Space regulation system, beta-alanine production strain and construction method and application thereof

Abstract

This invention provides a spatial regulation system, a β-alanine-producing strain, its construction method, and its applications. The spatial regulation system includes recombinant proteins RIDD-RGG-RGG and aspC-RIAD-RIAD-panD, and the strain is obtained by knocking out wild-type Escherichia coli genomes. adhE , ldhA , ackA , poxB , lacI , thrA , panC , cycA and yfbQ Gene, heterologous introduction of β-alanine transporter gene from Corynebacterium glutamicum. NCgl0580 Overexpression of phosphoenolpyruvate carboxylase gene ppc By integrating a spatial dynamic regulatory system containing the aspartate transaminase gene aspC and the aspartate decarboxylase gene panD, the resulting strain exhibited significantly improved synthesis efficiency and production intensity.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and fermentation engineering technology, and in particular to a spatial control system, a β-alanine producing strain, its construction method and application. Background Technology

[0002] β-alanine is one of the few naturally occurring β-type amino acids and has wide applications in pharmaceuticals, food, cosmetics, environmental science, and materials science.

[0003] Currently, β-alanine is commonly produced industrially using chemical synthesis, enzymatic methods, and fermentation. While chemical methods offer advantages such as high efficiency and short cycle times, they require extreme reaction conditions, including high temperature, high pressure, and strong acids or bases, and also produce numerous byproducts and high energy consumption. Enzymatic methods suffer from low enzyme activity and are prone to mechanistic inactivation. In contrast, fermentation offers advantages such as low cost, environmental friendliness, and ease of cultivation, and has become the preferred method for β-alanine production.

[0004] Traditional metabolic engineering strategies often rely on static and global regulation of gene expression (such as promoter substitution, gene knockout / overexpression). These methods have limitations such as continuous high expression of exogenous genes leading to severe metabolic burden, inability to achieve precise spatiotemporal localization of metabolic enzymes, and lack of reversibility. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a space control system.

[0006] Another technical problem to be solved by the present invention is to provide an application of the above-mentioned space control system.

[0007] Another technical problem to be solved by the present invention is to provide a β-alanine producing strain that utilizes the above-mentioned spatial regulation system.

[0008] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned β-alanine producing strain.

[0009] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned β-alanine producing strain.

[0010] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A spatial regulation system includes recombinant proteins RIDD-RGG-RGG and aspC-RIAD-RIAD-panD, wherein the recombinant protein RIDD-RGG-RGG has a 5 ’ The disordered protein tandem RGG-RGG with RIDD short peptides added to its ends, wherein the recombinant protein aspC-RIAD-RIAD-panD has a concentration of 3.’ The recombinant proteins RIDD-RGG-RGG and aspartate transaminase gene aspC, derived from the aspartate decarboxylase gene panD from Pseudomonas aeruginosa, were sequentially added to the ends. The recombinant proteins RIDD-RGG-RGG and aspC-RIAD-RIAD-panD self-assembled through RIAD / RIDD interaction short peptides.

[0011] In the aforementioned recombinant protein RIDD-RGG-RGG, a disordered protein tandem RGG-RGG was constructed and integrated into the recombinant E. coli genome, forming a soluble protein-rich phase, i.e., a membraneless organelle. To achieve precise spatial localization of key enzymes within this membraneless organelle, RIAD / RIDD interacting short peptide pairs were introduced as a recruitment system.

[0012] Preferably, in the above-mentioned spatial regulation system, the nucleotide sequence of the disordered protein tandem RGG-RGG is shown in SEQ ID NO.3 of the sequence listing; wherein, the nucleotide sequence of the disordered protein RGG is shown in SEQ ID NO.1 of the sequence listing, and the disordered proteins RGG are linked by a linker, the nucleotide sequence of which is shown in SEQ ID NO.2 of the sequence listing.

[0013] Preferably, in the above-mentioned spatial regulation system, the nucleotide sequence of RIDD in the interacting short peptide is shown in SEQ ID NO.4, and the nucleotide sequence of RIAD is shown in SEQ ID NO.5.

[0014] Preferably, in the above-mentioned spatial regulation system, the nucleotide sequence of the fusion protein of the RIDD short peptide and the disordered protein (i.e., the recombinant protein RIDD-RGG-RGG) is shown in SEQ ID NO.6 of the sequence listing.

