, RBS, and RepA-tag, and the gene to be regulated.

[0005] Another aspect of this invention protects an engineered strain of Bacillus amyloliquefaciens containing a theophylline-ribose switch control system, wherein the engineered strain is Bacillus amyloliquefaciens HZ-12. hemX Gene (SEQ ID NO.2) and mtnN The theophylline riboswitch regulatory element is integrated upstream of the gene (SEQ ID NO.3). The theophylline riboswitch regulatory element consists of the P43 promoter, theophylline aptamer TC, RBS, and RepA-tag, and its sequence is shown in SEQ ID NO.1.

[0006] This invention also protects the application of the aforementioned engineered Bacillus amyloliquefaciens containing the theophylline riboside switch control system in the production of heme and 5-adenosylmethionine (SAM). Cultivating the engineered bacteria in a fermentation medium supplemented with theophylline can simultaneously increase the yield of heme and SAM. In a specific embodiment of this invention, when the theophylline concentration is 2 mM, the yields of heme and SAM reach their maximum, at 1.75 mg / L and 12.21 mg / L, respectively, representing increases of 33.1 times and 0.5 times compared to the starting strain HZ-12.

[0007] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Based on the riboside switch gene regulatory element, the theophylline riboside switch regulatory system of Bacillus amyloliquefaciens was constructed for the first time.

[0008] (2) It was discovered for the first time that the system based on theophylline ribose switch regulation system can significantly increase the production of heme and SAM from Bacillus amyloliquefaciens. Attached Figure Description

[0009] Figure 1 This is a schematic diagram of the theophylline-ribose switching system.

[0010] Figure 2 In order to be in hemX Validation gel image of upstream integration of the P43TCR regulatory element. Lane M: DL5000 DNAMaker; Lane 1: PCR results of control strain HZ-12; Lane 2: PCR results of engineered strain HZ-A.

[0011] Figure 3 In order to be in mtnN Validation gel image of upstream integration of the P43TCR regulatory element. Lane M: DL5000 DNAMaker; Lane 1: PCR results of control strain HZ-A; Lane 2: PCR results of engineered strain HZ-B.

[0012] Figure 4 The results show the heme and SAM production of the dual-regulated strain HZ-B. Detailed Implementation

[0013] The present invention will be described in detail below through embodiments, but all embodiments do not constitute any limitation on the present invention.

[0014] Example 1: Construction and regulation of theophylline riboswitch in Bacillus amyloliquefaciens hemX Gene A riboswitch is a gene regulatory element with a structured non-coding RNA domain. The theophylline riboswitch consists of a theophylline aptamer and a downstream gene expression platform, functioning based on the presence of theophylline in the cellular environment. For example... Figure 1As shown, the secondary structure of theophylline aptamer is stem-like RNA. In the absence of theophylline, the formation of the intrinsic terminator is inhibited, leading to normal transcription readout and downstream gene expression. When theophylline binds to the aptamer, an endogenous terminator is formed and transcription is prematurely stopped, resulting in the inhibition of downstream gene expression. The theophylline riboswitches sequence reported in the literature (WANG X, FANG C, WANG Y, et al. Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression [J]. 2023, 11(1): e02752-02722.) was selected, and the P43 promoter was fused with the theophylline aptamer TC, RBS and RepA-tag to obtain gene regulatory elements ( Figure 1 It was named P43TCR, and its gene sequence is shown in SEQ ID NO.1.

[0015] HemX HemA is a complete membrane protein that negatively regulates the intracellular homeostatic concentration of HemA protein. HemA is a key enzyme in the synthesis of ALA (5-aminolevulinic acid). It is regulated by the theophylline-riboside switch. hemX Gene expression promotes ALA synthesis, thereby enhancing heme synthesis. This is based on the regulatory genes... hemX Based on the sequence, primers for two homologous arms of the P43TCR gene were designed: AF / R (SEQ ID NO. 4 and 5) and BF / R (SEQ ID NO. 6 and 7). Using Bacillus amyloliquefaciens HZ-12 genomic DNA as a template, the upstream and downstream homologous arms (A, B) were amplified, and the gene sequences of homologous arms A and B are shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively.

[0016] Homologous arms A (SEQ ID NO.8), P43TCR (SEQ ID NO.1), and homologous arm B (SEQ ID NO.9) were sequentially ligated by SOE-PCR. After product recovery and purification, the SOE-PCR fragment was double-digested with restriction endonucleases XbaI and BamHI, followed by further purification and recovery. Simultaneously, the temperature-sensitive knockout plasmid T2(2)-ori was treated using the same double-digestion method. The digested fragments and plasmid were then ligated using T4 DNA ligase and incubated at 25°C for 2 h. After ligation, the ligation product was converted to calcium. E. coliDH5α competent cells. Colony PCR was performed on transformed single colonies using T2-F / R primers (SEQ ID NO. 10 and 11). After preliminary confirmation as positive transformants, plasmids were extracted for double enzyme digestion and DNA sequencing verification. The plasmid that was verified was named T2::P43TCR-hemX.

