Method for mining and analyzing transcription factor AcaR regulating synthesis of acarbose based on multi-omics

Abstract

The present application relates to the technical field of bioengineering, and discloses a method for mining and analyzing transcription factor AcaR regulation of acarbose synthesis based on multiomics, which comprises multiomics analysis and mining of transcription factor AcaR on mutant strains; target promoter and binding mechanism are verified by gel retardation analysis and molecular simulation; and recombinant bacteria are constructed by overexpression of AcaR to improve acarbose yield. acbJ With acbL The present application proves that AcaR directly and specifically binds to the promoter region of the gene by molecular docking, the residue of arginine (Arg48) at the 48th position forms a specific hydrogen bond network with the phosphate skeleton and bases of the promoter DNA through the side chain functional group to realize accurate recognition; the conserved binding motif NNGTACNNNN is identified by whole genome scanning, and the key role of the motif and Arg48 in transcription activation is verified by virtual saturation mutation and reporter gene experiment, thereby providing theoretical support for directional regulation of acarbose synthesis.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for multi-omics mining and analysis of the regulation of acarbose synthesis by transcription factor AcaR. Background Technology

[0002] Acarbose is a natural product containing an aminocyclic alcohol structure. It controls glucose production by competitively binding to intestinal amylase and α-glucosidase, thus slowing polysaccharide hydrolysis. It is widely used in the clinical treatment of type II diabetes. Currently, industrial production of acarbose mainly relies on fermentation of zooplankton and its derivative strains. With advancements in research on the whole genome information of these microorganisms and related acarbose synthesis gene clusters, systematic modifications based on the anabolic pathway have made some progress, providing a foundation for the development of acarbose biomanufacturing processes.

[0003] Existing acarbose-producing actinomycetes still exhibit low fermentation yields, failing to fully meet the growing demand for acarbose. Traditional strain improvement strategies primarily rely on random mutagenesis and empirical screening, methods that are largely indiscriminate and lack a clear understanding of the underlying molecular mechanisms. Furthermore, current metabolic engineering interventions often focus on modifying single enzymatic reactions or local pathway genes, neglecting the exploration of the intracellular global transcriptional regulatory network and failing to grasp the overall metabolic flux distribution pathways. In addition, despite advancements in individual omics technologies, a systematic implementation framework is still lacking for integrating multi-omics data to guide the rational design of high-yield strains.

[0004] Current technologies lack in-depth elucidation of the molecular mechanisms by which global regulatory elements such as transcription factors recognize promoter sequences and their targeted regulation. This results in a lack of clear biological targets and underlying theoretical support for the targeted modification of zooplankton. Conventional modification methods are insufficient to effectively redirect intracellular carbon metabolism flux towards acarbose synthesis, and the resulting recombinant engineered bacteria are prone to genetic degradation and unstable synthetic performance during continuous subculturing. Simple mutagenesis screening has not been deeply integrated with multi-omics dynamic analysis, failing to systematically demonstrate and illustrate the patterns of increase in target products and precursors and decrease in competing substrates during fermentation. Therefore, establishing a scheme that integrates multi-omics analysis, elucidation of transcription factor regulatory mechanisms, and strain targeted modification technology to improve acarbose fermentation yield and stability is a problem that needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for increasing acarbose production based on multi-omics analysis and transcription factor AcaR regulation. This method solves the problems of low yield in existing acarbose production by zooxygenesis fermentation, blind application of traditional modification strategies with unclear underlying mechanisms, and difficulty in achieving carbon metabolism flux redirection and genetic trait instability in recombinant strains.

[0006] To address the above problems, the present invention provides the following technical solution: This invention provides a method for regulating acarbose synthesis based on multi-omics mining and analysis of transcription factor AcaR, employing the following technical solution: A method based on multi-omics mining and analysis of the regulation of acarbose synthesis by transcription factor AcaR includes the following steps: Multi-omics analysis of the mutant strain of Actinobacterium vegetans confirmed that the transcription factor AcaR was continuously upregulated throughout the fermentation process and that the acarbose synthesis gene cluster and transport protein gene were significantly upregulated during the peak yield period, thus identifying the regulatory target. The transcription factor AcaR protein was cloned and expressed to target the aforementioned regulatory mechanism. The binding of the transcription factor AcaR protein to the promoters related to acarbose synthesis and the core binding residues of the transcription factor AcaR protein were verified to determine the mechanism by which AcaR regulates acarbose synthesis.