[0015] Preferably, in the above spatial regulation system, the recombinant proteins aspC-RIAD-RIAD-panD are all linked by a linker, and the nucleotide sequence of the linker is shown in SEQ ID NO.2 of the sequence listing.

[0016] Preferably, in the above-mentioned spatial regulation system, the nucleotide sequence of the aspartate transaminase gene aspC is shown in SEQ ID NO.7, and the nucleotide sequence of the aspartate decarboxylase gene panD from Pseudomonas aeruginosa is shown in SEQ ID NO.8.

[0017] Preferably, in the above-mentioned spatial regulation system, the Ptrc promoter is linked to the first position of the aspartate transaminase gene aspC, followed by two RIADs, and finally linked to the aspartate decarboxylase gene panD from Pseudomonas aeruginosa, forming a recombinant protein sequence named Ptrc-aspC-RIAD-RIAD-panD. The nucleotide sequence of this recombinant protein sequence is shown in SEQ ID NO. 9 of the sequence listing.

[0018] In the aforementioned spatial regulatory system, the Ptrc promoter is linked first to the aspartate transaminase gene aspC, followed by two RIADs, and finally linked to the aspartate decarboxylase gene panD from Pseudomonas aeruginosa. panD and the rrnB T1 terminator form the recombinant protein Ptrc-aspC-RIAD-RIAD-panD. The nucleotide sequence of the Ptrc promoter is shown in SEQ ID NO.21 of the sequence listing, and the nucleotide sequence of the rrnB T1 terminator is shown in SEQ ID NO.22 of the sequence listing.

[0019] The application of the above-mentioned spatial regulation system in the construction of β-alanine-producing strains and / or the production of β-alanine.

[0020] A β-alanine-producing strain, named E. coli Al3-2 is a recombinant E. coli chassis constructed using gene editing technology, specifically based on wild-type E. coli. Escherichia coli W3110 Knockout of acetaldehyde dehydrogenase gene in the genome adhE lactate dehydrogenase gene ldhA Acetylkinase gene ackA pyruvate dehydrogenase gene poxB The gene of lactose operon repressor protein lacI Aspartate kinase gene thrA pantothenic acid synthase gene panC Alanine transaminase gene cycA Alanine transaminase gene yfbQ Heterogeneous introduction of Corynebacterium glutamicum ( Corynebacterium glutamicum β-alanine transporter gene derived from K051) NCgl0580 Overexpression of phosphoenolpyruvate carboxylase gene ppc Integrates recombinant protein RIDD-RGG-RGG and recombinant protein aspC-RIAD-RIAD-panD.

[0021] Preferably, the above-mentioned β-alanine producing strains all use the Ptrc promoter.

[0022] Preferably, the above-mentioned β-alanine-producing strains, respectively, in the genome mbhA site andycdN Integrating the phosphoenolpyruvate carboxylase gene at the site ppc (NCBI-Gene ID: 948457), and initiated by the Ptrc promoter; in the genome rph Integrating the β-alanine transporter gene from Corynebacterium glutamicum at the site NCgl0580 (NCBI-Gene ID: 1018609), and is started by the Ptrc promoter.

[0023] Preferably, in the above-mentioned β-alanine-producing strain, the acetaldehyde dehydrogenase gene... adhE The nucleotide sequence is shown in SEQ ID NO. 10 of the sequence listing; lactate dehydrogenase gene ldhA The nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing; Acetylkinase gene ackA The nucleotide sequence is shown in SEQ ID NO. 12 of the sequence listing; pyruvate dehydrogenase gene poxB The nucleotide sequence is shown in SEQ ID NO. 13 of the sequence listing; aspartate kinase gene. thrA The nucleotide sequence is shown in SEQ ID NO. 14 of the sequence listing; pantothenic acid synthase gene panC The nucleotide sequence is shown in SEQ ID NO.15 of the sequence listing; alanine transaminase gene. cycA The nucleotide sequence is shown in SEQ ID NO.16 of the sequence listing; alanine transaminase gene. yfbQ The nucleotide sequence is shown in SEQ ID NO.17 of the sequence listing; phosphoenolpyruvate carboxylase gene ppc The nucleotide sequence is shown in SEQ ID NO.18 of the sequence listing; β-alanine transporter gene from Corynebacterium glutamicum. NCgl0580 The nucleotide sequence is shown in SEQ ID NO.19 of the sequence listing; the gene for the lactose operon repressor protein. lacI The nucleotide sequence of the aspartate transaminase gene aspC is shown in SEQ ID NO.20; the nucleotide sequence of the aspartate decarboxylase gene aspC is shown in SEQ ID NO.7; and the nucleotide sequence of the aspartate decarboxylase gene panD from Pseudomonas aeruginosa is shown in SEQ ID NO.8.

