Self-induction regulated bacillus amyloliquefaciens and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a self-induction regulated bacillus amyloliquefaciens engineering strain and application thereof. According to the engineering strain, a quorum sensing QS-based activation module and a bifunctional molecular switch of an SspB-ssrA protein degradation closing module are constructed, wherein the activation module is integrated with a QS system element from bacillus subtilis, and sequential activation of a target gene is realized by sensing a thallus density threshold value; the closing module introduces Pantoea alhagi sourced SspB protein to specifically recognize target protein carrying an ssrA degradation tag, and target protein degradation is mediated by a ClpXP protease complex to close an expression pathway. The invention provides an efficient and low-cost gene dynamic regulation and control platform technology, and a technical foundation is laid for industrial production of high-added-value biological products such as natural active substances, functional polysaccharides and recombinant proteins.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a self-induced regulated Bacillus amyloliquefaciens and its applications. Background Technology

[0002] In microbial metabolic networks, dynamic regulation at the protein level offers advantages such as directness, reversibility, and rapid response. This is primarily achieved through allosteric regulation of enzyme molecules and degradation regulation based on protease systems. Protein degradation regulation relies on the bacterial transport-messenger RNA (tmRNA) system: after a target protein is fused with an ssrA tag at its C-terminus, it is recognized and degraded by the ClpXP protease, guided by the SspB protein, and the degradation rate can be precisely controlled by optimizing the tag sequence. Compared to transcriptional regulation dependent on promoter-transcription factor interactions, protein degradation regulation can directly degrade enzyme proteins, achieving instantaneous termination of metabolic reactions. This is particularly crucial for primary metabolic pathways requiring rapid switching of carbon flux allocation. Therefore, the SspB-ClpXP-based proteolysis system has become an important tool for dynamic metabolic flux regulation. Furthermore, in synthetic biology, where spatiotemporal decoupling of cell growth and product synthesis is required, population density-mediated dynamic regulation strategies (such as quorum sensing systems) offer another advantage: they operate in a process / pathway-independent manner and have broad applicability. Quorum sensing (QS) systems do not require exogenous inducers: bacteria secrete self-inducers (AIs), triggering changes in gene expression when the population density is high and the AI ​​concentration reaches a threshold. QS systems not only deepen our understanding of microbial community behavior but also provide a wealth of regulatory elements for synthetic biology.

[0003] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Bacillus amyloliquefaciens is a Gram-positive bacterium that efficiently secretes a variety of proteins and metabolites without producing endotoxins. It is a generally recognized as safe (GRAS) food-grade microbial system with a clear genetic background, simple operation, and strong secretory expression capabilities. It is frequently used as an excellent chassis cell in medicine, healthcare, and food industries, and is an ideal host for prokaryotic expression systems that secrete and express exogenous proteins. However, because Bacillus amyloliquefaciens is a derived bacterium, its metabolic byproducts can affect the expression of the target product to some extent, and currently, a self-regulating system is lacking.

[0004] With the continuous expansion of the application fields of high-end hyaluronic acid (HA) products, related studies have shown that the biological functions of HA are related to its molecular weight (M). WHyaluronic acid (HA) is closely related to its molecular weight. Compared to larger molecular weight HA, HA oligosaccharides (O-HA, ≤ 10 kDa) exhibit higher physiological activity, promoting bone and angiogenesis, and enhancing immune regulation, making them highly valuable for research in the fields of food, health, and medicine. Therefore, the efficient synthesis of hyaluronic acid with a specific molecular weight is of great significance for academic research and application exploration. However, currently, strains synthesizing hyaluronic acid still require IPTG induction for expression and secretion.

[0005] Bovine lactoferrin (LF) is a non-heme iron-binding glycoprotein with a molecular weight of approximately 78-80 kDa. This is because lactoferrin from different sources may exist in different molecular forms due to the influence of its glycosylation properties. Lactoferrin was first discovered in milk whey. As an iron-binding glycoprotein, its three-dimensional structure and amino acid sequence are now established. The most significant physicochemical characteristic of lactoferrin is its high affinity for iron, which can increase the bioavailability of iron in intestinal cells, stabilize iron ions, and reduce gastrointestinal irritation. In fact, it participates in many physiological functions, including regulating iron absorption and immune responses. It also exhibits antioxidant, antibacterial, antiviral, immunomodulatory, anticancer, and prebiotic activities. This makes it one of the most attractive drug candidates in the biomedical and biotechnology fields. However, current research shows that bovine lactoferrin yields low levels in Bacillus amyloliquefaciens.

[0006] Therefore, using genetic engineering techniques to explore different expression systems and delve into a strain with self-induced dual-switch regulation is an important research direction now and in the future. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a self-induced regulated Bacillus amyloliquefaciens and its application.

[0008] To address the aforementioned technical problems, this invention discloses a self-induced regulated Bacillus amyloliquefaciens and its applications. The specific technical solution is as follows: In a first aspect, the present invention provides a Bacillus amyloliquefaciens promoter P ispA Its nucleotide sequence is shown in SEQ ID NO.15.

[0009] In a second aspect, the present invention provides a self-induced regulatory system based on Bacillus amyloliquefaciens, comprising a quorum sensing element and the Bacillus amyloliquefaciens promoter described in the first aspect. The quorum sensing element comprises the Degu gene, the rapG gene, and the phrG gene, which are encoded by the amino acid sequences shown in SEQ ID NO. 9-11, respectively. The Bacillus amyloliquefaciens promoter and the quorum sensing element drive the expression of a first target protein according to cell density.

[0010] The self-inducible regulatory system further includes a closure element; the closure element includes the SspB gene and the SsrA tag, wherein the SspB gene is derived from *Panthera philoxeroides*. Pantoea alhagi The SspB gene is encoded by the amino acid sequence shown in SEQ ID NO.7; its expression is driven by the Bacillus amyloliquefaciens promoter described in the first aspect; the SsrA tag is encoded by the amino acid sequence shown in SEQ ID NO.2, and is linked to the C-terminus of the second target protein. The SspB gene and the SsrA tag jointly drive the degradation of the second target protein. Specifically, through the SspB-mediated target protein degradation system, the ssrA tag is specifically recognized, and the ClpXP protease complex is guided to complete the degradation of the substrate protein, thereby achieving dynamic shutdown of the gene expression circuit.

