Method for improving activity of beta-galactosidase by fusing biological aggregate element and recombinant engineering strain

Abstract

The invention belongs to the technical field of biological engineering, and particularly relates to a method for improving the activity of beta-galactosidase (LacZ) by fusing a biological aggregate element and a recombinant engineering strain. According to the method disclosed by the invention, a biological aggregate protein element with phase separation capacity is introduced and is subjected to fusion expression with beta-galactosidase, and a target enzyme is induced to form a biological condensed state structure with catalytic activity in cells, so that the biological aggregate is highly enriched in a cell polar region; the local concentration and the reaction efficiency of the beta-galactosidase are effectively improved. According to the strategy provided by the invention, the enzyme activity of the beta-galactosidase is obviously improved and can be about 8 times. The method has the advantages of simplicity and convenience in operation, high universality, capability of being expanded to other functional proteins and the like, is suitable for a biosynthesis system combining high-efficiency expression and activity optimization, and has a wide industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for enhancing β-galactosidase activity by fusing biological condensate elements and a recombinant engineered strain. Background Technology

[0002] In recent years, with the deepening research on bioaggregate elements (such as liquid-liquid phase separation (LLPS)), researchers have discovered that fusing protein tags with coagulation-promoting capabilities with target proteins can promote the formation of enriched aggregates of the target protein within cells, thereby enhancing its activity. However, existing studies have shown that although this type of fusion strategy has been reported to increase metabolite yields in various systems, research on the activity of key enzymes in metabolic pathways is relatively limited. This may stem from the significant uncertainty in the effectiveness of this strategy: in some cases, the activity of the target protease after fusion is significantly increased, while in others, fusion leads to a decrease in enzyme activity or no significant change. The reasons for this difference may include: potential interference with protein conformation by aggregate formation, redistribution of cellular metabolic resources, and low synthesis efficiency of the fusion protein within the host cell. Furthermore, factors such as the physiological state of the host cell (e.g., growth stage, culture conditions, intracellular pH and temperature) and biomass (OD value) can all affect enzyme coagulation effects, further increasing the uncertainty of coagulation outcomes.

[0003] Therefore, there is an urgent need for a technical solution that can stably and controllably achieve spatial enrichment of target proteins within host cells, and significantly enhance the activity of target enzymes without significantly affecting host physiological growth, in order to improve biocatalytic efficiency and enhance application stability. Furthermore, by rationally designing and fusing these bio-aggregate elements, it is possible not only to effectively increase the local concentration of target proteins, but also to avoid protein degradation that could reduce the overall activity of the enzyme.

[0004] β-galactosidase (LacZ) is an enzyme widely used in biological research, industrial fermentation, and biopharmaceuticals, playing a crucial role, especially in carbohydrate metabolism and lactose hydrolysis. β-galactosidase has significant industrial application value. To improve β-galactosidase activity, researchers have attempted to enhance its performance by optimizing expression systems, improving enzyme stability, and increasing catalytic activity. Existing technologies include protein engineering (such as mutation and directed evolution), molecular chaperone-assisted folding, and the addition of exogenous cofactors.

[0005] Based on this, the present invention aims to provide a novel and efficient strategy and method for enhancing enzyme activity. Summary of the Invention

[0006] The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing a method for enhancing β-galactosidase activity by incorporating bio-aggregate elements. Specifically, by introducing protein elements with phase-separation capabilities to link β-galactosidase, the target enzyme is induced to form a catalytically active bio-aggregate structure within the cell, thereby highly enriching the bio-aggregate in the cell polar region and effectively improving the local concentration of the enzyme and reaction efficiency.

[0007] Another technical problem to be solved by the present invention is to provide a recombinant strain that enhances β-galactosidase activity by incorporating bioaggregate elements.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a method for enhancing β-galactosidase activity by fusing a bio-aggregate element. The fusion gene fragment obtained by linking β-galactosidase lacZ with a bio-aggregate element is heterologously expressed in Escherichia coli, thereby enabling β-galactosidase to form a spatially enriched aggregated structure within the cell, thus enhancing β-galactosidase activity.

[0010] The sources of the β-galactosidase include, but are not limited to, Escherichia coli, Bacillus subtilis, Exiguobacterium acetylicum, Lactic streptococcal, Pediococcus pentosaceus, Thermus strain, Pseudoalteromonas haloplanktis, Aspergillus niger, Aspergillus oryzae, and Kluyveromyces lactis.

[0011] Preferably, the β-galactosidase is derived from Escherichia coli.

[0012] The amino acid sequence of the β-galactosidase lacZ is shown in SEQ ID NO.69.

[0013] The aforementioned "biopolymer elements" possess the property of promoting the aggregation of various biomolecules and regulating various intracellular physiological activities within cells. By utilizing these biopolymer elements, proteins can form aggregates at the cell terminals.

[0014] The bio-aggregate element includes any one of Erα, PAB1, Pbp1, Galectin-3, eIF4GII, U2AF65, RPB1, DDX3, SYN2, TAF15, Std1, FMRP, DYRK1A, BRD4, LplA, VAE1, VAE2, VAE3, VAE4, VAE5, VAE6, VAE7, VAE8, VAE9, VAE10, VAE11, VAE12, VAE13, Y145, Y2, Y4, and Y15, and their amino acid sequences are shown in SEQ ID NO.1~32.

[0015] Specifically, Erα, PAB1, Pbp1, Galectin-3, eIF4GII, U2AF65, RPB1, DDX3, SYN2, TAF15, Std1, FMRP, DYRK1A, BRD4, and LplA are natural tags; VAE1, VAE2, VAE3, VAE4, VAE5, VAE6, VAE7, VAE8, VAE9, VAE10, VAE11, VAE12, and VAE13 are synthetic tags; and Y145, Y2, Y4, and Y15 are bioaggregate tags.