[0024] The specific steps for constructing the above-mentioned β-alanine-producing strain are as follows: (1) Wild-type Escherichia coli Escherichia coli W3110 For the starting strain, the acetaldehyde dehydrogenase gene was knocked out of its genome. adhE lactate dehydrogenase gene ldhA Acetylkinase gene ackA pyruvate dehydrogenase gene poxBThe gene of lactose operon repressor protein lacI Aspartate kinase gene thrA pantothenic acid synthase gene panC Alanine transaminase gene cycA Alanine transaminase gene yfbQ ; (2) Overexpression of phosphoenolpyruvate carboxylase gene twice ppc ; (3) Heterogeneous introduction of Corynebacterium glutamicum ( Corynebacterium glutamicum β-alanine transporter gene derived from K051) NCgl0580 ; (4) Integrate the RIDD-RGG-RGG gene and the aspC-RIAD-RIAD-panD gene respectively.

[0025] Application of the above-mentioned β-alanine-producing strains in the production of β-alanine.

[0026] Preferably, the above-mentioned application uses a fermentation method to produce β-alanine.

[0027] Preferably, in the above application, shake-flask fermentation is carried out: seed liquid is inoculated into the fermentation medium at an inoculation rate of 10%-15%, and cultured by shaking. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water, and glucose solution is added to maintain the fermentation.

[0028] Preferably, the specific steps for shake-flask fermentation in the above application are as follows: (1) Activation of bacterial strain: Inoculate the bacterial solution from the preservation tube into the slant medium for activation culture; use a sterilized inoculation loop to scrape off a loopful of colonies from the slant medium and transfer it into a shaker containing seed culture medium for seed culture; (2) Pour the bacterial culture from the seed culture medium shaker into the Erlenmeyer flask containing the fermentation culture medium, seal the flask with nine layers of gauze, and culture at 37°C and 220 r / min. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water; 60% glucose solution is added to maintain the fermentation; the fermentation cycle is 24-32h (preferably 24h).

[0029] Preferably, the slant culture medium used in the above application consists of: glucose 2 g / L, yeast extract 5 g / L, peptone 10 g / L, NaCl 2.5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.2 g / L, agar powder 2.5 g / L, and the remainder being water.

[0030] Preferably, in the above application, the seed culture medium used is: yeast extract 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, VB1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water.

[0031] Preferably, the fermentation medium used in the above application is: glucose 30.0 g / L, yeast powder 4.0 g / L, peptone 2.0 g / L, ammonium sulfate 2.0 g / L, citric acid 2.0 g / L, monosodium glutamate 2.0 g / L, MgSO4·7H2O 1.2 g / L, KH2PO4 3.0 g / L, threonine 0.5 g / L, MnSO4·H2O 5.0 mg / L, FeSO4·7H2O 15 mg / L, phenol red 2.5%, and the remainder is water.

[0032] All of the above-mentioned culture media can be prepared using standard methods.

[0033] Beneficial effects: The aforementioned spatial regulation system, serving as an artificial membraneless organelle spatial recruitment system, utilizes a dynamic spatial metabolic regulation strategy based on liquid-liquid phase separation (LLPS) technology to spatially locate key enzymes in the β-alanine synthesis pathway. This achieves compartmentalized reconstruction of the metabolic pathway, significantly improving the throughput and efficiency of the target pathway while reducing cellular metabolic load. It overcomes bottlenecks in traditional metabolic engineering, such as metabolic flux dispersion, intermediate product loss, and competition from side reactions. The resulting β-alanine-producing strain exhibits significantly improved synthesis efficiency and production intensity, shortening the β-alanine production cycle. Specifically: This invention optimizes the disordered protein RGG and then repeats individual RGG modules in tandem (RGG-RGG) to enhance its multivalentity and phase-separation ability, making the formed aggregates more stable. Furthermore, this invention assembles the aspartate transaminase gene aspC and the aspartate decarboxylase gene panD from *Pseudomonas aeruginosa* through RIAD / RIDD interactions to form a multi-enzyme condensate. Key enzymes in the β-alanine synthesis pathway are precisely located within membraneless organelles, achieving spatial co-assembly and compartmentalized distribution of metabolic enzymes. This strategy effectively increases the concentration of local reactants, promotes the directional transport of substrates and the efficient conversion of intermediates, thereby significantly enhancing the synthesis efficiency of β-alanine. This system not only provides a new pathway for the green production of β-alanine but also offers a feasible approach for constructing a synthetic biology and metabolic engineering platform based on phase-separation regulation. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the terminal metabolic pathway of β-alanine and its modification.