[0011] In some embodiments of the present invention, the *Bacillus amyloliquefaciens* is *Bacillus amyloliquefaciens*. CF It is a genetically modified strain of NX-2S (CCTCC NO: M 2016346), which was created by knocking out the γ-polyglutamate synthase gene cluster from the original strain. PgsBCA Extracellular polysaccharide synthesis gene cluster epsA-O lipopolysaccharide synthesis gene lps and fructanase gene sacB For details, please refer to Chinese patent CN116004496A.

[0012] In some embodiments of the present invention, the self-inducible regulatory system includes a quorum sensing element, the *Bacillus amyloliquefaciens* promoter and a closure element as described in the first aspect, and is capable of autonomously inducing a bimolecular switch in response to cell density to finely regulate the expression of metabolic pathway enzymes or proteins to achieve the targeted synthesis of the target product, without relying on exogenous inducers. The self-inducible regulatory system forms a gene regulatory network based on a quorum sensing threshold, triggering the temporal expression of the target gene when the cell density reaches the logarithmic growth phase critical value.

[0013] Thirdly, the present invention provides a self-induced regulated Bacillus amyloliquefaciens engineered strain, comprising the self-induced regulated system described in the second aspect. In some embodiments of the present invention, the originating strain of the Bacillus amyloliquefaciens engineered strain is Bacillus amyloliquefaciens. CF .

[0014] Fourthly, the present invention provides the application of the engineered strain of Bacillus amyloliquefaciens described in the third aspect in the induction-free fermentation production of functional polysaccharides and / or recombinant proteins.

[0015] The functional polysaccharide mentioned above includes hyaluronic acid, and the recombinant protein mentioned above includes lactoferrin.

[0016] The method for producing functional polysaccharides and / or recombinant proteins by fermentation without an inducer using engineered strains of Bacillus amyloliquefaciens includes the following steps: (1) The rapG gene, phrG gene and Degu gene are respectively integrated into Bacillus amyloliquefaciens, and the Bacillus amyloliquefaciens promoter described in the first aspect is linked to the first target protein gene and then introduced into Bacillus amyloliquefaciens to obtain an engineered strain of Bacillus amyloliquefaciens; the Bacillus amyloliquefaciens promoter and quorum sensing element described in the first aspect drive the expression of the first target protein according to cell density; (2) Inoculate the engineered strain of Bacillus amyloliquefaciens into the culture system and incubate at 30~37 ℃ for 24~96 h to obtain the culture.

[0017] In some embodiments of the present invention, in step (1), the rapG gene, phrG gene, Degu gene, and the second target protein gene with an SsrA tag linked to its C-terminus are respectively integrated into Bacillus amyloliquefaciens, and the Bacillus amyloliquefaciens promoter described in the first aspect is respectively linked to the first target protein and the SspB gene and then introduced into Bacillus amyloliquefaciens to obtain an engineered strain of Bacillus amyloliquefaciens; the Bacillus amyloliquefaciens promoter described in the first aspect drives the expression of the first target protein gene and the SspB gene, and the SspB gene and the SsrA tag jointly drive the degradation of the second target protein; the SsrA tag is encoded by the amino acid sequence shown in SEQ ID NO.2, and the SspB gene is encoded by the amino acid sequence shown in SEQ ID NO.7.

[0018] In some embodiments of the present invention, when the engineered strain of *Bacillus amyloliquefaciens* produces hyaluronic acid without an inducer through fermentation, in step (1), the first target protein gene includes the hyaluronic acid (HA) synthase gene SthHL (which was previously successfully expressed in conjunction with two novel hyaluronic acid synthases and published in the reference *De novo synthesis of hyaluronic acid with tailored molecular weights using a new hyaluronidase*, with HA yield of 10.79 ± 0.13 g / L in SthHL), and its sequence is shown in SEQ ID NO. 18; the SthHL is cloned into the expression vector pMA5 for expression; the *Bacillus amyloliquefaciens* is based on the previously established... B. amyloliquefaciens CRISPR-Cas9n editing system modifies byproduct synthesis pathways (lactate dehydrogenase gene) ldh acetolactate synthase gene als Phosphoacetyltransferase gene pta(any one or more combinations thereof) and / or precursor competitive bypass (UDP-GlcNAc dehydrogenase gene) wbpA Mannitol-1-phosphate-5-dehydrogenase gene mtld Glc-6-phosphate deaminase gene nagB Glc-1-phosphate cytidine transferase gene yfnH Glc-1-phosphothymidine transferase gene spsI Glc-4-uridine diphosphate epimerase gene galE (any one or more combinations thereof). In some embodiments of the present invention, the *Bacillus amyloliquefaciens* is a gene that knocks out the competing HA precursor pathway gene. wbpA , nagB and ldh The obtained Bacillus amyloliquefaciens CF3 Furthermore, using Bacillus amyloliquefaciens... CF3 Using this as the chassis, a protein degradation system (closing element) and a Degu-rapG-phrG QS system were introduced, and selected... pfkA and fruA A dual-switch system was constructed using the gene as the secondary target protein, combining a protein degradation system with a quorum sensing system to successfully build a self-induced regulatory system, achieving self-induced HA expression. The fermentation medium used by the *Bacillus amyloliquefaciens* in fermenting hyaluronic acid (HA) included: 40 g / L sucrose, 9.6 g / L yeast extract, 7 g / L K₂HPO₄·3H₂O, 3 g / L KH₂PO₄, 6.2 g / L MgSO₄, and pH 7.0.

[0019] Specifically, the activated recombinant Bacillus amyloliquefaciens seed culture is inoculated into a fermentation medium to produce hyaluronic acid. The inoculation amount is 1-10% v / v; the hyaluronic acid fermentation conditions are: 30-37 ℃, 180-220 rpm for 24-96 h.

[0020] In some embodiments of the present invention, when the engineered strain of *Bacillus amyloliquefaciens* produces bovine lactoferrin through fermentation without an inducer, in step (1), the first target protein gene includes the BLF gene, which is cloned into pHT01 and introduced into *Bacillus amyloliquefaciens* for expression. Specifically, recombinant *Bacillus amyloliquefaciens* is used. CF2 Using a chassis containing a recombinant plasmid of BLF, BLF expression was successfully induced autonomously without the addition of an inducer. The fermentation medium for Bacillus amyloliquefaciens fermenting bovine lactoferrin (BLF) was either LB or TB medium.