[0016] Furthermore, the natural labels Erα, Galectin-3, U2AF65, DDX3, SYN2, TAF15, FMRP, DYRK1A, and BRD4 are derived from Homo sapiens; the natural labels PAB1, Pbp1, eIF4GII, RPB1, and Std1 are derived from Saccharomyces cerevisiae; and the natural label LplA is derived from Escherichia coli.

[0017] Furthermore, the artificially synthesized protein tags VAE1, VAE2, VAE3, VAE4, VAE5, VAE6, VAE7, VAE8, VAE9, VAE10, VAE11, VAE12, and VAE13 were genetically synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0018] Furthermore, Y145 is derived from the prion protein (PrP) by a mutation of atyrosine residue at 145 to a stop codon. Y2 and Y4 are truncated versions of Y145, and their gene synthesis was completed by Suzhou Genewise Biotech Co., Ltd. Y15 is derived from De novo designed self-assembling peptides (SAPs).

[0019] The β-galactosidase lacZ is connected to a biological condensate element, either directly, via a linker, or via a recruitment tag.

[0020] Specifically, the Linker is a flexible peptide composed of glycine G and serine S, including any one of (GGS)n, (GGGS)n, (GGGGS)n, and (GGGSSS)n, where n = 1 to 8.

[0021] Preferably, the Linker is (GGS)4 or (GGGGS)2.

[0022] Specifically, the recruitment tags are RIAD and RIDD, wherein the amino acid sequence of RIAD is shown in SEQ ID NO.33, and the amino acid sequence of RIDD is shown in SEQ ID NO.34.

[0023] The β-galactosidase is connected to the N-terminus or C-terminus of the bio-condensate element.

[0024] In some embodiments of the present invention, the β-galactosidase is linked to the N-terminus or C-terminus of the bio-condensate element Y145 via a linker.

[0025] The *Escherichia coli* strain is *Escherichia coli* BL21(DE3), or a recombinant strain B-ΔL of *Escherichia coli* BL21(DE3) lacking the lacZ gene (the inactivation of the lacZ gene is to eliminate the interference of *Escherichia coli*'s own β-galactosidase enzyme activity), or a recombinant strain B-ΔLC of *Escherichia coli* BL21(DE3) lacking both the lacZ and minC genes.

[0026] Specifically, the recombinant strain B-ΔL is obtained by knocking out the lacZ gene from Escherichia coli BL21(DE3) as the chassis strain; the recombinant strain B-ΔLC is obtained by further knocking out the minC gene from the recombinant strain B-ΔL as the chassis strain.

[0027] Specifically, the recombinant strain B-ΔLC is a recombinant Escherichia coli capable of producing minicells.

[0028] Wherein, the β-galactosidase activity is increased by at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8 times.

[0029] Secondly, the present invention provides a recombinant engineered strain that enhances β-galactosidase activity by fusing a bioconfluence element. The strain is constructed using Escherichia coli as a chassis strain and by heterologously expressing β-galactosidase lacZ and linking it to a bioconfluence element to obtain a fusion gene fragment.

[0030] The *Escherichia coli* strain is *Escherichia coli* BL21(DE3), or a recombinant strain B-ΔL of *Escherichia coli* BL21(DE3) lacking the lacZ gene, or a recombinant strain B-ΔLC of *Escherichia coli* BL21(DE3) lacking both the lacZ and minC genes.

[0031] The amino acid sequence of the β-galactosidase lacZ is shown in SEQ ID NO.69.

[0032] The bio-aggregate element includes any one of Erα, PAB1, Pbp1, Galectin-3, eIF4GII, U2AF65, RPB1, DDX3, SYN2, TAF15, Std1, FMRP, DYRK1A, BRD4, LplA, VAE1, VAE2, VAE3, VAE4, VAE5, VAE6, VAE7, VAE8, VAE9, VAE10, VAE11, VAE12, VAE13, Y145, Y2, Y4, and Y15, and their amino acid sequences are shown in SEQ ID NO.1~32.

[0033] The β-galactosidase lacZ is connected to the N-terminus or C-terminus of the bio-condensate element via direct linkage, linker linkage, or recruitment tag.

[0034] Specifically, the Linker is a flexible peptide composed of glycine G and serine S, including any one of (GGS)n, (GGGS)n, (GGGGS)n, and (GGGSSS)n, where n = 1 to 8.

[0035] Preferably, the Linker is (GGS)4 or (GGGGS)2.

[0036] Specifically, the recruitment tags are RIAD and RIDD, wherein the amino acid sequence of RIAD is shown in SEQ ID NO.33, and the amino acid sequence of RIDD is shown in SEQ ID NO.34.

[0037] Specifically, the recombinant strain that enhances β-galactosidase activity by fusing bioconglomerate elements is first synthesized by codon optimization of the amino acid sequence of the bioconglomerate element according to *E. coli*. Simultaneously, β-galactosidase is fused at the N-terminus or C-terminus via a linker such as (GGS)4, forming a bioconglomerate element gene fusion fragment (the above synthesis process was completed by Suzhou Genewiz Biotechnology Co., Ltd.). Then, the plasmid containing the bioconglomerate element gene fusion fragment is introduced into *E. coli* cells to obtain different recombinant engineered strains capable of expressing the bioconglomerate element and β-galactosidase complex.