[0007] By employing the above technical solutions, multi-omics analysis was used to analyze the gene and metabolic level changes of the high-yielding strain AH-2 obtained by atmospheric pressure and room temperature plasma mutagenesis, and the transcription factor AcaR and key catalytic genes were identified as regulatory targets. The key binding sites were determined by gel retardation analysis, molecular dynamics simulation, and in vivo and in vitro binding fluorescence experiments, thus clarifying the molecular mechanism by which AacR regulates acarbose.

[0008] Preferably, the specific process of performing multi-omics analysis on the motile actinomycete mutant strain includes: Genomic DNA was extracted from the mutant strain of *Actinomyces leuciscus* and whole-genome sequencing was performed to identify single nucleotide variant sites. Transcriptome sequencing was performed on cells from different fermentation time points during the fermentation process of the mutant strain to analyze differences. Fermentation broth and cells were collected during the fermentation process for metabolomics analysis to clarify the changes in the content of acarbose and its precursors and competing substrates.

[0009] By employing the above-mentioned technical solutions, combining whole-genome single nucleotide variant alignment, transcriptome time-series differential analysis, and dynamic monitoring of metabolomics substance content, the changes in the gene and metabolic levels of the mutant strain of *Actinomyces leucoma* obtained by atmospheric pressure and room temperature plasma mutagenesis were analyzed. The dynamic relationship between the accumulation of the acarbose precursor and the consumption of competing substrates was clarified, providing multi-dimensional data support for identifying transcriptional and metabolic regulatory targets.

[0010] Preferably, the amino acid sequence of the transcription factor AcaR protein is shown in SEQ ID NO:1, and the corresponding nucleotide sequence of the transcription factor AcaR protein is shown in SEQ ID NO:2; the core binding residue is arginine at position 48 of the transcription factor AcaR protein, and site-directed mutation of the core binding residue leads to a decrease in the binding ability and transcriptional activation ability of the transcription factor AcaR protein.

[0011] By employing the above technical solution, the sequence information of the transcription factor AcaR protein was defined. Combined with molecular dynamics simulations and in vivo and in vitro binding fluorescence experiments, it was revealed that arginine at position 48 is a key amino acid site for exerting transcriptional activation function, providing an accurate molecular biological basis for establishing the gene recombination modification strategy and regulating the activity of the target protein.

[0012] Preferably, the acarbose synthesis-related promoter is promoter P. acbJ and promoter P acbL Furthermore, gel retardation analysis confirmed that the transcription factor AcaR protein interacts with the promoter P. acbJ and the promoter P acbL Specific binding.

[0013] By employing the above technical solution, the downstream target of the transcription factor AcaR in exerting its positive regulatory effect is identified as promoter P. acbJ and promoter P acbL The specific binding physical interaction between the two was objectively confirmed in vitro using gel retardation analysis, revealing the intrinsic regulatory process by which the transcription factor AcaR directly activates the expression of the acarbose synthesis gene cluster.

[0014] Preferably, the process of obtaining the motile actinomycete mutant strain includes: Wild-type original strain of Actinobacterium moniliforme Actinoplanessp. SE50 / 110 was inoculated and activated to prepare a bacterial suspension, which was then evenly spread on a sterile metal slide and placed in the operating chamber of an atmospheric pressure, room temperature plasma mutagenesis system for multiple rounds of irradiation mutagenesis treatment. The irradiated and mutagenized bacterial cells were washed and collected to obtain a mutagenized bacterial suspension. The mutagenized bacterial suspension was spread on a plate containing streptomycin for resistance screening to obtain streptomycin-resistant mutant single colonies. The streptomycin-resistant mutant single colonies were picked for shake-flask fermentation for re-screening and continuous multi-generation subculturing to screen for acarbose-producing mutant strain AH-2 as the motile actinomycete mutant strain.

[0015] By adopting the above technical solution, the original strain was subjected to multiple rounds of physical induction mutations under normal pressure conditions using atmospheric pressure room temperature plasma mutagenesis technology. Combined with targeted streptomycin resistance isolation and screening, the acarbose high-yield mutant strain AH-2 with stable genetic characteristics and high initial yield was enriched and screened, providing recipient cell resources for subsequent multi-omics analysis and targeted gene recombination modification.