[0021] Specifically, the activated recombinant Bacillus amyloliquefaciens seed culture is inoculated into a fermentation medium to produce bovine lactoferrin. The inoculation amount is 1-10% v / v; the fermentation conditions for bovine lactoferrin are: 30-37℃, 180-220 rpm for 24-48 h.

[0022] Beneficial effects: To address the technical bottlenecks of low product synthesis efficiency and byproduct inhibition of cell growth in existing Bacillus strains, this application develops a self-induced dual-switch regulatory system that significantly reduces competitive synthesis of byproducts and enhances the target product synthesis pathway by dynamically balancing strain growth and product synthesis metabolic flux. This engineered strain constructs a bifunctional molecular switch based on a quorum sensing (QS) activation module and an SspB-ssrA protein degradation closure module: the activation module integrates QS system elements derived from Bacillus subtilis, including… rapG / phrG signaling molecules, degU Transcription regulators and P ispA A promoter-regulated red fluorescent protein reporter gene (RFP) activates the target gene sequentially by sensing a bacterial cell density threshold. A closure module introduces an SspB protein derived from *Umbrella cambogia*, which specifically recognizes the RFP-ssrA* fusion protein carrying an ssrA degradation tag (with a degradation efficiency of 86%). The target protein is then degraded via the ClpXP protease complex to shut down the expression pathway. Specific beneficial effects are as follows: This invention utilizes *Bacillus amyloliquefaciens* as the chassis cell and employs genetic engineering technology to construct a self-induced dual-switch regulated recombinant *Bacillus amyloliquefaciens* strain. This *Bacillus amyloliquefaciens* strain successfully and simultaneously heterologously expresses hyaluronic acid (HA) and bovine lactoferrin (BLF) of different molecular weights, providing a chassis strain capable of more efficient product expression and meeting food safety standards. Specifically, this study constructed a self-induced molecular regulatory switch system to autonomously induce the expression and secretion of hyaluronic acid of different molecular weights, synthesizing 13.5 ± 0.19 g / L of hyaluronic acid without the need for an inducing agent. Furthermore, this study provides an effective solution for the construction of industrial microbial cell factories by introducing a quorum sensing system, and achieves a breakthrough in BLF production based on shake-flask technology. Attached Figure Description

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0024] Figure 1 The time process of RFP degradation under the regulation of SspB from different sources.

[0025] Figure 2Plasmid construction and genome editing for SspB-ssrA*-regulated RFP synthesis.

[0026] Figure 3 The fluorescence-to-cell density ratios for different promoters predicted in the literature.

[0027] Figure 4 OD of promoter-regulated QS systems predicted by literature 600 The relationship between A and fluorescence intensity change trends, where A is promoter P aprE B is the promoter P flgB C is the promoter P srfA .

[0028] Figure 5 The relative fluorescence and OD of different promoters in CF5 or CF6 strains 600 Analysis of the graphs: A shows the difference in fluorescence expression levels, with CF5 on the left and CF6 on the right in each promoter group; B shows the relative fluorescence analysis; C shows the expression levels of promoter P. ispA Regulating the OD of the rapG-phrG-Degu system 600 A graph showing the relationship between the trend of RFP changes.

[0029] Figure 6 This study investigates the pathway and results of HA biosynthesis in Bacillus amyloliquefaciens. In this study, A represents the pathway research, and B represents the HA production and cell mass of strain CF4 after pMA5-StAB-SthHL2 was transformed into it.

[0030] Figure 7 The images show the plasmid map of bovine lactoferrin expressed in the QS system and the electrophoresis gel image for nucleic acid verification. In the image, A is the plasmid map of pHT-PispA-SPamyQ-BLF, and B is the nucleic acid verification image of the recombinant bacteria.

[0031] Figure 8 The graph shows the results of bovine lactoferrin expression using the QS system. In the graph, A is the SDS-PAGE plot, and B is the OD value at 24 h. 600 Bar chart of Wagyu lactoferrin production. Detailed Implementation

[0032] 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. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0033] In the following examples, the yield of hyaluronic acid (HA) was determined by high-performance liquid chromatography (HPLC). Specifically, the fermentation broth was diluted 5-fold, centrifuged, and the supernatant was collected after filtration through a 0.22 μm filter membrane and then collected in a chromatographic injection vial. Chromatographic analysis was performed using an Agilent HPLC system with a Shodex Ohpak SB-806M HQ gel permeation column. The mobile phase was 0.1 M NaSO4 solution (pH 4.0, adjusted with acetic acid), the column temperature was 35 °C, and the flow rate was 0.8 mL / min. Peak areas were recorded using a differential refractive index detector, combined with HA standards (molecular weight approximately 2 × 10⁻⁶). 6 HA yield was calculated using the concentration-peak area standard curve of Da. DCW was determined using the constant weight method: fermentation cells were collected by centrifugation, washed 2-3 times with distilled water, dried at 100 °C to constant weight, and weighed using an analytical balance. The purified HA product was structurally characterized by Hangzhou Yanqu Information Technology Co., Ltd., using Fourier transform infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance spectroscopy (NMR). 1 The chemical structure was verified by ¹H-NMR. All experiments were repeated three times, and the data were statistically analyzed using SPSS 19.0 software. Graphs were plotted using Origin 2021 software. In the following examples, the yield of bovine lactoferrin was detected using an ELISA kit purchased from AmyJet Scientific, following the manufacturer's instructions.

[0034] In the following embodiments, the Bacillus amyloliquefaciens... CF The method of using the CRISPE-Cas9n editing system has been disclosed in Chinese patent CN116004496A. The following examples demonstrate the introduction of the pHT01-Cas9n plasmid using electroporation. CF (The construction method of pHT01-Cas9n plasmid is the same as that of pNX-Cas9n in Chinese patent CN116004496A, except that the expression vector pNX01 is replaced with pHT01, and the restriction sites are BmhⅠ and SmaⅠ.) Gene-edited competent cells were prepared after colony PCR verification. CF (Cas9n), used for subsequent gene editing.

[0035] The specific information of the strains constructed in the following examples is shown in Table 1.