[0038] Furthermore, the plasmid includes any one of the following: pET series plasmids, pBAD series plasmids, pJ23119 series plasmids, pTrc99a series plasmids, pRSFDuet-1 series plasmids, pACYCDuet-1 series plasmids, pCDFDuet-1 series plasmids, and pCOLADuet-1 series plasmids.

[0039] Preferably, the plasmid is pET28a plasmid or pCTDuet-1 plasmid.

[0040] The β-galactosidase reaction system was prepared by mixing 100 μL of induced protein sample with 4 μL of FDG (1 mg / mL) in 10 mM PBS as the reaction buffer. Measurements were taken every few minutes (excitation wavelength 488 nm, measurement wavelength 535 nm). The 1 mg / mL FDG solution was resuspended in 98% water, 1% DMSO, and 1% ethanol. After detecting the fluorescence value in the reaction system using a microplate reader, simple linear regression analysis was performed to calculate the growth rate. Note: Due to different bacterial cell OD or storage conditions, there are some differences between batches of reactions. However, the results of the same batch are consistent, and the experimental group showed a significant difference in enzyme activity enhancement compared to the control group.

[0041] Beneficial Effects: Compared with existing technologies, this invention optimizes the design of bioconfluent elements and heterologously expresses the fusion gene fragment obtained by linking β-galactosidase lacZ to bioconfluent elements in *E. coli*, achieving a more efficient phase separation process and significantly improving the activity and stability of the β-galactosidase target protein. This invention can precisely regulate β-galactosidase aggregation within cells, thereby enhancing its functionality. Through the strategy provided by this invention, the enzyme activity of β-galactosidase is significantly increased, reaching approximately 8-fold, showing broad application prospects. This invention not only overcomes the limitations of existing technologies in the regulation of β-galactosidase activity and function but also provides innovative solutions for fields such as efficient biocatalysis and gene regulation. The method of this invention has the advantages of simple operation, strong universality, and extensibility to other functional proteins, making it suitable for biosynthetic systems that combine efficient expression and activity optimization, and has broad industrial application prospects. Attached Figure Description

[0042] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0043] Figure 1 This image shows the formation of aggregates induced by EGFP fusion with natural tags (Erα, PAB1, Pbp1, Galectin-3, eIF4GII, U2AF65, RPB1, DDX3). The image includes grayscale values ​​of the cellular regions marked by white dashed lines, with red arrows indicating the locations of the aggregate grayscale values.

[0044] Figure 2 This image shows the formation of aggregates induced by EGFP fusion with natural tags (TAF15, FMRP, DYRK1A, BRD4, LplA). Grayscale values ​​were statistically analyzed for the cellular regions marked by white dashed lines, and red arrows indicate the locations of the aggregate grayscale values.

[0045] Figure 3 Diagram showing the formation of uninduced aggregates of natural tags (SYN2, Std1) fused with EGFP.

[0046] Figure 4 This study used SDS-PAGE to analyze aggregate proteins formed by the fusion of natural tags with EGFP at both ends of cells. Wherein, P: precipitate; S: supernatant.

[0047] Figure 5 This image shows the formation of aggregates induced by EGFP fusion with artificially synthesized protein tags (VAE1, VAE2, VAE3, VAE4, VAE5, VAE6, VAE7, VAE8). The image includes grayscale values ​​of the cellular regions marked by white dashed lines, with red arrows indicating the grayscale locations of the aggregates.

[0048] Figure 6 This diagram illustrates the formation of aggregates induced by the fusion of artificially synthesized protein tags (VAE9, VAE10, VAE11, VAE12, VAE13) with EGFP. The diagram shows the grayscale values ​​of the cellular regions marked by white dashed lines, with red arrows indicating the grayscale locations of the aggregates.

[0049] Figure 7 SDS-PAGE analysis was performed on the aggregate proteins formed by the fusion of synthetically produced tags with EGFP. Wherein, P: precipitate; S: supernatant.

[0050] Figure 8 Diagram showing the formation of EGFP-induced aggregates fused with Y145, Y2, Y4, and Y15 tags.

[0051] Figure 9 SDS-PAGE analysis was performed on the aggregate proteins and unit OD of Y145, Y2, and Y4 tags fused with EGFP. 600 Average fluorescence intensity. Where P: precipitation; S: supernatant. White arrows indicate the location of aggregate proteins.

[0052] Figure 10 SDS-PAGE analysis of condensate proteins and unit OD formed by Y15 tag fusion with EGFP. 600 Average fluorescence intensity. Where P: precipitation; S: supernatant. White arrows indicate the location of aggregate proteins.

[0053] Figure 11 The figure shows how fusing DDX3 and TAF15 tags, respectively, to β-galactosidase enhances enzyme activity in recombinant E. coli.

[0054] Figure 12 The figure shows how the fusion of VAE10 and VAE12 tags, respectively, with β-galactosidase enhances enzyme activity in recombinant E. coli.

[0055] Figure 13 The figure shows how fusing Y2 and Y4 tags, respectively, to β-galactosidase enhances enzyme activity in recombinant Escherichia coli.

[0056] Figure 14 The figure shows how fusing the Y15 tag with β-galactosidase enhances enzyme activity in recombinant Escherichia coli.