[0016] Preferably, the overexpressed transcription factor AcaR The gene process includes: transcription factors AcaR The gene was ligated into an expression vector with a promoter to construct a recombinant expression vector, which was then transformed into donor bacteria. The donor bacteria were co-cultured with the mutant strain of Actinobacterium molluscum as the recipient bacteria for conjugation transfer. Transformants were screened by covering the bacteria with sterile water containing antibiotics, and the transformants were extracted for identification and verification to obtain gene-overexpressing recombinant bacteria.

[0017] By adopting the above technical solution, the transcription factor AcaR The gene was introduced into motile actinomycetes for overexpression, which upregulated the activity of synthetic pathways at the transcriptional level and promoted acarbose synthesis.

[0018] Preferably, the specific process of activating the recombinant bacteria and inoculating it into the culture medium for fermentation is as follows: The recombinant bacteria were streaked and activated, then inoculated into seed culture medium for two-stage shake-flask expansion, and subsequently inoculated into fermentation medium. Fermentation was carried out for 7 days at 28°C and 220 rpm with shaking. After 48 hours of fermentation, a mixture of glucose and maltose with a final concentration of 3% was added.

[0019] By adopting the above technical solution, the operation standards from strain activation to fermentation culture are standardized, and a mixture of glucose and maltose is added at 48h of fermentation, the starting point of large-scale product synthesis, to supplement the precursor substrate and cellular carbon source required for the construction of the acarbose molecular skeleton, thereby meeting the material requirements of the recombinant bacteria during the peak production period and maintaining long-term fermentation momentum.

[0020] Preferably, the seed culture medium formula specifically comprises, by mass concentration: The seed culture medium contains 40 g / L soybean meal, 20 g / L glycerol, 10 g / L corn starch, and 2 g / L calcium carbonate, with the pH value controlled between 6.4 and 7.0. The fermentation medium formula, by mass concentration, specifically includes: The fermentation medium contains 23 g / L glucose, 70 g / L maltose, 10 g / L soybean meal, 3.5 g / L CaCl2, 5 g / L monosodium glutamate, 1 g / L K2HPO4·3H2O, 5 g / L glycerol, 3 g / L calcium carbonate, and 0.5 g / L FeCl3·6H2O, with the pH value controlled between 6.4 and 6.7.

[0021] By adopting the above technical solution, a carbon-nitrogen source ratio system adapted to the amplification of the recombinant bacteria and the synthesis of acarbose is provided. Combined with a strictly controlled inorganic salt buffer network and pH range, the fermentation state and secondary metabolic process of the engineered strain are guaranteed to be stable.

[0022] Preferably, the specific operation of collecting, extracting, and testing the fermentation broth after fermentation is completed is as follows: After the fermentation is completed, the fermentation broth is centrifuged, and the supernatant is used to detect the acarbose content by high performance liquid chromatography.

[0023] By adopting the above technical solution, a standardized process for product liquid-solid separation extraction and instrumental analysis quantification is established, eliminating the interference of fermentation cells and impurities on the detection results, and ensuring the objectivity and consistency of the acarbose yield assessment of the recombinant bacteria.

[0024] This invention provides a method for analyzing the regulation of acarbose synthesis by the transcription factor AcaR based on multi-omics. It has the following beneficial effects: 1. This invention, through multi-omics analysis of a mutant strain of Actinobacterium praecox and cloning and expressing the transcription factor AcaR protein, clarified the specific binding physical role of the arginine core binding residue with the promoter related to acarbose synthesis. It revealed the key amino acid sites of transcription factor AcaR in exerting its transcriptional activation function and the intrinsic molecular mechanism of directly activating the expression of acarbose synthesis gene clusters at the molecular structure level, providing an accurate molecular biological basis for establishing gene recombination modification strategies and regulating the activity of target proteins.