[0036] Table 1. Strain Information

[0037] Example 1: Validation of the SspB-ClpXP protein degradation system in fluorescent protein expression 1. Screening and construction of the protein degradation SspB-ssrA system The SspB-ssrA system, a widely distributed protein regulatory system in prokaryotes and some fungi, can achieve precise post-translational regulation by specifically recognizing ssrA-tagged target proteins through the SspB protein and mediating their binding to the ClpXP protease complex for degradation. The construction method of the SspB-ssrA system of this invention is as follows: First, using the shuttle plasmid pMA5 as a vector, the constitutive strong promoter P was employed. HpaII The expression of the fluorescent protein RFP (nucleotide sequence shown in SEQ ID NO.1) was driven, and an ssrA (amino acid sequence AANDENYALAA) tag was fused to its C-terminus. The RFP sequence was amplified using primers phpa-RFP-F / pMA5-RFP-R and then ligated to a restriction enzyme. Nde I and BamH The recombinant plasmid pMA5-RFP was obtained by linearizing the pMA5 vector and introduced into Bacillus amyloliquefaciens CF. No fluorescent protein expression was detected. Subsequently, a novel tag ssrA* (amino acid sequence shown in SEQ ID NO.2, nucleotide sequence shown in SEQ ID NO.3) was obtained using the Linker optimization method and fused to the C-terminus of RFP (RFP protein fused with the ssrA* tag is abbreviated as RFP*). The ssrA* tag was further fused to the C-terminus of the RFP gene using long primers ssrA*-pMA5-F / ssrA*-RFP-R, constructing the fluorescent reporter plasmid pMA5-RFP*. This plasmid was introduced into Bacillus amyloliquefaciens CF, successfully achieving RFP expression unaffected by the tag. Furthermore, no significant degradation of RFP was observed in the later stages of fermentation, indicating that Bacillus amyloliquefaciens... B. amyloliquefaciens The endogenous SspB homologs cannot specifically recognize the ssrA* marker.

[0038] Constructing xylose-inducible promoter P HpaII Expression vectors were used to initiate the degradation of RFP* by SspB proteins from four different sources, and cloned using primers SspB-F1 / R1, SspB-F2 / R2, SspB-F3 / R3, and SspB-F4 / R4, respectively. B. amyloliquefaciens, B.subtilis, E.coli, Pantoea alhagi SspB homolog from the source SspB 1-4 (their sequence order is shown in SEQ ID NO.4-7) are ligated to restriction enzymes. Nde I and BamH Linearized pMA5-RFP* vector was used to construct recombinant plasmid pMA5-RFP*-SspB1~4. After DNA sequencing verification, recombinant plasmid pMA5-RFP*-SspB1~4 was electroporated into competent Bacillus amyloliquefaciens. CFAfter shaking and recovery, the culture was plated onto LB agar plates containing 25 mg / L kanamycin sulfate (Km) and incubated overnight for 12 h. Single colonies with a red phenotype were selected and inoculated into 5 mL of liquid medium containing LB + Km. After shaking and incubation for 10 h, the culture was inoculated into 50 mL of liquid medium containing LB + Km at a 6% v / v inoculation rate. After shaking and incubation for 12 h, 10 g / L xylose was added for induction culture for 48 h. The results are as follows. Figure 1 All four groups of SspB proteins from different sources caused varying degrees of decrease in fluorescence intensity, with the group from [specific source not provided] showing the most significant decrease. P.alhagi The SspB4 protein exhibited the highest degradation efficiency, with a 37% decrease in fluorescence intensity that persisted until the end of fermentation. The SspB4 protein is encoded by the nucleotide sequence shown in SEQ ID NO. 8.

[0039] 2. Construction of strains with SspB integrated into the genome Based on the above screening results, SspB4 was integrated into CF The strain genome was analyzed, and the RFP protein with an ssrA* tag was expressed freely using the plasmid pMA5, such as... Figure 2 As shown, the specific operation is as follows: using plasmid pSTOP as a template, P is amplified using primers WCPxylA-F / R to obtain P. xylA , put SspB4 and P xylA P was obtained using overlap PCR with primers PxylA-F / SR-SspB4-R. xylA - SspB Using the Bacillus amyloliquefaciens CF genome as a template, fragments were amplified using primer pairs sgRNA-SspB4-F / SspB4-gRNA-R, L-SspB4-F1 / R1, and R-SspB4-F1 / R1, respectively. sgRNASspB Upstream homologous arm SL and downstream homologous arm SR Combine these three segments with P xylA -SspB Obtained via overlap PCR sgRNASspB-SL-P xylA -SspB-SR The fragment was ligated into the pDR244 vector, which was linearized with Sal I and Xho I, to construct the recombinant plasmid pDR-sgRNA-InSspB4. After sequencing verification, the plasmid was transformed into competent cells. CF In (Cas9n), strains were obtained CF1The recombinant plasmid pMA5-RFP* was then introduced, and positive clones were selected and cultured at 32 °C with shaking for 12 h. Then, 10 g / L xylose was added to induce sspB4 protein expression. The RFP* protein degradation efficiency reached a peak of 86%. The results indicate that the SspB4-ssrA* degradation system can not only effectively destroy pre-existing proteins but also inhibit the accumulation of newly synthesized proteins, providing important technical support for the subsequent decoupling of cell growth and target product synthesis through selective degradation of key growth proteins. The primers used in this example are shown in Table 2.

[0040] Table 2 Primers for protein degradation systems

[0041] Example 2: Functional validation of the quorum sensing system Degu-rapG-phrG in red fluorescent protein expression. The dynamic regulatory mechanism of the QS system has become a fundamental tool for fine-grained gene expression regulation, and its dependence on cell density changes avoids the need for exogenous inducers. Based on this, the rapG-phrG-Degu QS system was constructed to achieve dynamic regulation of target product expression, and the performance of the Degu-rapG-phrG QS system was evaluated. CF The system comprises Degu (a regulatory factor), rapG, and phrG (a signaling molecule), whose amino acid sequences are shown in SEQ ID NO. 9-11, and whose nucleotide sequences encoding the genes are shown in SEQ ID NO. 12-14. rapG inhibits the activity of phosphorylated Degu (Degu-P) by binding to Degu, while phrG competitively binds to the rapG protein, relieving its inhibition of Degu-P and thus partially restoring the transcriptional activation function of Degu-P on target genes.