[0057] Figure 15 This figure illustrates how fusion expression of the β-galactosidase gene at the N-terminus or C-terminus of the Y145 gene in biological condensate elements enhances enzyme activity. Detailed Implementation

[0058] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0059] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0060] Example 1: Verification of Intracellular Molecular Agglutination Using Natural Protein Agglutination Tags

[0061] 1. Construction and induction of expression of natural tag fusion gene fragments and corresponding recombinant engineered strains

[0062] The natural tag (Erα, PAB1, Pbp1, Galectin-3, eIF4GII, U2AF65, RPB1, DDX3, SYN2, TAF15, Std1, FMRP, DYRK1A, BRD4, LplA) with amino acid sequences as shown in SEQ ID NO. 1~15 was used to synthesize a gene after codon optimization for E. coli (the codon-optimized nucleotide sequences are shown in SEQ ID NO. 35~49). At the same time, the natural tag and enhanced green fluorescent protein (EGFP) were linked together by linkers such as flexible peptide (GGS)4 and (GGGGS)2 to form a natural tag fusion gene fragment. That is, enhanced green fluorescent protein (EGFP) was linked to the C-terminus of the natural tag by linker, or directly linked to the C-terminus of the natural tag. The natural tag fusion gene fragments digested with EcoRI and XhoI were ligated with plasmid pET28a to obtain the corresponding natural tag fusion EGFP plasmids. All of the above plasmids were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0063] The synthesized natural tag fusion EGFP plasmid was transformed into *E. coli* BL21(DE3) competent cells via heat shock to obtain recombinant engineered strains expressing different natural tag fusion EGFPs. Single colonies of the recombinant engineered strains were picked and placed in 50 mL centrifuge tubes containing 5 mL LB medium (containing 100 mg / L Amp) and incubated overnight at 37°C and 200 rpm for 12 h. The activated bacterial culture was then inoculated at 2% v / v into 250 mL Erlenmeyer flasks containing 100 mL of TB for expansion culture. When OD... 600 When the expression level reached 0.8, IPTG inducer was added at a final concentration of 0.2 mM, and protein expression was induced overnight at 22°C and 200 rpm.

[0064] The natural tag fusion gene fragment and the corresponding recombinant engineered strain in this embodiment are shown in Table 1.

[0065] Table 1 Natural tag fusion gene fragments and corresponding recombinant engineered strains

[0066]

[0067] The formation of intracellular induced aggregates is as follows: Figures 1-3 As shown, the natural tags Erα, PAB1, Pbp1, Galectin-3, eIF4GII, U2AF65, RPB1, DDX3, TAF15, FMRP, DYRK1A, BRD4, and LplA, when fused with EGFP, can induce aggregate formation. Figure 1 and Figure 2 However, some natural tags SYN2 and Std1, when fused with EGFP, still did not induce the formation of aggregates. Figure 3 Among them, EGFP fused with Pbp1, Galectin-3, RPB1, DDX3, TAF1, FMRP, and DYRK1A tags all showed significant aggregation effects at the cell ends. Interestingly, the presence of the TAF1 tag significantly increased the length of E. coli; EGFP fused with eIF4GII and FMRP tags showed aggregation at both ends and the center of the cell.

[0068] 2. SDS-PAGE analysis of aggregate proteins formed by the fusion of natural tags with EGFP at both ends of cells exhibiting aggregation.

[0069] Further investigation was conducted on tags exhibiting aggregation at both ends of cells. SDS-PAGE analysis was used to analyze aggregates formed by the fusion of Pbp1, Galectin-3, eIF4GII, RPB1, DDX3, TAF1, FMRP, and DYRK1A tags with EGFP. Specifically, the bacterial culture was sonicated, and the supernatant and precipitate were resuspended and subjected to SDS-PAGE protein gel electrophoresis. The results showed ( Figure 4 All strains B-N3-EGFP, B-N4-EGFP, B-N5-EGFP, B-N7-EGFP, B-N8-EGFP, B-N10-EGFP, B-N12-EGFP, and B-N13-EGFP successfully expressed soluble agglutinin proteins. Among them, the soluble agglutinin protein expressed by strain B-N3-EGFP appeared in the precipitate, indicating that the agglutinin protein is insoluble. The soluble agglutinin proteins expressed by strains B-N5-EGFP, B-N10-EGFP, B-N12-EGFP, and B-N13-EGFP appeared in both the supernatant and the precipitate. The agglutinin proteins formed by the fusion of the B-N4-EGFP, B-N7-EGFP, and B-N8-EGFP tags with EGFP were completely present in the supernatant, indicating that the agglutinin protein is soluble.

[0070] Example 2: Verification of Intracellular Molecular Agglutination Using Artificially Synthesized Protein Agglutination Tags

[0071] 1. Construction and induced expression of artificially synthesized protein aggregation tag fusion gene fragments and corresponding recombinant engineered strains.

[0072] The natural proteins in Example 1 were replaced with synthetic tags VAE1, VAE2, VAE3, VAE4, VAE5, VAE6, VAE7, VAE8, VAE9, VAE10, VAE11, VAE12, and VAE13 (the amino acid sequences of synthetic tags VAE1-13 are shown in SEQ ID NO. 16-28, and the codon-optimized nucleotide sequences are shown in SEQ ID NO. 50-62). All other steps remained unchanged. The corresponding recombinant engineered strains were constructed and induced to express the proteins. The synthetic tag fusion gene fragments and corresponding recombinant engineered strains of this example are shown in Table 2.

[0073] Table 2. Artificially synthesized tag fusion gene fragments and corresponding recombinant engineered strains

[0074]

[0075] The results are as follows Figures 5-6 As shown, the artificially synthesized proteins VAE1, VAE2, VAE3, VAE4, VAE5, VAE6, VAE7, VAE8, VAE9, VAE10, VAE11, VAE12, and VAE13, when fused with EGFP as aggregation tags, can all induce the formation of aggregates within the cell. Furthermore, the aggregation effect at the cellular end can be observed with all VAE-designed tags fused with EGFP.