[0025] 2. This invention utilizes atmospheric pressure and room temperature plasma mutagenesis combined with streptomycin resistance isolation and screening to obtain a stable genetic mutant strain of actinomycetes as the recipient cell for targeted gene recombination modification. During fermentation culture, a mixture of glucose and maltose is added to supplement the precursor substrate and cellular carbon source required for the construction of the acarbose molecular backbone. Combined with seed culture medium and fermentation culture medium with precise carbon and nitrogen source ratio control, the material requirements of the recombinant bacteria are met, ensuring the stability of secondary metabolic processes and fermentation state. Attached Figure Description

[0026] Figure 1 This is a heatmap of differentially expressed genes in key metabolic pathways in the multi-omics analysis of this invention; Figure 2 This is a diagram showing the docking of the AcaR protein with DNA molecules and its three-dimensional structure in the transcription factor regulation mechanism of this invention. Figure 3 This is a diagram showing the conserved binding motif of AcaR in the target recognition mechanism of this invention. Figure 4 This is a graph showing the change in the binding free energy between the AcaR mutant and the target promoter in the molecular simulation calculations of this invention. Figure 5 This is a graph showing the results of fluorescent reporter gene detection during the in vivo functional verification of this invention. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The customized raw materials and related culture medium formulations used in the embodiments and test examples of this invention are specifically described below. Reagents not specifically described are all commercially available analytical grade or higher grade products: Plasmid PPR is formed by introducing the strong promoter P into the expression vector pSET152. ermF* Recombinant-customized vectors were constructed from the downstream RBS sequence.

[0029] The SM medium formula, by mass concentration, specifically includes: 30 g / L sucrose, 5 g / L peptone, 1 g / L L-tyrosine, 0.5 g / L KCl, 0.5 g / L MgSO4·7H2O, and 1 g / L K2HPO4·3H2O, with the pH value controlled between 7.0 and 7.3.

[0030] The formulation of the isolation and slant culture medium, by mass concentration, specifically includes: 30 g / L sucrose, 5 g / L peptone, 1 g / L L-tyrosine, 0.5 g / L KCl, 0.5 g / L MgSO4·7H2O, 1 g / L K2HPO4·3H2O, and 18–20 g / L agar, with the pH value controlled at 7.0–7.3.

[0031] The SM mycelial culture medium formula, by mass concentration, specifically includes: 10 g / L maltose, 15 g / L glucose, 5 g / L tryptone, 5 g / L yeast extract, 10 g / L malt extract, 10 g / L glycerol, 1 g / L casein hydrolysate, and 1 g / L K2HPO4, with the pH value controlled at 7.2.

[0032] The seed culture medium formula, by mass concentration, specifically includes: 40 g / L soybean meal, 20 g / L glycerol, 10 g / L corn starch, and 2 g / L calcium carbonate, with the pH value controlled between 6.4 and 7.0.

[0033] The fermentation medium formula, by mass concentration, specifically includes: 23 g / L glucose, 70 g / L maltose, 10 g / L soybean meal, 3.5 g / L CaCl2, 5 g / L sodium glutamate, 1 g / L K2HPO4·3H2O, 5 g / L glycerol, 3 g / L calcium carbonate, and 0.5 g / L FeCl3·6H2O, with the pH value controlled between 6.4 and 6.7.

[0034] Preparation Example 1: This preparation example provides a method for obtaining the high-yield acarbose mutant strain AH-2, including the following steps: Wild-type original strain of Actinobacterium moniliforme Actinoplanes sp. SE50 / 110 was inoculated into the culture medium for activation culture, the bacterial cells were collected and thoroughly washed with sterile physiological saline, and then broken up and dispersed to prepare a bacterial suspension with uniform concentration for later use.

[0035] An appropriate amount of the bacterial suspension prepared above was evenly coated onto a sterile metal slide and placed in the operating chamber of an atmospheric pressure, room temperature plasma mutagenesis system. Suitable radio frequency power and gas flow rate parameters were set, and the bacteria on the slide underwent multiple rounds of irradiation to induce mutagenesis. After treatment, the bacteria on the slide were eluted with sterile physiological saline, and the mutagenized bacterial suspension was collected.

[0036] The mutagenic bacterial suspension was serially diluted and spread onto isolation medium plates containing a specific concentration of streptomycin for resistance screening. The plates were then incubated at a suitable temperature until single colonies appeared. Well-grown, robust streptomycin-resistant mutant single colonies were selected and inoculated into seed medium and fermentation medium for shake-flask fermentation for secondary screening. High-yielding strains were selected by detecting the acarbose titer in the fermentation broth.

[0037] The selected high-yielding strains were subjected to multiple generations of subculturing, and the fermentation products were analyzed simultaneously to verify their production capacity and genetic stability. Finally, a genetically stable mutant strain was obtained and named the acarbose high-yielding mutant strain AH-2.

