Halomonas engineering system for production of difficult-to-express recombinant proteins and application thereof

Abstract

This invention relates to the field of recombinant protein biomanufacturing, and discloses a Halomonas strain based on a coupled low-temperature response switch. Halomonas A method for producing recombinant proteins, engineered bacteria, and expression vectors. The low-temperature response switch includes a cold-inactivated transcriptional regulatory protein and a heat-inactivated TEV protease (TEV: tobacco erosion virus protease). At a first temperature, it inhibits target gene expression and promotes bacterial growth; upon cooling to a second temperature, it relieves transcriptional inhibition and enhances the expression of the target recombinant protein. This system is particularly suitable for recombinant proteins whose continuous, premature, or high-level expression would adversely affect host cell growth, protein production capacity, or genetic stability, preferably *Bacillus thuringiensis* (Bt). Bacillus thuringiensis Insecticidal proteins. The system preferably triggers expression conversion through temperature changes, without the need for exogenous chemical inducers, and can be optimized by combining host engineering and fermentation engineering to achieve efficient production of bioactive target recombinant proteins.

Description

Technical Field

[0001] This invention relates to the fields of biomanufacturing, genetic engineering, and synthetic biology. More specifically, this invention relates to a method for producing recombinant proteins that are difficult to express, and the construction of recombinant engineered bacteria for this method. Background Technology

[0002] Recombinant proteins are widely used in medicine, food, agriculture, feed, biomaterials, and industrial catalysis. With the increasing demand for recombinant proteins, developing efficient, stable, and scalable microbial expression systems has become an important research direction in the field of biomanufacturing.

[0003] However, some recombinant proteins, when expressed continuously, prematurely, or at high levels, can consume large amounts of the host cell's transcriptional, translational, energy, and metabolic resources, and may cause protein folding stress, membrane stress, or metabolic imbalance, thereby inhibiting cell growth, reducing bacterial biomass, and decreasing the yield of the target protein. A strong expression burden may also lead to the loss of expression plasmids or mutations, deletions, or inactivation of expression modules, thus affecting the genetic stability of the producing strain (Borkowski O, Ceroni F, Stan GB, ...). et al. 2016. Overloaded and stressed: whole-cell considerations for bacterial synthetic biology. Current Opinion in Microbiology, 33: 123-130).

[0004] To reduce the impact of target protein expression on host growth, the culture process can be divided into a cell growth phase and a protein production phase. During the cell growth phase, target gene expression is inhibited or reduced, allowing the host to preferentially accumulate biomass; once the cells reach a suitable state, target gene expression is then initiated or enhanced (Jenzsch M, Gnoth S, Beck M, ...). et al.2006. Open-loop control of the biomass concentration within the growth phase of recombinant protein production processes. Journal of Biotechnology, 127:84-94. By decoupling cell growth and target protein production in time, it is beneficial to alleviate the host burden caused by premature expression of the target protein (De Baets J, De Paepe B, De Mey M. 2024. Delaying production with prokaryotic inducible expression systems. Microbial Cell Factories, 23: 249).

[0005] Existing induction expression systems typically use IPTG, arabinose, or other chemical inducers to initiate target gene expression. The use of chemical inducers may increase production costs and operational steps; their addition time, amount, and mixing uniformity may also affect the induction effect in large-scale fermentation (Restrepo-Pineda S, Pérez NO, Valdez-Cruz NA, et al. 2021. Thermoinducible expression system for producing recombinantproteins in Escherichia coli Advances and insights. FEMS Microbiology Reviews, 45: fuab023). In contrast, culture temperature is a process parameter that is easy to monitor and adjust during fermentation. Utilizing temperature changes to trigger expression conversion eliminates the need for additional chemical inducers, simplifying the induction process and demonstrating good potential for large-scale application (Valdez-Cruz NA, Caspeta L, Pérez NO, et al. 2010. Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and / or pR promoters. Microbial Cell Factories, 9: 18).

[0006] Therefore, it is necessary to develop a low-temperature-responsive dynamic expression system that allows the target gene to remain unexpressed or expressed at low levels at higher temperatures, and to initiate or enhance the expression of the target protein by lowering the culture temperature after sufficient biomass has accumulated in the bacterial cells. This would alleviate the host burden caused by continuous expression, premature expression, or high-level expression, and improve the production level of recombinant proteins and the genetic stability of the production strain. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a dynamic expression system suitable for Halomonas bacteria, which enables the recombinant protein that is difficult to express to be maintained at no expression or at a low level during the bacterial growth stage, and to initiate or enhance expression by lowering the culture temperature after the bacterial biomass has accumulated, thereby alleviating the growth inhibition and genetic instability caused by continuous expression, premature expression or high level expression of the target protein, and reducing the dependence on exogenous chemical inducers.