[0076] 2. SDS-PAGE analysis of aggregate proteins formed by fusing artificially synthesized tags with EGFP

[0077] SDS-PAGE was used to analyze the aggregate proteins formed by fusing artificially synthesized tags with EGFP. The results showed that the recombinant engineered strains B-V1-EGFP, B-V2-EGFP, B-V3-EGFP, B-V4-EGFP, B-V5-EGFP, B-V6-EGFP, B-V7-EGFP, B-V8-EGFP, B-V9-EGFP, B-V10-EGFP, B-V11-EGFP, B-V12-EGFP, and B-V13-EGFP all successfully expressed aggregate proteins. Among them, the aggregate proteins expressed by B-V1-EGFP, B-V2-EGFP, B-V4-EGFP, B-V5-EGFP, B-V6-EGFP, B-V8-EGFP, B-V9-EGFP, and B-V13-EGFP appeared in the precipitate, indicating that these aggregate proteins are insoluble; the aggregate proteins expressed by B-V7-EGFP, B-V10-EGFP, and B-V12-EGFP appeared in both the supernatant and the precipitate; and the aggregate protein expressed by B-V3-EGFP appeared in the supernatant, indicating that these aggregate proteins are soluble.

[0078] Example 3: Verification of EGFP-induced aggregate formation induced by Y145, Y2, Y4 and Y15 tags.

[0079] The amino acid sequences of the bioaggregate tags (Y145, Y2, Y4, Y15) shown in SEQ ID NO. 29-32 were optimized according to the codons of *E. coli* to synthesize genes (the optimized nucleotide sequences are shown in SEQ ID NO. 63-66). Simultaneously, the bioaggregate tags were linked to enhanced green fluorescent protein (EGFP) using linkers such as flexible peptide (GGS)4 and (GGGGS)2, respectively, to form bioaggregate tag fusion gene fragments. This involved linking EGFP to the C-terminus of the bioaggregate tag via a linker, or directly linking EGFP to the C-terminus of the bioaggregate tag. The bioaggregate tag fusion gene fragments, after being double-digested with NcoI and SalI, were ligated with plasmid pETDuet-1 to obtain the corresponding bioaggregate tag fusion EGFP plasmids. All plasmids were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0080] The synthesized bioconglomerate tag fused EGFP plasmid was transformed into *E. coli* BL21(DE3) competent cells via heat shock to obtain recombinant engineered strains expressing different bioconglomerate tag fused EGFP. Single colonies of the recombinant engineered strains were picked and placed in 50 mL centrifuge tubes containing 5 mL LB medium (containing 100 mg / L Amp) and incubated overnight at 37°C and 200 rpm for 12 h. The activated bacterial culture was then inoculated at 2% v / v into 250 mL Erlenmeyer flasks containing 100 mL of TB for expansion culture. When OD... 600 When the expression level reached 0.8, IPTG inducer was added at a final concentration of 0.2 mM, and protein expression was induced overnight at 22°C and 200 rpm.

[0081] The bioaggregate tag fusion gene fragment and the corresponding recombinant engineered strain in this embodiment are shown in Table 3.

[0082] Table 3. Tag fusion gene fragments of bioaggregates Y145, Y2, Y4, and Y15 and corresponding recombinant engineered strains

[0083]

[0084] The results showed that B-Y145-EGFP, B-Y2-EGFP, B-Y4-EGFP, and B-Y15-EGFP could all induce the formation of aggregates in cells (e.g., Figure 8 (As shown). Among them, B-Y145-EGFP, B-Y2-EGFP, and B-Y4-EGFP all form soluble fusion proteins with a unit OD of [missing information]. 600 The fluorescence intensity was higher than that of the control bacterium B-EGFP-1 (e.g. Figure 9 (As shown). B-Y15-EGFP forms an insoluble fusion protein with a unit OD. 600 The fluorescence intensity was approximately 300% higher than that of the control bacteria. (e.g.) Figure 10 (As shown).

[0085] Example 4: Construction of recombinant strains B-ΔL and B-ΔLC

[0086] 1. Construction of the target strain E. coli BL21(DE3) and B-ΔL containing the pEcCas plasmid

[0087] The pEcCas plasmid (gifted by Professor Yang Sheng of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences) was heat-shock transformed into the target strain E. coli BL21(DE3) or B-ΔL competent cells, respectively. After single colonies were picked and activated, the plasmid was extracted and PCR was performed using primers pEcCas-F and pEcCas-R. The correct target strains E. coli BL21(DE3) or B-ΔL containing the pEcCas plasmid were then verified by nucleic acid electrophoresis.

[0088] 2. Construction of B-ΔL and B-ΔLC recombinant strains

[0089] (1) Construction of recombinant strains with inactivated lacZ gene

[0090] Using the extracted E. coli BL21(DE3) genome as a template, PCR was performed using primers ΔlacZ-1-F and ΔlacZ-1-R to amplify the upstream homologous arm of the lacZ gene, and primers ΔlacZ-2-F and ΔlacZ-2-R ​​to amplify the downstream homologous arm of the lacZ gene. Then, using the upstream and downstream homologous arms as templates, PCR was performed using primers ΔlacZ1-F and ΔlacZ-2-R ​​to construct the Donor DNA fragment that knocks out the lacZ gene. Using plasmid pTarget F (given by Professor Yang Sheng of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences) as a template, PCR amplification was performed using primers pF-1-F and pF-1-R to obtain the linear recombinant plasmid pF-1. The linear recombinant plasmid pF-1 was then purified and recovered by gel electrophoresis. After eliminating the original template by DpnI enzyme digestion, it was introduced into the cloning host E. coli Trans1-T1 (purchased from Beijing TransGen Biotech Co., Ltd.) by heat shock transformation. It was incubated overnight at 37°C on LB solid culture medium containing streptomycin (40 mg / L). The next day, single colonies were picked for activation culture and finally sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing screening to obtain the correct pF-1 plasmid containing the N20 sequence of the lacZ gene knockout.