[0038] Example 1: This embodiment provides a method for increasing acarbose production based on multi-omics analysis and transcription factor AcaR regulation, including the following steps: S10. Genomic DNA of the mutant strain AH-2 obtained in Preparation Example 1 was extracted and whole-genome sequencing was performed. Single nucleotide variant sites were identified by comparison. Transcriptome sequencing was performed on bacterial cells collected at different fermentation time points. Differential analysis confirmed that the transcription factor AcaR was continuously upregulated throughout the fermentation process, and the acarbose synthesis gene cluster acb series and ABC transporter gene were significantly upregulated during the peak yield period. Metabolomics detection was performed on fermentation broth and bacterial cells, which showed that the content of acarbose and its precursors increased and the content of competing substrates decreased. S20. Cloning and expressing the transcription factor AcaR protein (its amino acid sequence is shown in SEQ ID NO:1, and its corresponding nucleotide sequence is shown in SEQ ID NO:2), and confirming its interaction with the promoter P by gel retardation analysis. acbJ P acbL Specific binding; molecular dynamics simulations and site-directed mutagenesis confirmed that arginine at position 48 of AcaR is the core binding residue, and mutations in it lead to a significant decrease in binding capacity and transcriptional activation capacity.

[0039] The detection conditions for high performance liquid chromatography were as follows: the chromatographic column was a Thermon Fisher Hypersil APS-2 NH2; the mobile phase was a mixture of phosphate buffer and acetonitrile at a volume ratio of 25:75, wherein the phosphate buffer was obtained by dissolving 0.6 g of potassium dihydrogen phosphate and 0.279 g of anhydrous disodium hydrogen phosphate in water and diluting it to 1 L; the flow rate was 1.0 mL / min, the column temperature was 40 ℃, the injection volume was 10 μL, and the detection wavelength of the ultraviolet detector was 210 nm.

[0040] Test Example 1-3: Test Example 1: Experimental description: This section aims to comprehensively analyze the genetic variations and metabolic differences between the original motile actinomycetes and the mutant and recombinant strains after mutagenesis and recombination using genomics, transcriptomics and metabolomics technologies, and then to discover and verify the changes in key regulatory targets and metabolic pathways affecting acarbose synthesis.

[0041] Experimental steps: Genomic DNA of the mutant strain AH-2 obtained in Preparation Example 1 and the original strain was extracted and sequenced at 175× depth using the Illumina sequencing platform, yielding 10,426,852 high-quality reads. Whole genome sequencing was performed using NC_017803.1 as the reference genome, and sequence alignment was performed. Cell samples of the original strain and the mutant strain AH-2 were collected at different time points (30h, 36h, 60h, 72h, 90h, 96h) during fermentation. Total RNA was extracted, a library was constructed, and transcriptome sequencing was performed to screen differentially expressed genes at different fermentation stages. Simultaneously, cell and fermentation broth samples were collected from each of the above fermentation stages. Metabolites and intermediates were detected using high performance liquid chromatography-mass spectrometry (HPLC-MS), and the relative abundance variation of metabolites in each pathway was calculated.

[0042] Experimental conclusion: Multi-omics analysis revealed the mechanism by which recombinant modification promotes acarbose production. Transcriptomic analysis showed that in the recombinant strains, the acarbose synthesis gene cluster (acb series genes) and ABC transporter genes... acbWXY The yield peaked at 72 hours of fermentation, showing a clear upward trend. For example... Figure 1 The differential gene expression heatmap of key metabolic pathways shows the fold change in the expression of related genes in the glycolysis pathway (EMP), tricarboxylic acid cycle (TCA), pentose phosphate pathway (HMP), and acarbose synthesis pathway. The changes in the different shades of gray in the legend reflect the significant upregulation or downregulation of gene expression.

[0043] Homology comparison between the mutant strain AH-2 from Example 1 and the original strain confirmed that... acaR Gene (Gene ID: ACPL_ RS31540 The expression levels of the transcription factor AcaR encoded by the strain were increased by 2.09-fold, 1.38-fold, and 1.45-fold at 36h, 72h, and 96h, respectively, compared with the original strain. The high expression level was maintained throughout the fermentation cycle, which clarified that the transcription factor AcaR has a positive regulatory effect on the downstream synthetic clusters and transport protein genes of the strain in the example.