[0042] First, to evaluate the regulation efficiency of the system, a promoter P reported in the literature was constructed. D4 Driven by RFP expression, constitutive strong promoter P HpaII Expression of Degu, xylose-inducible promoter P xylA Expressing rapG and phrG, recombinant plasmids pMA5-RFP*1, pMA5-Degu-RFP*1, pMA5-Degu-RFP*1-rapG, and pMA5-Degu-RFP*1-rapG-phrG were constructed and transformed into [various molecules]. CF Dynamic regulation tests were conducted. Experimental results showed that the presence of Degu significantly weakened the fluorescence signal intensity, indicating its influence on the promoter P. D4The activity exhibits an inhibitory effect; however, the introduction of rapG partially reverses this inhibition, resulting in a rebound in fluorescence signal intensity. Further introduction of phrG reactivates this inhibitory effect, leading to a significant decrease in RFP expression levels. These results indicate that the rapG-phrG-Degu QS system can dynamically regulate target genes in response to changes in cell density, but the promoter P remains unchanged in the presence of Degu. D4 Unable to effectively activate target gene expression. To screen promoters upregulated by Degu, six candidate promoters, P, were predicted based on literature. aprE P flgB P pgds P sacB P srfA P yvcA To replace the promoter driving RFP expression in pMA5-RFP*1, a series of recombinant plasmids pMA5-Degu-RFP*3~8 were constructed, using the promoter P... HpaII Functional validation was performed as a positive control (pMA5-Degu-RFP*2). Results are as follows: Figure 3 As shown, under the condition of Degu's presence, promoter P aprE P flgB P srfA The relative fluorescence intensity of the driven element was significantly higher than that of the P element. pgds P sacB P yvcA Furthermore, these three candidate promoters were used to replace the RFP* promoter P in the recombinant plasmid pMA5-Degu-RFP*-rapG-phrG. D4 The dynamic regulation characteristics are analyzed through fermentation. For example... Figure 4 As shown, the above promoters all exhibit a cell density-dependent expression pattern and cannot respond to dynamic changes in phrG.

[0043] To screen for highly efficient promoters activated by Degu, the method described in Example 1 was used. CF The CRISPR-Cas9n editing system is based on CF2 The strain was constructed Degu Deletion strain CF5 and Degu overexpression strains CF6 .in CF2 strain using promoter P cdd Expressions rapG and phrG, promoter P aprE Degu is expressed, with rapG and phrG integrated into the genome for expression. Degu is expressed via plasmid integration. CF2 The strain was constructed using the constitutive promoter P synthesized by the company. cddUsing the nucleotide sequence shown in SEQ ID NO.16 as a template, the promoter P was amplified using primer pairs L-Pcdd-F / Pcdd-R and rapG-Pcdd-F / phrG-Pcdd-R. cdd Fragment 1 and P cdd Fragment 2; Using the synthesized rapG and phrG as templates, respectively, rapG and phrG fragments were amplified using primer pairs rapG-F / R and phrG-F / R-phrG-R; the amplified fragments were then used to obtain P... cdd Fragment 1, P cdd Fragment 2, rapG and phrG fragments were amplified by overlap PCR using primer pair L-Pcdd-F / R-phrG-R to obtain P cdd -rapG-P cdd -phrG fragment. Using the CF strain genome as a template, sgRNA, upstream homologous arm L-RP, and downstream homologous arm R-RP fragments were amplified using primer pairs sgRNA-RP-F / HH-gRNA-R, L-RP-F / R, and R-RP-F / R, respectively. These three fragments were then combined with the P obtained from the amplification process. cdd -rapG-P cdd The -phrG fragment was amplified by overlap PCR using primer pair sgRNA-RP-F / R-RP-R to obtain sgRNA-L-RP-P. cdd -rapG-P cdd The -phrG-R-RP fragment was ligated into the pDR244 vector linearized with Sal I and Xho I to obtain a recombinant plasmid, which was then transformed into competent cells. CF Intermediate strains were obtained from (Cas9n); then, linearized pMA5 plasmids were amplified using primer pair WCBamHI-F / pMA5-Degu-R, and promoter P was obtained using primer pair L-PaprE-F / PaprE-R. aprE (The nucleotide sequence is shown in SEQ ID NO.17). The Degu fragment was obtained by amplification using the primer pair Degu-F / R, with promoter P. aprE P was obtained by overlapping PCR with the Degu fragment. aprE The Degu fragment was integrated into the linearized pMA5 plasmid obtained by reverse PCR using primers Degu-RF / WCpMA5-R, resulting in the recombinant plasmid pMA5-P. aprE -Degu, transformed into the above intermediate strain, yielded recombinant bacteria. CF2 .Will CF5 and CF6The bacterial strain was inoculated into LB liquid medium and cultured at 37 °C and 200 rpm until the stationary phase. The bacterial cells were then collected, flash-frozen in liquid nitrogen, and sent to Shanghai Paiseno Biotechnology Co., Ltd. for transcriptome sequencing. Based on differentially expressed genes, six promoters P with upregulated levels of thousands of times were identified. npr P ispA P apr P hp P cdhA P yvgO Recombinant plasmids pMA5-RFP*9~14 were constructed and transformed into CF5 and CF6 cells for expression verification. The results showed that P... npr and P cdhA Both strains exhibited high fluorescence intensity, but the fold difference was insufficient; P ispA and P apr The promoter exhibits Degu-dependent expression, where P ispA The fluorescence intensity of the promoter in the overexpression strain was relatively high. Degu The expression level of the missing strain increased more than 2-fold, and the red fluorescence expression level was as follows: Figure 5 As shown in A and B, promoter P, which exhibits the most significant differential expression, was selected. ispA Expressing RFP. Wherein, promoter P ispA The nucleotide sequence is shown in SEQ ID NO.15. The recombinant plasmid pMA5-RFP*10 was constructed as follows: P was amplified using primer pair P5-PispA-F / RFP-PispA-R to obtain P... ispA Promoter fragment, pMA5-RFP* plasmid via Nde I and BamH After double digestion at site I, the recombinant plasmid pMA5-RFP*10 was obtained through one-step ligation. The promoter P was then obtained through promoter screening. ispA The primers were upregulated by Degu and used in the subsequent QS system. The primers used in this embodiment are shown in Table 3.