[0008] To address the aforementioned problems, this invention employs a cold-inactivated transcriptional regulatory protein, TF. ts and heat-inactivated TEV protease TEV ts This constitutes a coupled low-temperature response control module. TF ts Includes the ability to be TEV ts Identifying and cleaving protease recognition sites; TEV ts The coding sequences and the coding sequences of the target recombinant protein are respectively affected by TF ts The identified responsive promoter regulation.

[0009] Compared to the situation at the lower second temperature, at the higher first temperature, TF ts It maintains strong DNA binding and transcriptional repression activity, while TEV ts It exhibits low cleavage activity due to thermal deactivation. TF ts Therefore, TEV is inhibited ts The expression of the target recombinant protein induces preferential accumulation of biomass in the host. After being lowered to the second temperature, TF... ts Reduced transcriptional repression ability, TEV ts Improved cutting activity; newly generated TEV ts Further cutting and inactivating TF ts This further lifts restrictions on TEV ts The inhibition of target recombinant protein expression forms a coupled regulation that enhances low-temperature expression.

[0010] A first aspect of the present invention provides a method for producing a target recombinant protein (a difficult-to-express recombinant protein) using *Haloxylon ammodendron*, comprising: (1) Provide recombinant Halomonas bacteria containing a coupled low-temperature response regulation module and the coding sequence of the target recombinant protein; (2) The recombinant Halomonas bacteria were cultured at a first temperature to maintain the target recombinant protein at no or low level of expression and to allow the bacterial cells to accumulate biomass. (3) Lower the culture temperature to a second temperature to initiate or enhance the expression of the target recombinant protein without adding an exogenous chemical inducer to initiate target gene expression; and (4) Cultivate and obtain the target recombinant protein.

[0011] In one implementation, the method includes the following steps: (1) Using recombinant halometabolites as expression chassis cells, said recombinant halometabolites comprising: a) Transcription regulatory protein expression module, which includes the cold-inactivated transcription regulatory protein TF. ts The encoding gene of the TF ts It contains a TEV protease (tobacco erosion virus protease) recognition site; b) TEV protease expression module, which contains heat-inactivated TEV protease TEV. ts The encoding gene of the TEV ts The encoding gene and the TF that can be used ts Suppressed first-response promoter operable connection; and c) A target recombinant protein expression module, comprising a gene encoding the target recombinant protein, wherein the gene encoding the target recombinant protein is expressed by the TF. ts The suppressed second-response promoter is operable; (2) The recombinant Halomonas bacteria were cultured at a first temperature to allow the TF to... ts Maintaining transcriptional repression activity, while the TEV ts The cutting activity is relatively low, thus making the TEV ts The target recombinant protein is not expressed or is expressed at a low level, and the bacterial cells accumulate biomass. (3) Lower the culture temperature to a second temperature below the first temperature, so that the TF ts Reduced transcriptional repression activity and decreased TEV activity ts The cutting activity of the TEV is improved. ts Cutting the TF ts Furthermore, by releasing the inhibition of the first and second responsive promoters, the expression of the target recombinant protein is induced or enhanced; and (4) Recover bacterial cells, bacterial cell lysates and / or the target recombinant protein containing the target recombinant protein.

[0012] In one embodiment, the halomonas includes Halomonas bluephagenesis,Halomonas aydingkolgenesis , Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas sp. KM1、 Halomonas elongata and Halomonas smyrnensis Preferred Halomonas bluephagenesis TD1.0 Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (culture preservation number CGMCC No. 19880) and Halomonas campaniensis LS21 (CGMCC No. 6593) or its derivative strains.

[0013] In another embodiment, the first temperature is 30–42°C (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42°C), and the second temperature is lower than the first temperature; preferably, the first temperature is 35–39°C (e.g., 35, 36, 37, 38, 39°C), and the second temperature is 14–30°C (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30°C); more preferably, the target recombinant protein is expressed by lowering the temperature to 16°C after culturing at 37°C.

[0014] In another specific implementation, after incubating at the first temperature for 12 to 16 hours, the temperature is lowered to the second temperature, and incubation continues for 28 to 40 hours.

[0015] The "difficult-to-express recombinant protein" as described in this invention refers to a recombinant protein that, when continuously expressed, prematurely expressed, or expressed at high levels, can adversely affect the growth, survival, protein production capacity, or genetic stability of host cells. These effects may manifest as one or more of the following: decreased growth rate, prolonged lag phase, reduced final biomass, decreased cell viability, decreased plasmid retention rate, decreased expression module integrity, or reduced target protein production capacity during continuous culture.

[0016] In some embodiments, the difficult-to-express recombinant proteins include insecticidal proteins, antimicrobial proteins or antimicrobial peptides, cell lysis-related proteins, membrane proteins, proteases, nucleases, phospholipases, and other recombinant proteins that affect host growth, metabolism, cell division or genetic stability when expressed at high levels.

[0017] Preferably, the difficult-to-express recombinant protein is selected from suicidal proteins, antimicrobial proteins or antimicrobial peptides, cell lysis-related proteins, membrane proteins, proteases, nucleases, phospholipases, proteins that interfere with host metabolism or cell division, as well as industrial enzymes, food proteins, feed proteins, cytokines, therapeutic proteins or biomaterial proteins whose continuous expression, premature expression or high-level expression will reduce host growth, productivity or genetic stability.