[0044] Metabolomics results were consistent with transcriptomics data. Comparative analysis of the mutant strain AH-2 (Example 1) and the original strain showed a significant increase in the content of the target product acarbose and its upstream precursors D-ribulose-5-phosphate (D-Ribulose-5-P) and D-ribose-5-phosphate (D-Ribose-5-P) (acarbose content was upregulated by 2.37-fold), while the content of substrates such as glucose was downregulated (maltose and glucose contents were downregulated by 0.50-fold and 0.47-fold, respectively). Differential metabolites were mainly enriched in pathways such as transport proteins and amino acid biosynthesis, including intermediates such as citrate and α-ketoglutarate in the tricarboxylic acid cycle, and metabolic flux rearrangement characteristics in the glutamate metabolism pathway. The above analysis demonstrates that the upregulation of the transcription factor AcaR drove the redirection of intracellular carbon flux towards acarbose synthesis in the example cells, achieving an increase in the fermentation level of the target product.

[0045] Test Example 2: Experimental description: To verify the impact of the core target transcription factor AcaR and its key mutation sites on the acarbose synthesis mechanism, in vivo and in vitro protein-DNA binding experiments were conducted, and molecular simulation calculations were used to elucidate the molecular mechanism of its targeted regulation.

[0046] Experimental steps: Based on transcription factors acaR Primers were designed based on the gene sequence (the forward primer sequence is shown in SEQ ID NO:3, containing the NotI restriction site; the reverse primer sequence is shown in SEQ ID NO:4, containing the EcoRV restriction site). PCR amplification was performed using the *Actinomyces foetida* genome as a template (PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; final extension at 72℃ for 10 min). The target gene was amplified and cloned into the pET28a expression vector. After constructing the recombinant plasmid, it was transformed into E. coli BL21 host, and expression was induced at 16℃ for 10-12 h with 0.5 mM IPTG. The expression was then analyzed using Ni... 2+ Wild-type AcaR protein was obtained by affinity chromatography, and the R48D mutant of AcaR protein was obtained by site-directed mutagenesis. The relevant promoter fragments in the acarbose synthesis gene cluster were amplified and incubated with purified wild-type AcaR and R48D mutants at room temperature, respectively, and then separated by non-denaturing polyacrylamide gel electrophoresis. A reporter vector was constructed to drive the expression of a fluorescent protein using a target promoter. This vector was then compared with wild-type AcaR expression vectors, R48D mutant expression vectors, and vectors containing no... acaREmpty expression vectors of the gene (as control group) were co-transformed into host strains and changes in fluorescence intensity were detected. The three-dimensional structure of AcaR was constructed using AlphaFold3 software. Molecular docking and molecular dynamics simulations were performed using Discovery Studio to calculate the free energy changes of each mutant binding to the DNA promoter. At the same time, sequence analysis tools were used to scan the conserved motifs in the promoter region.

[0047] Experimental conclusion: In vitro and in vivo binding experiments, along with molecular simulation calculations, corroborated each other, confirming the regulatory mechanism by which the overexpressed transcription factor AcaR in the examples activates downstream gene expression by binding to a specific promoter region. In vitro gel electrophoresis results showed that wild-type AcaR can specifically bind to the target promoter P. acbJ With P acbL The binding ability of the R48D mutant was significantly weakened, and the binding ability of the protein complex was clearly weakened, which confirms that arginine residues play an essential role in recognizing the target promoter.

[0048] like Figure 4 The calculated binding free energy (ΔΔG) between the mutant and the target promoter is shown in the figure, with "AcaR-P" in the legend. acbJ "and "AcaR-P acbL "Represents transcription factor AcaR and promoter P respectively" acbJ and P acbL The system of bound amino acid point mutations is shown on the horizontal axis. The horizontal axis represents different combinations of point mutations, including single-point mutations (such as R48D, R48D-2, etc.) and multi-point combined mutations involving sites such as L24D, R45E, R54E, and Q25D. The vertical axis represents the change in binding free energy, expressed in kcal / mol. When an R48D mutation occurs (whether a single-point or multi-point combined mutation), the binding free energy interacts with the target promoter (the left-hand grouped bars in the figure represent P). acbJ The grouped bar chart on the right represents P. acbL The free energy of the binding system showed a significant positive change (greater than 0), which thermodynamically confirms that residue mutations lead to unstable binding between the protein and the promoter, resulting in a significant weakening of binding ability. Figure 2 Molecular docking model and Figure 3 Conserved binding motif analysis clearly shows that arginine at position 48 (Arg48) is the core functional residue, and its side chain N η1 N η2 and N ε The functional group can bind to the phosphate backbone of promoter DNA (OP). 1 / OP 2 ) and bases (N 7 / O6 This forms a specific hydrogen bond network, thereby accurately identifying and combining conserved motifs NNGTACNNNN.