[0035] Figure 2 Image of pET-28a-WM plasmid. Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0037] Unless otherwise specified, the percentage sign "%" in the examples refers to volume percentage; the percentage of a solution "% (m / v)" refers to the number of grams of solute contained in 100 ml of solution.

[0038] The starting strain used in the examples is E. coli W3110 ATCC 27325; Codon-optimized Aspartate Decarboxylase Gene of Pseudomonas aeruginosa panD Synthesized by Suzhou Genewiz Biotechnology Co., Ltd.; the corresponding promoter and gene are shown in the sequence listing. Primers used in the strain construction process are shown in Table 1.

[0039] The gene editing method used is referenced in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). Unless otherwise specified, all technical terms used in this invention are explained in this article. In this invention, "knockout" refers to the inactivation of the target gene, and "integration" refers to the insertion of a foreign gene into the engineered bacterial genome after linking it with a promoter and terminator.

[0040] Table 1 Primers used in strain construction

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] Example 1 Constructing β-alanine chassis-producing strains, such as Figure 1 As shown, the specific process for constructing genetically engineered strains is as follows: E. coli UR12 / pRed-Cas9 electrocompetent cells were prepared using the starting strain E. coli W3110 ATCC 27325 according to the procedures described in CN 202411087442.4. Figure 1 As shown, the specific steps for constructing the genetically engineered strain E. coli Al1-12 are as follows: 1.1 Knockout of the thrA gene: Using the W3110 genome as a template, upstream and downstream homologous arms were amplified by PCR using thrA-US, thrA-UA and thrA-DS, thrA-DA primers, respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were amplified by overlapping PCR using thrA-US and thrA-DA primers. The gRNA fragment was annealed using pGRB-thrA-S and pGRB-thrA-A primers and ligated into the pGRB vector to obtain thrA-pGRB. Electroporation competent cells of the strain to be modified were prepared, and the overlapping fragment and thrA-pGRB were electroporated into competent cells together. Positive transformants were screened to obtain the thrA gene knockout, and the strain E. coli Al1-1 was finally obtained.

[0047] 1.2 Knockout of the adhE gene: The same operation method as in step 1.1 is used, except that the primers used are adhE-US, adhE-UA, adhE-DS, adhE-DA, pGRB-adhE-S, and pGRB-adhE-A. The cells are electroporated into E. coli Al1-1 / pRed-Cas9 electroporation competent cells to finally obtain strain E. coli Al1-2.

[0048] 1.3 Knockout of the ldhA gene: The operation method is the same as in step 1.1, except that the primers used are ldhA-US, ldhA-UA, ldhA-DS, ldhA-DA, pGRB-ldhA-S, and pGRB-ldhA-A. The cells are electroporated into E. coli Al1-2 / pRed-Cas9 electroporation competent cells to finally obtain strain E. coli Al1-3.

[0049] 1.4 Knockout of the ackA gene: The same operation method as in step 1.1 is used, except that the primers used are ackA-US, ackA-UA, ackA-DS, ackA-DA, pGRB-ackA-S, and pGRB-ackA-A. The cells are electroporated into E. coli Al1-3 / pRed-Cas9 electroporation competent cells to finally obtain strain E. coli Al1-4.

[0050] 1.5 Knockout of the poxB gene: The procedure is the same as in step 1.1, except that the primers used are poxB-US, poxB-UA, poxB-DS, poxB-DA, pGRB-poxB-S, and pGRB-poxB-A. The cells are electroporated into E. coli Al1-4 / pRed-Cas9 electroporation competent cells to finally obtain strain E. coli Al1-5.

[0051] 1.6 Knockout of the cycA gene: The operation method is the same as in step 1.1, except that the primers used are cycA-US, cycA-UA, cycA-DS, cycA-DA, pGRB-cycA-S, and pGRB-cycA-A. The cells are electroporated into E. coli Al1-5 / pRed-Cas9 electroporation competent cells to finally obtain strain E. coli Al1-6.