[0044] The recombinant plasmid pMA5-RFP*10 was transformed into... CF5 and CF6 Growth curves and fluorescence intensity were dynamically monitored. In the early stages of fermentation (OD... 600 <4.0), the bacterial cell density continued to increase but the fluorescence signal did not change significantly; when OD 600 After reaching 4.0, the fluorescence intensity increased significantly with increasing cell density; after entering the stationary phase, the fluorescence intensity maintained a steady increasing trend, while Degu Similar phenomena were not observed in the missing strains, such as Figure 5 As shown in C. The above results indicate that the rapG-phrG-Degu QS system can serve as a gene expression regulatory switch in response to changes in bacterial cell density.

[0045] Table 3 Primers for quorum sensing systems

[0046] Example 3: Application of self-induced molecular regulatory switches in heterologous synthesis of low molecular weight hyaluronic acid 1. Eliminating byproduct and precursor synthesis bypasses to improve hyaluronic acid synthesis. Hyaluronic acid (HA) biosynthesis strictly depends on two precursors, GlcUA and GlcNAc. GlcNAc, as an essential substrate for peptidoglycan synthesis in bacterial cell walls, leads to significant substrate competition between bacterial growth and HA synthesis. Furthermore, metabolic byproducts such as lactic acid and acetic acid produced during HA synthesis consume approximately 80% of the carbon source, severely reducing substrate conversion efficiency. To direct more carbon flow to the HA synthesis module, based on… CF The CRISPR-Cas9n editing system was used to construct engineered bacteria with various gene deletions. Specifically, this embodiment uses the CRISPOR online tool (https: / / crispor.gi.ucsc.edu / ) to design targeted byproduct synthesis pathways (genes). ldh , als or pta ) and / or precursor competition bypass (gene) wbpA , mtld , nagB , yfnH , spsI or galE The 20 bp complementary sequence of the target sequence was integrated into the sgRNA scaffold backbone of the thermosensitive vector pDR244 using fusion PCR technology. Taking the construction method of the knockout plasmid pDR-sgRNA-ldh as an example, using the CF genome as a template, primer pairs L-ldh-F / R, R-ldh-F / R, and sgRNA-ldh-F / ldh-gRNA-R were used to PCR amplify the sgRNA expression cassette into the upstream 800 bp homologous arm, the downstream 800 bp homologous arm, and the sgRNA expression cassette, respectively. Using primers L-ldh-F and sgRNA-ldh-R, overlap extension PCR was used to fuse the sgRNA expression cassette with the upstream and downstream homologous arms, constructing an overlapping fragment containing specific sgRNA. The thermosensitive vector pDR244 was then used... Sal I and XhoThe vector was digested with restriction endonuclease I to obtain a linear vector, which was then ligated with an overlapping fragment using a homologous recombinase to construct the knockout plasmid pDR-sgRNA-ldh. The knockout plasmids pDR-sgRNA-als, pDR-sgRNA-pta, pDR-sgRNA-wbpA, pDR-sgRNA-mtld, pDR-sgRNA-nagB, pDR-sgRNA-yfnH, pDR-sgRNA-spsI, and pDR-sgRNA-galE were constructed using the same method.

[0047] The nine recombinant knockout plasmids were introduced into the target cells using electroporation. CF1 (Cas9n) competent cells were recovered by shaking at 37 ℃ for 3 h and then plated on LB agar plates containing 10 mg / L chloramphenicol (Cm) and 200 mg / L spectinomycin (Spec), and cultured overnight for 12 h. Single colonies were picked and inoculated into 200 mL of LB+Cm+Spec liquid medium. After confirming the presence of the pHT01 and pDR244 double plasmids using universal primers, the cells were transferred to 5 mL of LB+Cm+Spec liquid medium at a 2% v / v inoculation rate and cultured at 32 ℃ with shaking for 1.5 h. Then, 0.6 mM IPTG was added for induction culture for 24 h. Second-pass culture was serially diluted and plated on LB+Cm+Spec agar plates for culture. Several transformants were randomly selected for colony PCR verification using knockout primers. The genome of the successfully verified strains was extracted and sent to General Biotechnology (Anhui) Co., Ltd. for DNA sequencing. Subsequently, the Cas9n and sgRNA expression plasmids in the edited strains were eliminated using a 45℃ high-temperature subculture method: single colonies of the edited strains were picked and inoculated into 10 mL LB liquid medium, cultured at 45℃ for 12 h, and then continuously subcultured. The cultures were serially diluted and plated on LB agar plates, and antibiotic susceptibility testing was used to screen for strains without antibiotic markers. Several transformants were randomly selected and identified by PCR using universal primers for pHT01 and pDR, yielding single-gene knockout strains. Based on this, a combined editing strategy was used to construct multi-gene deletion engineered bacteria. CF3 ( CF1 △nagB△wbpA△ldh ).

[0048] Amplification was performed from plasmid pSTOP using primer pair PxylA-F / R. P xylA The gene was simultaneously amplified using primers SthHL-F2 / R2. SthHL Gene (nucleotide sequence as shown in SEQ ID NO.18). Use Kpn I and Hind III. Treatment of plasmid pMA5-sthasA-cghasB with restriction endonucleases (details can be found in Chinese Patent CN116004496A), followed by ligation.P xylA Genes and SthHL The fragment was used to construct the recombinant plasmid pMA5-StAB-SthHL1, which was then transformed into the strain. CF3 After culturing at 32 ℃ with shaking for 12 h, xylose at 5 g / L to 15 g / L (10 g / L in this example) was added to induce sspB4 protein expression. After further culturing at 32 ℃ for 12 h, the HA yield reached 12.03 ± 0.11 g / L, which was 14% higher than that of the CF+pMA5-StAB-SthHL1 strain. The fermentation medium of the strain in this example included: 40 g / L sucrose, 9.6 g / L yeast extract, 7 g / L K2HPO4·3H2O, 3 g / L KH2PO4, 6.2 g / L MgSO4, and pH 7.0.

[0049] Table 4 Primer sequence listing for the construction of HA high-efficiency synthetic chassis strains

[0050] Table 5. Continued list of primer sequences for constructing HA high-efficiency synthetic chassis strains.