[0017] Subsequently, the T2::P43TCR-hemX plasmid was electroporated into Bacillus amyloliquefaciens HZ-12 competent cells. All cells were collected and plated on kanamycin (Kan) resistant plates and incubated at 37°C for 16-20 h. Universal plasmid validation primers T2-F / R and integration fragment validation primers AF / BR (SEQ ID NO. 4 and 7) were designed. The two primer pairs were cross-validated for colony transformation, and transformants with correct bands were selected for single and double cross-exchange screening. Using AF / BR as primers, the selected transformants were verified by PCR. PCR products were detected by agarose gel electrophoresis. Figure 2 As shown, the difference in band size between the constructed engineered strain and the control strain (HZ-12) is exactly the size of the P43TCR. The successfully constructed integrated engineered strain was named Bacillus amyloliquefaciens HZ-A.

[0018] Example 2: Regulation of Bacillus amyloliquefaciens using theophylline-riboside switches mtnN Gene SAM is an important cofactor in heme synthesis, participating in the catalytic reaction of HemN protein to generate protoporphyrinogen IX, an intermediate in heme biosynthesis. (Gene) mtnN Encoding 5'-methylthioadenosine / S-adenosine homocysteine ​​nucleosidase, regulated by theophylline riboswitch mtnN Genes reduce the consumption of SAM in downstream pathways, providing sufficient cofactors for heme synthesis.

[0019] according to mtnN For gene sequencing, primers for two homologous arms of the P43TCR gene were designed: CF / R (SEQ ID NO. 12 and 13) and DF / R (SEQ ID NO. 14 and 15). Using Bacillus amyloliquefaciens HZ-12 genomic DNA as a template, the upstream and downstream homologous arms (C, D) were amplified, respectively. The gene sequences of homologous arms C and D are shown in SEQ ID NO. 16 and SEQ ID NO. 17.

[0020] The P43TCR integration plasmid was constructed using the same method as in Example 1 and named T2::P43TCR-mtnN. Subsequently, T2::P43TCR-mtnN was transformed into HZ-A competent cells using the same electroporation method. Validation primers CF / DR (SEQ ID NO. 12 and 15) were designed to perform PCR validation on the final screened strains. The PCR products were detected by agarose gel electrophoresis. Figure 3 As shown, the band differences between the constructed engineered strain and the control strain (HZ-A) correspond to the size of the P43TCR. This proves that the P43TCR integration was successful, and the latest integrated engineered strain was named HZ-B.

[0021] Example 3: Application of the theophylline-ribose switch control system in increasing heme and SAM production The dual-regulated strain HZ-B was prepared in MRFe medium (20 g / L glucose, 5 g / L yeast extract, 5 g / L L-glutamic acid monohydrate, 4 g / L (NH4)2HPO4, 6.67 g / L KH2PO4, 0.8 g / L citric acid, 0.8 g / L MgSO4·7H2O, 20 mg / L FeSO4·7H2O, 5 mL / L metal solution (10 g / L FeSO4·7H2O, 2.2 g / L ZnSO4·7H2O, 1 g / L CuSO4·5H2O, 2 g / L CaCl2, 0.5 mol / L HCl, 0.5 g / L MnSO4·4H2O, 0.1 g / L (NH4)6Mo7O)). 24 Fermentation was carried out in 4H2O and 0.02 g / L Na2B4O7·10H2O for 60 h. Different concentrations (2, 4, 6, 8, 10 mM) of theophylline solution were added before fermentation to induce fermentation. Heme and SAM yields were detected by high-performance liquid chromatography. Results are as follows: Figure 4 The results showed that the production of heme and SAM reached its maximum at a theophylline concentration of 2 mM, which were 1.75 mg / L and 12.21 mg / L, respectively, representing increases of 33.1 times and 0.5 times compared to the original strain HZ-12.

[0022] In summary, an engineered Bacillus amyloliquefaciens strain with high heme and SAM production was obtained based on the theophylline-ribose switch control system. Different concentrations of theophylline can be used to regulate... hemX and mtnN Gene expression is regulated to varying degrees, thereby promoting heme synthesis and SAM accumulation. This also demonstrates the feasibility and effectiveness of the gene regulation system based on theophylline riboswitch in Bacillus amyloliquefaciens HZ-12.

Claims

1. A Bacillus amyloliquefaciens theophylline riboswitch regulatory system, characterized in that, The regulation system comprises a theophylline riboswitch regulatory element consisting of a P43 promoter, a theophylline aptamer TC, an RBS, a RepA-tag, and a gene to be regulated, and the sequence of the regulatory element is shown as SEQ ID NO.

1.

2. The B. amyloliquefaciens engineering bacteria containing the theophylline riboswitch regulatory system of claim 1.

3. An engineered Bacillus amyloliquefaciens bacterium, characterized in that, The engineered bacteria are in hemX and mtnN The theophylline riboswitch regulatory element is integrated upstream of the gene respectively, and the theophylline riboswitch regulatory element is composed of P43 promoter, theophylline aptamer TC, RBS, RepA-tag, and the sequence is shown as SEQ ID NO.

1.

4. The use of the B. amyloliquefaciens engineering bacteria of claim 3 in the production of hemin.

5. Use according to claim 4, characterized in that, 2-10 mM theophylline is added to the fermentation medium.

6. The use of the B. amyloliquefaciens engineering bacteria of claim 3 in the production of 5-adenosylmethionine.

7. The use of claim 6, wherein 2-4 mM theophylline is added to the fermentation medium.

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