[0052] 1.7 Knockout of the lacI gene: The same operation method as in step 1.1 is used, except that the primers used are lacI-US, lacI-UA, lacI-DS, lacI-DA, pGRB-lacI-S, and pGRB-lacI-A. The cells are electroporated into E. coli Al1-6 / pRed-Cas9 electroporation competent cells to finally obtain strain E. coli Al1-7.

[0053] 1.8 Knockout of the panC gene: The procedure is the same as in step 1.1, except that the primers used are panC-US, panC-UA, panC-DS, panC-DA, pGRB-panC-S, and pGRB-panC-A. The cells are electroporated into E. coli Al1-7 / pRed-Cas9 electroporation competent cells to finally obtain strain E. coli Al1-8.

[0054] 1.9 Knockout of the yfbQ gene: The procedure is the same as in step 1.1, except that the primers used are yfbQ-US, yfbQ-UA, yfbQ-DS, yfbQ-DA, pGRB-yfbQ-S, and pGRB-yfbQ-A. The cells are electroporated into E. coli Al1-8 / pRed-Cas9 electroporation competent cells to finally obtain strain E. coli Al1-9.

[0055] 1.10 Integration of the ppc gene at the mbhA site: Using the W3110 genome as a template, upstream and downstream homologous arms were amplified by PCR using mbhA-US, mbhA-UA and mbhA-DS, mbhA-DA primers, respectively. Using the E. coli genome as a template, the target gene fragment was amplified by PCR using ppc-trc-S and ppc-trc-A primers. Then, using the upstream and downstream homologous arms and the target gene fragment as templates, the overlapping fragment was amplified by overlapping PCR using mbhA-US and mbhA-DA primers. Using pGRB-mbhA-S and pGRB-mbhA-A as primers, gRNA fragments were annealed to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain mbhA-pGRB. Electroporation competent cells of the strain to be modified were prepared, and the overlapping fragments and mbhA-pGRB were electroporated into competent cells. Electroporation was then performed on E. coli Al1-9 / pRed-Cas9 competent cells, and positive transformants were screened. Thus, the integration of the ppc gene from E. coli at the mbhA site was completed, and the strain E. coli Al1-10 was finally obtained.

[0056] 1.11 Integration of the ppc gene at the ycdN site: The same operation method as in step 1.10 is used, except that the primers used are ycdN-US, ycdN-UA, ycdN-DS, ycdN-DA, pGRB-ycdN-S, and pGRB-ycdN-A. The cells are electroporated into E. coli Al1-10 / pRed-Cas9 electroporation competent cells to finally obtain strain E. coli Al1-11.

[0057] 1.12 Integration of the NCgl0580 gene at the rph site: The procedure is the same as in step 1.10, except that the primers used are rph-US, rph-UA, rph-DS, rph-DA, pGRB-rph-S, pGRB-rph-A, NCgl0580-trc-S, and NCgl0580-trc-A. The mixture is electroporated into E. coli Al1-11 / pRed-Cas9 electroporated competent cells to obtain strain E. coli Al1-12.

[0058] Example 2 A membraneless organelle spatial regulatory system was constructed in recombinant Escherichia coli via gene editing, as follows: Using the strain E. coli Al1-12 described in Example 1, genetically engineered strains were further constructed. E. coli Al2-2 (a strain with a dynamically regulated system and directional modification), the specific steps are as follows: 2.1 Integration of the RIDD-RGG-RGG gene at the ilvG gene locus: Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using ilvG-US, ilvG-UA, ilvG-DS, and ilvG-DA, respectively. Using the codon-optimized RIDD-RGG-RGG sequence as a template, and using RIDD-RGG-trc-S and RIDD-RGG-trc-A as primers, the target gene fragment was amplified by PCR. Then, using the upstream and downstream homologous arms and the target gene fragment as templates, and using ilvG-US and ilvG-DA as primers, the overlapping fragment was amplified by overlapping PCR amplification. Using pGRB-ilvG-S and pGRB-ilvG-A as primers, gRNA fragments were annealed and ligated into the pGRB vector to obtain ilvG-pGRB. Electroporation competent cells of the strain to be modified were prepared, and the overlapping fragments and ilvG-pGRB were electroporated into competent cells. Electroporation was then performed on E. coli Al1-13 / pRed-Cas9 competent cells, and positive transformants were screened to finally obtain strain E. coli Al2-1.