[0051] 2. Decoupling of bacterial growth and hyaluronic acid synthesis by dual-function switch engineered bacteria During microbial HA synthesis, carbon sources compete for allocation across multiple metabolic pathways, including cell growth, glycolysis, and pentose phosphate. Among these, the carbon source derived from genes... pfkA The encoded phosphofructokinase 6-phosphate, as the first rate-limiting enzyme in the glycolysis pathway, is a key node regulating carbon flux allocation. Based on KEGG pathway analysis results, the phosphofructokinase 6-phosphate gene was selected. pfkA (SEQ ID NO.19) and PTS-fructose-specific enzyme gene fruA (SEQ ID NO.20) serves as the ssrA* tag fusion target, integrating it into the genome. pfkA and genes fruA All are generated by their own promoter pfkA q and fruA q Driven expression, specific regulatory mechanisms such as Figure 6 The plasmid construction method for A in the example is as follows: Construction of recombinant plasmid pDR-sgRNA-InpfkA*: Using the CF genome as a template, the pfkA target fragment gRNApfkA*, upstream homologous arm PL, and pfkA were amplified using primer pairs sgRNA-pfkA-F / HH-gRNA-R, L-pfkA-F / SsrA*-L-pfkA-R, pfkA-in-F / R, and SsrA*-R-pfkA-F / R-pfkA-R.q The sgRNA-pfkA-SsrA* fragment and its downstream homologous arm BR were obtained using overlapping PCR with primers sgRNA-pfkA-F and R-pfkA-R. This fragment was then cloned into the pDR244 linear vector digested with Sal I and Xho I to construct the recombinant plasmid pDR-sgRNA-InpfkA*, which was used to knock in pfkA*.

[0052] Construction of recombinant plasmid pDR-sgRNA-InfruA*: Using the CF genome as a template, primer pairs sgRNA-fruA-F / HH-gRNA-R, L-fruA-F / R, fruA-in-F / R, and R-fruA-F / R were used to amplify the fruA target fragment gRNAfruA*, upstream homologous arm FL, and fruA. q -fruA-SsrA* and downstream homologous arm FR were obtained by combining overlap PCR technology to obtain the sgRNAfruA*-FL-fruA*-FR fragment, which was then cloned into the pDR244 linear vector digested with Sal I and Xho I to construct the recombinant plasmid pDR-sgRNA-InfruA*, which was used to knock in fruA*.

[0053] The repressor protein coding region and xylose-inducible promoter in plasmid pMA5-StAB-SthHL1 were deleted using reverse PCR with primers SthHL-F / ispA-pMA5-R. P xylA Replace with QS promoter P ispA The recombinant plasmid pMA5-StAB-SthHL2 was obtained and used to construct P ispA Driven by SthHL.

[0054] Then, the QS promoter P was constructed. ispA The rapG-phrG-Degu regulatory module, driven by xylose, achieves self-inducible regulation of SspB and SthHL by replacing the original xylose-inducible regulatory elements. The rapG-phrG-Degu regulatory module is integrated into the genome, resulting in recombinant strain CF4. Both the rapG-phrG-Degu regulatory module and the SspB4 expression module in CF4 are integrated into the genome. The specific method is as follows: CF3 For the basal bacteria, the Degu target fragment gRNADegu, upstream homologous arm DL, and downstream homologous arm DR were amplified using primers sgRNA-Degu-F / HH-gRNA-R, L-Degu-F / R, and R-Degu-F / R, and the gene fragment P was constructed in Example 2. aprEUsing Degu as a template, P was amplified using the primer pair Degu-out / in-F / R. aprE The Degu fragment was used to obtain the sgRNADegu-DL-Degu-DR fragment by overlap PCR, and then cloned into the linear vector pDR244 digested with Sal I and Xho I to construct the recombinant plasmid pDR-sgRNA-P. aprE Degu, by typing P aprE -Degu, and then continuously knock P into the genome cdd -rapG-P cdd -phrG (see Example 2) and pfkA*, continue knocking in fruA*, and finally amplify the fragments using primer pairs sgRNA-SspB4-F2 / SspB4-gRNA-R (see Table 2), L-SspB4-F2 / R2, L-PispA-F / PispA-R, SspB4-F / R, and R-SspB4-F2 / R2 respectively. sgRNASspB Upstream homologous arm SL, PispA, SspB4, and downstream homologous arms SR These 5 fragments were obtained through overlap PCR. sgRNASspB-SL-P ispA -SspB4-SR The fragment was ligated into the pDR244 vector linearized with Sal I and Xho I to construct the recombinant plasmid pDR-sgRNA-PispA-InSspB4 knock-in P ispA -SspB4, to obtain recombinant bacteria CF4 The primers used in this embodiment are listed in Tables 4-6.

[0055] Table 6 Primer sequences used to construct the self-inducible regulatory system in HA synthetic strains

[0056] Next, the recombinant plasmid pMA5-StAB-SthHL2 was transformed into... CF4 Subsequently, positive clones of the obtained recombinant strain were selected and cultured with shaking at 32 °C. The fermentation results are as follows: Figure 6 As shown in Figure B, in the early stage of fermentation, the cell biomass increased exponentially, but HA synthesis was low. After entering the transition phase, the cell growth rate slowed down, HA synthesis began in large quantities and gradually reached a bottleneck. When the biomass reached a critical concentration of 7 g / L, the cascade activation of the QS system was triggered, initiating the co-expression of SspB4 and SthHL. At this point, the cell concentration tended to stabilize, and the HA synthesis rate increased significantly. Ultimately, the recombinant strain achieved a HA yield of 13.5 ± 0.19 g / L, with a relative molecular weight of 2.89 × 10⁻⁶. 4Furthermore, the dual-function switch effectively avoids the use of exogenous inducers, reduces the production cost of customized HA with different molecular weights, and provides a more economical and convenient control strategy for industrial-scale production.

[0057] Example 4: Application of the self-induction system in the recombinant secretory expression of lactoferrin The cDNA sequence of bovine lactoferrin (BLF) was retrieved from NCBI (NCBI database gene accession number NP_851341.1). Specific primers for the BLF gene were designed for the expression vector pHT01. Using BLF as a template, PCR amplification was performed using primers BLF-F / R to obtain bovine lactoferrin BLF with homologous arms. ispA Using a template, a promoter with homologous arms was amplified using primers PHT-PispA-F / R. The amylase signal peptide (nucleotide sequence shown in SEQ ID NO. 21) was then amplified using SPamyQ-F / R, followed by overlap PCR to obtain a single fragment P. ispA -BLF, using the expression vector pHT01 Bam HⅠ and Sma I. Purification after double enzyme digestion. Using a single-fragment homologous recombinase, the target gene (BLF gene fragment) containing the promoter PispA, amylase signal peptide, and purified gene was ligated in one step with the enzyme-digested linear vector pHT01 to obtain the recombinant plasmid pHT-PispA-SPamyQ-BLF, as shown in the diagram. Figure 7 A in the middle.