[0091] The successfully constructed Donor DNA and pF-1 plasmid were simultaneously electroporated into the target strain *E. coli* BL21(DE3) containing the pEcCas plasmid at a concentration of 5:1 (100 ng / μL). The electroporator was set to 2500 V for 5–6 ms. After electroporation, LB liquid medium was immediately added, and the culture was incubated at 37°C and 250 rpm for 2 h. After incubation, the culture was spread onto LB solid medium containing streptomycin (40 mg / L) and kanamycin (50 mg / L) and incubated overnight at 37°C. After activation, single colonies were picked and colony PCR was performed using primers ΔlacZ-Scr-F and ΔlacZ-Scr-R to amplify the mutant fragment. Preliminary verification was performed based on nucleic acid electrophoresis bands. Finally, the PCR product with the correct nucleic acid band size was sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing for further confirmation, obtaining the recombinant strain B-ΔL.

[0092] (2) Construction of recombinant strains for producing minicells with inactivated minC gene

[0093] Using the extracted recombinant strain B-ΔL genome as a template, PCR amplification of the upstream homologous arm of the minC gene was performed using primers ΔC-1-F and ΔC-1-R, and PCR amplification of the downstream homologous arm of the minC gene was performed using primers ΔC-2-F and ΔC-2-R. Then, using the upstream and downstream homologous arms as templates, PCR was performed using primers ΔC-1-F and ΔC-2-R to construct the Donor DNA fragment with the C gene knocked out. Using plasmid pTarget F (given by Professor Yang Sheng of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences) as a template, PCR amplification was performed using primers pF-2-F and pF-2-R to obtain the linear recombinant plasmid pF-2. The linear recombinant plasmid pF-2 was then purified and recovered by gel electrophoresis. After eliminating the original template by DpnI enzyme digestion, it was introduced into the cloning host E. coli Trans1-T1 (purchased from Beijing TransGen Biotech Co., Ltd.) by heat shock transformation. It was incubated overnight at 37°C on LB solid culture medium containing streptomycin (40 mg / L). The next day, single colonies were picked for activation culture and finally sent to Suzhou Genewise Biotech Co., Ltd. for sequencing screening to obtain the correct pF-2 plasmid containing the N20 sequence of the knockout minC gene.

[0094] The successfully constructed Donor DNA and pF-2 were simultaneously electroporated into the target strain B-ΔL containing the pEcCas plasmid at a concentration of 5:1 (100 ng / μL). The electroporator was set to an electroporation voltage of 2500 V and an electroporation time of 5-6 ms. After electroporation, LB liquid medium was quickly added, and the culture was incubated at 37°C and 250 rpm for 2 h. After incubation, the culture was spread on LB solid medium containing streptomycin (40 mg / L) and kanamycin (50 mg / L) and incubated overnight at 37°C. After activation culture, single colonies were picked and colony PCR was performed using primers ΔC-Scr-F and ΔC-Scr-R to amplify the mutant fragment. Preliminary verification was performed based on nucleic acid electrophoresis bands. Finally, the PCR product with the correct nucleic acid band size was sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing for further confirmation, obtaining the recombinant strain B-ΔLC.

[0095] The primers used in this embodiment are shown in Table 4.

[0096] Table 4 Primer sequences used in this embodiment

[0097]

[0098] Example 5: Application of natural label in the production of galactosidase LacZ

[0099] In Example 1, the enhanced green fluorescent protein EGFP was replaced with galactosidase LacZ (the amino acid sequence of galactosidase LacZ is shown in SEQ ID NO. 69, and the codon-optimized nucleotide sequence is shown in SEQ ID NO. 70). E. coli BL21(DE3) competent cells were replaced with B-ΔL or B-ΔLC competent cells, while the rest of the process remained unchanged. Recombinant engineered strains expressing different natural tags (Erα, PAB1, Pbp1, Galectin-3, eIF4GII, U2AF65, RPB1, DDX3, TAF15, FMRP, DYRK1A, BRD4, LplA) fused with LacZ were obtained. The expression of each recombinant engineered strain was induced, and the enzyme activity of β-galactosidase was measured using an enzyme catalytic reaction system.

[0100] The enzyme-catalyzed reaction system for β-galactosidase was prepared by mixing 100 μL of the induced protein sample with 4 μL of FDG (1 mg / mL) in 10 mM PBS. Measurements were taken every few minutes (excitation wavelength 488 nm, measurement wavelength 535 nm). The 1 mg / mL FDG solution was resuspended in 98% water, 1% DMSO, and 1% ethanol. After detecting the fluorescence value in the reaction system using a microplate reader, simple linear regression analysis was performed to calculate the growth rate.

[0101] Specifically, the natural tag fusion with the LacZ gene fragment, the corresponding recombinant engineered strain, the simple linear regression equation (the slope represents the relative enzyme activity), and the enzyme activity of β-galactosidase ("fold" = experimental group slope value / control slope value - 1) are shown in Table 5. Figure 11 The figure shows how fusing DDX3 and TAF15 tags, respectively, to β-galactosidase enhances enzyme activity in recombinant E. coli.