[0059] 2.2 Integration of the aspC-RIAD-RIAD-panD gene at the yjiT gene site: The procedure is the same as in 1.1, except that the primers used are yjiT-US, yjiT-UA, yjiT-DS, yjiT-DA, pGRB-yjiT-S, pGRB-yjiT-A, aspC-RIAD-trc-S, and panD-trc-A. The mixture is electroporated into E. coli Al2-1 / pRed-Cas9 electroporation competent cells, and finally strain E. coli Al2-2 is obtained.

[0060] Example 3 like Figure 2 As shown, the plasmid pET-28a-WM was constructed using the following steps: 3.1 Using an artificially synthesized genome as a template, and the codon-optimized RIDD-RGG-RGG sequence as a template, the target gene fragment was amplified by PCR using RIDD-RGG-trc-S and RIDD-RGG-trc-A primers; then, using the codon-optimized aspC-RIAD-RIAD-panD sequence as a template, the target gene fragment was amplified by PCR using aspC-RIAD-trc-S and panD-trc-A primers.

[0061] 3.2 Using the PET28a(+) plasmid genome as a template, and xz-S and xz-A as primers, the DNA fragment of the linearized vector was obtained by PCR amplification.

[0062] 3.3 The linearized vector and the target gene fragment were ligated using recombinase and transformed into DH5α competent cells. Positive transformants were screened and plasmid pET-28a-WM was extracted. The nucleotide sequence of plasmid pET-28a-WM is shown in SEQ ID NO.23 of the sequence listing.

[0063] Example 4 The effectiveness of a membrane-free organelle spatial regulation system in the fermentation production of β-alanine was verified using a whole-cell catalysis method. 4.1 The plasmid pET-28a-WM constructed in Example 3 was electroporated into BL21(DE3) ELECTRO competent cells, and positive transformant YZ-1 was obtained by screening.

[0064] 4.2 Spread the YZ-1 strain evenly on the activated slant, incubate at 37℃ for 12h, scrape off a loopful of colonies from the slant culture medium using a sterilized inoculation loop, and then transfer it to a shaker containing 5ml of seed culture medium for seed culture for 10h at 37℃.

[0065] The slant culture medium used was: glucose 2 g / L, yeast extract 5 g / L, peptone 10 g / L, NaCl 2.5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.2 g / L, agar powder 2.5 g / L, and the remainder was water.

[0066] The seed culture medium used was: yeast extract 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water.

[0067] 4.3 Fermentation culture: Pour the bacterial culture from the shaker containing the seed culture medium into a 500mL Erlenmeyer flask containing the fermentation culture medium (final volume 30mL), seal with nine layers of gauze, and culture at 37℃ with shaking at 220r / min. During fermentation, maintain the pH at 7.0-7.2 by adding ammonia water; add 60% (m / v) glucose solution to maintain fermentation (using phenol red as an indicator, when the color of the fermentation broth no longer changes, it is considered as sugar deficiency, and 1-2 mL of 60% (m / v) glucose solution is added when sugar deficiency occurs).

[0068] The fermentation medium used was as follows: glucose 30.0 g / L, yeast extract 4.0 g / L, peptone 2.0 g / L, ammonium sulfate 2.0 g / L, citric acid 2.0 g / L, monosodium glutamate 2.0 g / L, MgSO4·7H2O 1.2 g / L, KH2PO4 3.0 g / L, threonine 0.5 g / L, MnSO4·H2O 5.0 mg / L, FeSO4·7H2O 15 mg / L, phenol red 2.5%, and L-aspartic acid 5 g / L. After weighing the solid components, they were dissolved in 1 L of water and stored at 4℃. IPTG was added to induce fermentation after 8 hours, and fermentation was stopped after 24 hours. The yield of β-alanine synthesized by catalysis reached 3.43 g / L.

[0069] Example 5 The effectiveness of the membraneless organelle spatial regulation system in the fermentation production of β-alanine was verified using a bacterial fermentation method. Using the strain E. coli Al1-12 described in Example 1, genetically engineered strains were further constructed. E. coli Al3-2 (a strain specifically modified using a traditional static control system), the specific operation is as follows: 5.1 Integration of the aspC gene at the yeeP site: The procedure is the same as in step 1.1, except that the primers used are yeeP-US, yeeP-UA, yeeP-DS, yeeP-DA, pGRB-yeeP-S, pGRB-yeeP-A, aspC-trc-S, and aspC-trc-A. The cells are electroporated into E. coli Al1-12 / pRed-Cas9 electroporated competent cells to obtain strain E. coli Al3-1.