[0058] The recombinant plasmid was first transformed into E. coli GM2163: 100 μL of E. coli was taken. E. coli GM2163 competent cells were placed on ice for 5–10 min. After thawing, 50–200 ng of recombinant plasmid pHT-PispA-SPamyQ-BLF was added, and the cells were incubated on ice for 30 min, followed by heat shock at 42 °C for 1.5 min, and then on ice for another 5–10 min. 1 mL of LB medium was added, and the cells were incubated at 37 °C with shaking at 200 rpm for 1 h. The bacterial culture was centrifuged at 5000 rpm for 3 min, and a portion of the supernatant was discarded. The resuspended cells were spread onto LB agar plates containing 100 mg / L ampicillin resistance. The plates were inverted and incubated at 37 °C for 16–24 h. Single positive clones were picked and inoculated into 3–5 mL of LB medium containing 100 mg / L ampicillin antibiotic. The cells were incubated overnight at 37 °C with shaking at 200 rpm. The recombinant plasmid was extracted for positive clone identification. The recombinant plasmid was then transformed into [a specific culture medium] using electroporation. CF2In competent cells, after recovery with shaking at 37 ℃ for 3 h, the cells were plated onto LB agar plates containing 10 mg / L chloramphenicol (Cm) and incubated overnight for 12 h. Single colonies were picked and inoculated into 200 mL of LB+Cm liquid medium. Verification was performed using universal primers pHT01-F / R. Figure 7 As shown in B in the figure. The primers used are shown in Table 7.

[0059] Table 7 Primers for self-induced heterologous expression of BLF

[0060] Verified positive single colonies were inoculated into 200 mL of LB+Cm liquid medium and fermented for 24 h. SDS-PAGE was used to detect bovine lactoferrin synthesis. Figure 8 As shown in A, lane 1 contains the recombinant bacteria, which successfully expressed the BLF gene. The fermentation yield of the recombinant bacteria, as measured by an enzyme-linked immunosorbent assay (ELISA) kit, was 93.1 ± 0.8 mg / L. Figure 8 As shown in B in the figure. This self-inducible molecule-controlled switching system avoids the use of exogenous inducers and increases the yield by 16.37% compared to the shake flask yield in patent CN116987653A.

[0061] This invention provides a self-induced regulated recombinant Bacillus amyloliquefaciens and its application, along with related ideas and methods. 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 promoter for Bacillus amyloliquefaciens, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

15.

2. A self-inducing regulation system based on Bacillus amyloliquefaciens, characterized in that, The invention includes a quorum sensing element and the Bacillus amyloliquefaciens promoter as described in claim 1, wherein the quorum sensing element includes the Degu gene, the rapG gene, and the phrG gene; wherein the Bacillus amyloliquefaciens promoter and the quorum sensing element drive the expression of a first target protein according to cell density.

3. The self-inducing regulation system according to claim 2, characterized in that, The Degu gene, rapG gene, and phrG gene are encoded by the amino acid sequences shown in SEQ ID NO. 9-11, respectively.

4. The self-inducing regulation system according to claim 2, characterized in that, The self-induced regulatory system includes a closure element; the closure element includes the SspB gene and the SsrA tag.

5. The self-inducing regulation system according to claim 4, characterized in that, The SspB gene mentioned above is derived from Pantotheca acuminata. Pantoea alhagi The SspB gene is encoded by the amino acid sequence shown in SEQ ID NO.7; its expression is driven by the Bacillus amyloliquefaciens promoter as described in claim 1. The SsrA tag is encoded by the amino acid sequence shown in SEQ ID NO.

2. The SsrA tag is attached to the C-terminus of the second target protein. The SspB gene and the SsrA tag together drive the degradation of the second target protein.

6. A self-induced regulated engineered strain of Bacillus amyloliquefaciens, characterized in that, Includes the self-inducing regulation system as described in any one of claims 2 to 5.

7. The use of the engineered Bacillus amyloliquefaciens strain according to claim 6 in the induction-free fermentation production of functional polysaccharides and / or recombinant proteins.

8. The application according to claim 7, characterized in that, The functional polysaccharide includes hyaluronic acid, and the recombinant protein includes lactoferrin.

9. The application according to claim 7, characterized in that, A method for producing functional polysaccharides and / or recombinant proteins by fermentation without an inducer using engineered strains of Bacillus amyloliquefaciens includes the following steps: (1) The rapG gene, phrG gene and Degu gene are respectively integrated into Bacillus amyloliquefaciens, and the Bacillus amyloliquefaciens promoter described in claim 1 is linked to the first target protein gene and then introduced into Bacillus amyloliquefaciens to obtain an engineered strain of Bacillus amyloliquefaciens; the Bacillus amyloliquefaciens promoter and quorum sensing element described in claim 1 drive the expression of the first target protein according to cell density; (2) Inoculate the engineered strain of Bacillus amyloliquefaciens into the culture system and incubate at 30~37 ℃ for 24~96 h to obtain the culture.

10. The application according to claim 9, characterized in that, In step (1), the rapG gene, phrG gene, Degu gene and the second target protein gene with an SsrA tag linked to the C-terminus are respectively integrated into Bacillus amyloliquefaciens, and the Bacillus amyloliquefaciens promoter described in claim 1 is respectively linked to the first target protein and the SspB gene and then introduced into Bacillus amyloliquefaciens to obtain an engineered strain of Bacillus amyloliquefaciens. The Bacillus amyloliquefaciens promoter and quorum sensing element of claim 1 drive the expression of the first target protein gene and the SspB gene according to cell density, wherein the SspB gene and the SsrA tag jointly drive the degradation of the second target protein. The SsrA tag is encoded by the amino acid sequence shown in SEQ ID NO.2, and the SspB gene is encoded by the amino acid sequence shown in SEQ ID NO.7.

Images