[0102] As shown in Table 5, most natural tags, after achieving soluble expression in *E. coli*, can enhance β-galactosidase activity. Figure 11 After fusion of the TAF15 tag with β-galactosidase, the enzyme activity in recombinant *E. coli* B-ΔL and B-ΔLC increased by approximately 1.4-fold and 4-fold, respectively, indicating that further inactivation of the MinC gene can enhance enzyme activity. Similarly, after fusion of the DDX3 tag with β-galactosidase, the enzyme activity in recombinant *E. coli* B-ΔL decreased, but the enzyme activity in recombinant *E. coli* B-ΔLC increased by approximately 0.6-fold. Therefore, some aggregate elements have a negative impact on β-galactosidase activity in the B-ΔL-LacZ strain, but further inactivation of the minC gene can significantly improve β-galactosidase activity.

[0103] Table 5 Natural tag fusion LacZ gene fragment, corresponding recombinant engineered strains and enzyme activities

[0104]

[0105] Example 6: Application of artificially synthesized tags in the production of galactosidase LacZ

[0106] In Example 2, the enhanced green fluorescent protein EGFP was replaced with galactosidase LacZ, and the E. coli BL21(DE3) competent cells were replaced with B-ΔL or B-ΔLC competent cells, while the rest of the process remained unchanged. Recombinant engineered strains expressing different artificially synthesized tags fused with LacZ were obtained. The expression of each recombinant engineered strain was induced, and the enzyme activity of β-galactosidase was measured using an enzyme catalytic reaction system.

[0107] As shown in Table 6, most of the synthetic tags increased the enzyme activity of LacZ. Figure 12 No increase in enzyme activity was observed when the VAE10 tag was fused alone to β-galactosidase or in the recombinant E. coli expression system with further inactivated minC. After fusion of the VAE12 tag with β-galactosidase, enzyme activity decreased by 2.4-fold in recombinant E. coli B-ΔL, and increased by 0.25-fold in recombinant E. coli B-ΔLC with further inactivated minC, but the increase was not significant. Therefore, a modular phase separation platform based on the recruitment peptide pair RIAD-RIDD was used to optimize the enzyme activity (the amino acid sequences of RIAD and RIDD are shown in SEQ ID NO. 33-34, and the codon-optimized nucleotide sequences are shown in SEQ ID NO. 67-68), constructing the recombinant engineered strain B-ΔLC-V12-RIAD-LacZ. The construction process was based on Example 5 of Chinese Patent CN118291510A. The results showed that the recruitment peptide pair RIAD-RIDD could further improve the efficient recruitment of β-galactosidase in the condensed state, increasing enzyme activity by 1.2-fold.

[0108] Table 6. Synthetic tag fusion with LacZ gene fragment, corresponding recombinant engineered strain and enzyme activity.

[0109]

[0110] Example 7: Application of bioaggregate tags Y145, Y2, Y4 and Y15 in the production of galactosidase LacZ

[0111] I. Application of bioaggregate tags Y2 and Y4 in the production of galactosidase LacZ

[0112] In Example 1, the natural proteins were replaced with bioconglomerate tags Y2 and Y4, plasmid pETDuet-1 was replaced with plasmid pET28a, enhanced green fluorescent protein EGFP was replaced with galactosidase LacZ, and E. coli BL21(DE3) competent cells were replaced with B-ΔL or B-ΔLC competent cells, while the rest of the process remained unchanged. Recombinant engineered strains expressing Y2 or Y4 bioconglomerate tags fused with LacZ were obtained. The expression of each recombinant engineered strain was induced, and the enzyme activity of β-galactosidase was determined using an enzyme catalytic reaction system.

[0113] The results are shown in Table 7. Figure 13 As shown, the β-galactosidase fusion with the Y2 tag increased the enzyme activity in recombinant Escherichia coli B-ΔLC by 1.5 times; the β-galactosidase fusion with the Y4 tag increased the enzyme activity in recombinant Escherichia coli B-ΔLC by 7.2 times.

[0114] Table 7. Y2 and Y4 bioaggregate tag fusion with LacZ gene fragment, corresponding recombinant engineered strains and enzyme activities

[0115]

[0116] II. Application of bioaggregate label Y15 in the production of galactosidase LacZ

[0117] Following the application of bioconfluence tags Y2 and Y4 in the production of galactosidase LacZ, recombinant engineered strains expressing the Y15 bioconfluence tag fused with LacZ were obtained. These recombinant strains were induced to express β-galactosidase, and the enzyme activity of β-galactosidase was measured using an enzyme catalytic reaction system. The results are shown in Table 8. Figure 14 As shown.

[0118] After β-galactosidase was fused with the Y15 tag, the enzyme activity was increased in both B-ΔL and B-ΔLC of recombinant Escherichia coli, with B-ΔLC increasing the enzyme activity by 7.6 times.

[0119] Table 8. Y15 bioaggregate tag fusion with LacZ gene fragment, corresponding recombinant engineered strain and enzyme activity

[0120]

[0121] III. Application of bioaggregate tag Y145 in the production of galactosidase LacZ

[0122] The amino acid sequence of the bioaggregate tag Y145 was codon-optimized according to *E. coli* and the gene was synthesized. Simultaneously, the Y145 tag was linked to the galactosidase LacZ at the N-terminus or C-terminus using linkers such as flexible peptide (GGS)4 and (GGGGS)2, forming a Y145 tag fusion LacZ gene fragment. That is, galactosidase LacZ is linked to the N-terminus or C-terminus of the Y145 tag via linkers. The Y145 tag fusion LacZ gene fragments, after being double-digested with EcoRI and XhoI, were ligated to plasmid pET28a to obtain the corresponding Y145 tag fusion LacZ plasmids. All plasmids were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0123] The synthesized Y145 tag fusion LacZ plasmid was transformed and introduced into E. coli B-ΔL and B-ΔLC competent cells by heat shock method to obtain recombinant engineered strains expressing different natural tag fusion LacZ. The expression of each recombinant engineered strain was induced, and the enzyme activity of β-galactosidase was measured using an enzyme catalytic reaction system.