[0070] 5.2 Integration of the panD gene from Pseudomonas aeruginosa at the ylbE site: The procedure is the same as in step 1.1, except that the primers used are ylbE-US, ylbE-UA, ylbE-DS, ylbE-DA, pGRB-ylbE-S, pGRB-ylbE-A, panD-trc-S, and panD-trc-A. The mixture is electroporated into E. coli Al1-13 / pRed-Cas9 electroporated competent cells to obtain strain E. coli Al3-2.

[0071] 5.3 The bacterial strain was evenly spread on the activation slant and incubated at 37℃ for 12 hours. Then, using a sterilized inoculation loop, the colonies from the slant were transferred to a shaker containing 5 ml of seed culture medium for seed culture for 10 hours at 37℃. The slant culture medium used consisted of: glucose 2 g / L, yeast extract 5 g / L, peptone 10 g / L, NaCl 2.5 g / L, KH₂PO₄ 1 g / L, MgSO₄·7H₂O 0.2 g / L, agar powder 2.5 g / L, and the remainder being water. The seed culture medium used consisted of: yeast extract 8.0 g / L, peptone 2.0 g / L, (NH₄)₂SO₄ 2.0 g / L, KH₂PO₄ 3.0 g / L, and V₂SO₄ 2.0 g / L. B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water.

[0072] 5.4 Fermentation culture: Pour the bacterial culture from the shaker containing the seed culture medium into a 500mL Erlenmeyer flask containing the fermentation culture medium (final volume 30mL), seal with nine layers of gauze, and incubate at 37℃ with shaking at 220r / min. During fermentation, maintain the pH at 7.0-7.2 by adding ammonia water; add 60% (m / v) glucose solution to maintain fermentation (using phenol red as an indicator, when the color of the fermentation broth no longer changes, it is considered as sugar deficiency, and 1-2 mL of 60% (m / v) glucose solution is added when sugar deficiency occurs). The fermentation medium used was as follows: glucose 30.0 g / L, yeast extract 4.0 g / L, peptone 2.0 g / L, ammonium sulfate 2.0 g / L, citric acid 2.0 g / L, monosodium glutamate 2.0 g / L, MgSO4·7H2O 1.2 g / L, KH2PO4 3.0 g / L, threonine 0.5 g / L, MnSO4·H2O 5.0 mg / L, FeSO4·7H2O 15 mg / L, phenol red 2.5%, and the remainder was water. The solid components were weighed and dissolved in 1 L of water, and stored at 4℃.

[0073] 5.5 The production of Al2-2 and Al3-2 strains was compared to verify whether the membraneless organelle spatial regulation system had a significant impact on β-alanine production. The results are shown in Table 2. After 12 hours of shake-flask fermentation, the yield of Al2-2 was as high as 9.98 g / L, while the β-alanine yield of Al3-2 was 6.27 g / L. The experimental results indicate that, compared to traditional overexpression strains, the engineered strain based on artificial membraneless organelles constructed in this invention significantly improved the β-alanine production rate, demonstrating the significant advantages of the dynamic control system in increasing metabolic flux and shortening the production cycle.

[0074] Table 2 Comparison of Al2-2 and Al3-2 yields

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A space control system, characterized in that: This includes recombinant proteins RIDD-RGG-RGG and aspC-RIAD-RIAD-panD, wherein the recombinant protein RIDD-RGG-RGG has a content of 5. ’ A disordered protein tandem RGG-RGG with a short RIDD peptide added to its end is described in the nucleotide sequence of the recombinant protein RIDD-RGG-RGG, as shown in SEQ ID NO. 6 of the sequence listing. The recombinant protein aspC-RIAD-RIAD-panD has a nucleotide sequence of 3. ’ The recombinant proteins aspC, aspartate decarboxylase gene panD derived from Pseudomonas aeruginosa, were sequentially added to the ends of the protein. Linkers were used to connect aspC-RIAD-RIAD-panD. The nucleotide sequences of the linkers are shown in SEQ ID NO.2, as in SEQ ID NO.7, as in SEQ ID NO.8, and as in SEQ ID NO.