[0049] like Figure 5 The results of fluorescent reporter gene assays, with fluorescence intensity on the ordinate, show that co-expression of wild-type AcaR significantly increased the transcriptional level of downstream reporter genes of the target promoter (fluorescence intensity increased 3.2-fold compared to the control group). However, induction of the R48D mutant significantly reduced its transcriptional activation capacity compared to the wild type (fluorescence intensity was only 27.3% of that of the wild type). This in vivo functional analysis confirms the crucial role of this core residue and its binding motif in mediating the transcriptional activation of acarbose synthesis genes, demonstrating that the transcription factor AcaR possesses a positive regulatory function of directly binding to target gene promoters and activating the acarbose synthesis pathway.

[0050] Appendix: AcaR amino acid sequence: SEQ ID No:1: MPSEYAKSLGARLRSIRQQQGLSLQGVEEKSNGRWKAVVVGSYERGDRAVTVSRLAELADFYRVPVSELLPDGSGIRLEATNKIVLDLEKLYDTTGEDLAYVARYARAIQQQRGDYNGRVLSIRADDLRALAIVYDISPSGLIERLTEQGVLVADPRAFFAS.

[0051] acaR Nucleotide sequence: SEQ ID No:2: ATGCCGTCTGAGTACGCGAAGTCACTGGGCGCTCGTCTGCGCTCCATTCGCCAGCAGCAGGGCCTGTCCCTGCAGGGCGTCGAAGAGAAGTCCAACGGCCGCTGGAAGGCCGTCGTGGTCGGCTCGTACGAGCGTGGCGACCGTGCCGTGACCGTCTCGCGCCTGGCCGAGTTGGCCGACTTCTACCGGGTGCCCGTCTCCGAGCTGCTGCCCGACGGCAGCGGCATCCGCCTCGAGGCCACCAACAAGATCGTGCTGGACCTCGAGAAGCTGTACGACACCACCGGTGAGGACCTCGCCTACGTGGCGCGGTACGCCCGGGCGATCCAGCAGCAGCGTGGCGACTACAACGGCCGGGTCCTCTCGATCCGCGCCGACGACCTGCGCGCGCTCGCCATCGTGTACGACATCTCGCCGTCCGGCTTGATCGAGCGGCTCACCGAGCAGGGCGTCCTGGTGGCCGACCCGCGGGCGTTCTTCGCGAGCTGA。

[0052] Nucleotide sequence of primer AcaRF: SEQ ID No: 3: ACCCCAGGATCCGCGGCCGCATGCCGTCTGAGTACGCG。

[0053] Nucleotide sequence of primer AcaRR: SEQ ID No: 4: ATGATTACGAATTCGATATCTCAGCTCGCGAAGAACGC。

Claims

1. A method for mining and analyzing the regulation of acarbose synthesis by transcription factor AcaR based on multi-omics, characterized in that, Includes the following steps: Multi-omics analysis of the mutant strain of Actinobacterium vegetans confirmed that the transcription factor AcaR was continuously upregulated throughout the fermentation process and that the acarbose synthesis gene cluster and transport protein gene were significantly upregulated during the peak yield period, thus identifying the regulatory target. The transcription factor AcaR protein was cloned and expressed to target the aforementioned regulatory mechanism. The binding of the transcription factor AcaR protein to the promoters related to acarbose synthesis and the core binding residues of the transcription factor AcaR protein were verified to determine the molecular mechanism by which AcaR regulates acarbose.

2. The method for regulating acarbose synthesis based on multi-omics mining and analysis of transcription factor AcaR according to claim 1, characterized in that, The specific process of performing multi-omics analysis on the mutant strain of motile actinomycetes includes: Genomic DNA was extracted from the mutant strain of *Actinomyces leuciscus* and whole-genome sequencing was performed. Single nucleotide variant sites were identified by comparison. Transcriptome sequencing was performed on cells collected at different fermentation time points during the fermentation process of the mutant strain of *Actinomyces leuciscus* to analyze differential morphological differences. The fermentation broth and bacterial cells were collected during the fermentation process for metabolomics analysis to clarify the changes in the content of acarbose and its precursors and competing substrates.