[0124] The results are shown in Table 9. Figure 15 As shown, this section systematically compared the effects of different fusion sites of the Y145 tag on β-galactosidase activity. The experimental results showed that when β-galactosidase was linked to the C-terminus of the Y145 tag, the enzyme activity in B-ΔL strains remained unchanged, while the enzyme activity in B-ΔLC strains increased by 0.4-fold. However, when β-galactosidase was linked to the N-terminus of the Y145 tag, the enzyme activity in recombinant E. coli B-ΔL strains increased by 1.5-fold, and the enzyme activity in B-ΔLC strains increased by 1.03-fold. Therefore, the fusion site (N-terminus or C-terminus) of the bioconcentration element with the target enzyme is also an important factor affecting enzyme activity.

[0125] Table 9. Y145 bioaggregate tag fusion with LacZ gene fragment, corresponding recombinant engineered strain and enzyme activity

[0126]

[0127] This invention provides a method for enhancing β-galactosidase activity by fusing biological aggregate elements, as well as a concept and method for recombinant engineered strains. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for enhancing β-galactosidase activity by incorporating bio-aggregate elements, characterized in that, The fusion gene fragment obtained by linking β-galactosidase lacZ to a bioconcentration element was heterologously expressed in Escherichia coli, thereby increasing β-galactosidase activity. The amino acid sequence of the β-galactosidase lacZ is shown in SEQ ID NO. 69; the bio-aggregate element includes any one of Erα, PAB1, Pbp1, Galectin-3, eIF4GII, U2AF65, RPB1, DDX3, SYN2, TAF15, Std1, FMRP, DYRK1A, BRD4, LplA, VAE1, VAE2, VAE3, VAE4, VAE5, VAE6, VAE7, VAE8, VAE9, VAE10, VAE11, VAE12, VAE13, Y145, Y2, Y4, and Y15, and their amino acid sequences are shown in SEQ ID NO. 1 to 32 respectively.

2. The method according to claim 1, characterized in that, The β-galactosidase lacZ is linked to biological condensate elements, either directly, via a linker, or via a recruitment tag.

3. The method according to claim 2, characterized in that, The Linker is a flexible peptide composed of glycine G and serine S, including any one of (GGS)n, (GGGS)n, (GGGGS)n, and (GGGSSS)n, where n = 1 to 8; the recruitment tags are RIAD and RIDD, wherein the amino acid sequence of RIAD is shown in SEQ ID NO. 33, and the amino acid sequence of RIDD is shown in SEQ ID NO.

34.

4. The method according to any one of claims 1 to 3, characterized in that, The β-galactosidase is linked to the N-terminus or C-terminus of the bio-aggregate element.

5. The method according to claim 1, characterized in that, The *Escherichia coli* strain is *Escherichia coli* BL21(DE3), or a recombinant strain B-ΔL of *Escherichia coli* BL21(DE3) lacking the lacZ gene, or a recombinant strain B-ΔLC of *Escherichia coli* BL21(DE3) lacking both the lacZ and minC genes.

6. The method according to claim 5, characterized in that, The recombinant strain B-ΔL was obtained by knocking out the lacZ gene from Escherichia coli BL21(DE3) as the chassis strain; the recombinant strain B-ΔLC was obtained by further knocking out the minC gene from the recombinant strain B-ΔL as the chassis strain.

7. The method according to claim 1, characterized in that, The β-galactosidase activity is increased by at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8 times.

8. A recombinant engineered strain that enhances β-galactosidase activity by fusing bioaggregate elements, characterized in that, The fusion gene fragment was constructed using Escherichia coli as the chassis strain and obtained by heterologously expressing β-galactosidase lacZ and linking it to a bioconcentration element.

9. The recombinant engineered strain according to claim 8, characterized in that, The *Escherichia coli* strain is *Escherichia coli* BL21(DE3), or a recombinant strain B-ΔL of *Escherichia coli* BL21(DE3) lacking the lacZ gene, or a recombinant strain B-ΔLC of *Escherichia coli* BL21(DE3) lacking both the lacZ and minC genes.

10. The recombinant engineered strain according to claim 8, characterized in that, The amino acid sequence of the β-galactosidase lacZ is shown in SEQ ID NO. 69; the bio-aggregate element includes any one of Erα, PAB1, Pbp1, Galectin-3, eIF4GII, U2AF65, RPB1, DDX3, SYN2, TAF15, Std1, FMRP, DYRK1A, BRD4, LplA, VAE1, VAE2, VAE3, VAE4, VAE5, VAE6, VAE7, VAE8, VAE9, VAE10, VAE11, VAE12, VAE13, Y145, Y2, Y4, and Y15, and their amino acid sequences are shown in SEQ ID NO. 1 to 32 respectively; the β-galactosidase lacZ is linked to the N-terminus or C-terminus of the bio-aggregate element by direct linkage, linker linkage, or recruitment tag; The Linker is a flexible peptide composed of glycine (G) and serine (S), including any one of (GGS)n, (GGGS)n, (GGGGS)n, and (GGGSSS)n, where n = 1 to 8; the recruitment tags are RIAD and RIDD, wherein the amino acid sequence of RIAD is shown in SEQ ID NO. 33, and the amino acid sequence of RIDD is shown in SEQ ID NO. 34.

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