5. The recombinant proteins RIDD-RGG-RGG and aspC-RIAD-RIAD-panD self-assembled via RIAD / RIDD interaction peptides.

2. The application of the spatial regulation system of claim 1 in the construction of β-alanine-producing strains and / or the production of β-alanine.

3. A β-alanine-producing strain, characterized in that: It was created using gene editing technology: in wild-type Escherichia coli Escherichia coli W3110 Knockout of acetaldehyde dehydrogenase gene in the genome adhE lactate dehydrogenase gene ldhA Acetylkinase gene ackA pyruvate dehydrogenase gene poxB The gene of lactose operon repressor protein lacI Aspartate kinase gene thrA pantothenic acid synthase gene panC Alanine transaminase gene cycA Alanine transaminase gene yfbQ Heterogeneous introduction of the β-alanine transporter gene from Corynebacterium glutamicum. NCgl0580 Overexpression of phosphoenolpyruvate carboxylase gene PPC Integrating the recombinant protein RIDD-RGG-RGG of claim 1 and the recombinant protein aspC-RIAD-RIAD-panD of claim 1, wherein the phosphoenolpyruvate carboxylase gene PPC The nucleotide sequence is shown in SEQ ID NO.18 of the sequence listing, representing the β-alanine transporter gene from Corynebacterium glutamicum. NCgl0580 The nucleotide sequence is shown in SEQ ID NO.19 of the sequence listing.

4. The β-alanine-producing strain according to claim 3, characterized in that: The acetaldehyde dehydrogenase gene adhE The nucleotide sequence is shown in SEQ ID NO. 10 of the sequence listing; lactate dehydrogenase gene ldhA The nucleotide sequence is shown in SEQ ID NO. 11 of the sequence listing; Acetylkinase gene ackA The nucleotide sequence is shown in SEQ ID NO. 12 of the sequence listing; pyruvate dehydrogenase gene poxB The nucleotide sequence is shown in SEQ ID NO. 13 of the sequence listing; aspartate kinase gene. thrA The nucleotide sequence is shown in SEQ ID NO.14 of the sequence listing; pantothenic acid synthase gene. panC The nucleotide sequence is shown in SEQ ID NO.15 of the sequence listing; alanine transaminase gene. cycA The nucleotide sequence is shown in SEQ ID NO.16 of the sequence listing; alanine transaminase gene. yfbQ The nucleotide sequence is shown in SEQ ID NO.17 of the sequence listing; the gene for the lactose operon repressor protein. lacI The nucleotide sequence is shown in the sequence listing SEQ ID NO.

20.

5. The method for constructing the β-alanine-producing strain according to claim 3 or 4, characterized in that: The specific steps are as follows: (1) Wild-type Escherichia coli Escherichia coli W3110 For the starting strain, the acetaldehyde dehydrogenase gene was knocked out of its genome. adhE lactate dehydrogenase gene ldhA Acetylkinase gene ackA pyruvate dehydrogenase gene poxB The gene of lactose operon repressor protein lacI Aspartate kinase gene thrA pantothenic acid synthase gene panC Alanine transaminase gene cycA Alanine transaminase gene yfbQ ; (2) Overexpression of phosphoenolpyruvate carboxylase gene twice PPC ; (3) Heterologous introduction of β-alanine transporter gene from Corynebacterium glutamicum NCgl0580 ; (4) Integrate the RIDD-RGG-RGG gene and the aspC-RIAD-RIAD-panD gene respectively.

6. The use of the β-alanine-producing strain according to claim 3 or 4 in the production of β-alanine.

7. The application according to claim 6, characterized in that: β-alanine was produced by fermentation.

8. The application according to claim 7, characterized in that: Shake flask fermentation: Inoculate the seed culture into the fermentation medium at an inoculation rate of 10%-15%, and culture with shaking. During the fermentation process, maintain the pH at 7.0-7.2 by adding ammonia water and add glucose solution to maintain the fermentation.

9. The application according to claim 8, characterized in that: The specific steps for shake-flask fermentation are as follows: (1) Activation of bacterial strain: Inoculate the bacterial solution from the preservation tube into the slant medium for activation culture; use a sterilized inoculation loop to scrape off a loopful of colonies from the slant medium and transfer it into a shaker containing seed culture medium for seed culture; (2) Pour the bacterial solution in the shaker containing the seed culture medium into the Erlenmeyer flask containing the fermentation culture medium, seal the flask with gauze, shake and culture. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water; glucose solution is added to maintain the fermentation.

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