3. The method for regulating acarbose synthesis based on multi-omics mining and analysis of transcription factor AcaR according to claim 1, characterized in that, The amino acid sequence of the transcription factor AcaR protein is shown in SEQ ID NO:1, and the nucleotide sequence corresponding to the transcription factor AcaR protein is shown in SEQ ID NO:2; The core binding residue is arginine at position 48 of the transcription factor AcaR protein. Site-directed mutation of the core binding residue leads to a decrease in the binding ability and transcriptional activation ability of the transcription factor AcaR protein.

4. The method for regulating acarbose synthesis based on multi-omics mining and analysis of transcription factor AcaR according to claim 1, characterized in that, The acarbose synthesis-related promoter is promoter P. acbJ and promoter P acbL Furthermore, gel retardation analysis confirmed that the transcription factor AcaR protein interacts with the promoter P. acbJ and the promoter P acbL Specific binding.

5. The method for regulating acarbose synthesis based on multi-omics mining and analysis of transcription factor AcaR according to claim 1, characterized in that, The process of obtaining the mutant strain of the motile actinomycete includes: Wild-type original strain of Actinobacterium moniliforme Actinoplanes sp. SE50 / 110 was inoculated and activated to prepare a bacterial suspension, which was then evenly coated onto a sterile metal slide and placed in the operating chamber of an atmospheric pressure, room temperature plasma mutagenesis system for multiple rounds of irradiation mutagenesis treatment. After elution and collection of irradiated mutagenesis-treated bacterial cells, a mutagenized bacterial suspension was obtained. The mutagenized bacterial suspension was then spread on a streptomycin-containing isolation medium plate for resistance screening to obtain streptomycin-resistant mutant single colonies. Single colonies of the streptomycin-resistant mutant were selected for shake-flask fermentation and repeated screening and multiple generations of subculturing to obtain the acarbose-producing mutant strain AH-2 as the motile actinomycete mutant strain.

6. The method for regulating acarbose synthesis based on multi-omics mining and analysis of transcription factor AcaR according to claim 1, characterized in that, The overexpressed transcription factor acaR Genetic processes include: transcription factors acaR The gene was ligated into an expression vector with a promoter to construct a recombinant expression vector, which was then transformed into donor bacteria. The donor bacteria were co-cultured with the mutant strain of Actinobacterium molluscum as the recipient bacteria for conjugation transfer. Transformants were screened by covering the bacteria with sterile water containing antibiotics, and the transformants were extracted for identification and verification to obtain gene-overexpressing recombinant bacteria.

7. The method for regulating acarbose synthesis based on multi-omics mining and analysis of transcription factor AcaR according to claim 1, characterized in that, The specific process of activating the recombinant bacteria and inoculating them into the culture medium for fermentation is as follows: The recombinant bacteria were streaked and activated, then inoculated into seed culture medium for two-stage shake-flask expansion, and subsequently inoculated into fermentation medium. Fermentation was carried out for 7 days at 28°C and 220 rpm with shaking. After 48 hours of fermentation, a mixture of glucose and maltose with a final concentration of 3% was added.

8. The method for multi-omics mining and analysis of transcription factor AcaR regulating acarbose synthesis according to claim 8, characterized in that, The seed culture medium formula, by mass concentration, specifically includes: The seed culture medium contains 40 g / L soybean meal, 20 g / L glycerol, 10 g / L corn starch, and 2 g / L calcium carbonate, with the pH value controlled between 6.4 and 7.

0. The fermentation medium formula, by mass concentration, specifically includes: The fermentation medium contains 23 g / L glucose, 70 g / L maltose, 10 g / L soybean meal, 3.5 g / L CaCl2, 5 g / L monosodium glutamate, 1 g / L K2HPO4·3H2O, 5 g / L glycerol, 3 g / L calcium carbonate, and 0.5 g / L FeCl3·6H2O, with the pH value controlled between 6.4 and 6.

7.

9. The method for regulating acarbose synthesis based on multi-omics mining and analysis of transcription factor AcaR according to claim 1, characterized in that, The specific steps for collecting, extracting, and testing the fermentation broth after fermentation are as follows: After the fermentation is completed, the fermentation broth is centrifuged, and the supernatant is used to detect the acarbose content by high performance liquid chromatography.

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