[0018] Preferably, the recombinant protein that is difficult to express is an insecticidal protein; more preferably, it is an insecticidal protein of the Cry, Cyt or Vip family; more preferably, it is Cry1Ac or Vip3Aa16.

[0019] In some embodiments, the present invention triggers the expression conversion of the target recombinant protein simply by lowering the culture temperature, and in step (3), IPTG (isopropyl-β-D-1-thiogalactopyranoside), arabinose, formaldehyde, AHL (N-acylhomoserine lactone), oleic acid or other exogenous chemical inducers for initiating the expression of the target recombinant protein are not added.

[0020] In some embodiments, the low-temperature response regulation module and the target recombinant protein expression module are disposed on the genome of *Haloxylon ammodendron*, on a plasmid, or on an expression vector that can be stably maintained in *Haloxylon ammodendron*, preferably on pHbPBC or a vector derived therefrom; more preferably, the endogenous plasmid in the recombinant *Haloxylon ammodendron* that has the same or incompatible replication maintenance system as the expression vector is knocked out, removed, or inactivated.

[0021] A second aspect of the invention provides a recombinant expression vector for *Halomonas*, the vector comprising: (1) TF ts Expression unit; (2) Subject to TF ts Regulated TEV ts Expression unit (TEV) ts Encoded sequences under TF ts (Regulated expression under promoter); and (3) Subject to TF ts The target recombinant protein expression unit (i.e., the coding sequence of the target recombinant protein under TF regulation) is regulated. ts (Expression under the promoter of regulation).

[0022] In one implementation, the TF ts Derived from CI434 transcriptional repressor protein or a functional variant thereof, preferably with the amino acid sequence shown in SEQ ID NO: 1, and more preferably with the gene coding sequence shown in SEQ ID NO: 7. The TF tsIt includes a DNA-binding domain and a dimerization domain, with a cleavage site between the two domains that can be recognized by the TEV protease.

[0023] Preferably, the TEV protease recognition site includes the ENLYFQG sequence; more preferably, the recognition site is located between amino acids 69 and 70 of CI434.

[0024] In one implementation, the TEV ts TEV protease or a functional variant thereof derived from Tobacco etch virus (TEV), said TEV ts This is a temperature-sensitive TEV protease variant; its protease activity decreases or becomes inactive at higher temperatures, while it can recognize and cleave TF at lower temperatures. ts The TEV protease recognition site in the protein. Preferably, the TEV... ts The amino acid sequence is shown in SEQ ID NO: 2, and more preferably the gene coding sequence is shown in SEQ ID NO: 8.

[0025] In one implementation, the responsive promoter is one that can be TF-mediated. ts The promoter to be identified and inhibited is preferably P derived from the phage 434 regulatory system. R promoter, P L A promoter or a functional variant thereof, more preferably a responsive promoter is a P R The promoter is shown in SEQ ID NO: 9.

[0026] In one implementation scheme, by adjusting TF ts expression intensity, TEV ts The translation intensity of the target recombinant protein, the regulatory intensity of the responsive promoter, and the basic leakage expression, induction fold, and temperature response range of the cryogenic response system are regulated.

[0027] Preferably, by adjusting TEV ts The strength of the ribosome binding site enables the low-temperature response regulation module to exhibit both low high-temperature leakage expression and high low-temperature induced expression ability in Halomonas.

[0028] In one implementation, the transcriptional regulatory protein expression module, the TEV ts The expression module and the target recombinant protein expression module are located on the same expression vector, on different expression vectors, or on the genome of the recombinant halometa.

[0029] In one embodiment, the expression vector is a pSEVA plasmid (preferably pSEVA321 or pSEVA341, see Martinez-Garcia E, Aparicio T, Goni-Moreno A, et al. 2015. SEVA 2.0: an update of the Standard European Vector Architecture for de- / re-construction of bacterial functionalities. Nucleic Acids Research 43: D1183-1189), and / or the expression vector is a toxin-antitoxin system plasmid, more preferably the toxin-antitoxin system plasmid is pHbPBC or a derivative vector thereof (see Ren K, Zhao YQ, Chen GQ, et al. 2023. Construction of a StableExpression System Based on the Endogenous hbpB / hbpC Toxin-Antitoxin System of Halomonas bluephagenesis . ACS Synthetic Biology 13: 61-67).

[0030] A third aspect of the present invention provides a recombinant halometabolite containing the above-described low-temperature responsive expression module and used for producing recombinant proteins that are difficult to express. The recombinant halometabolite comprises: (1) The cold-inactivated transcriptional regulatory protein TF ts and heat-inactivated TEV protease TEV ts The coupled low-temperature response control module constitutes the structure; and (2) A target recombinant protein expression unit, wherein the target recombinant protein expression unit comprises a protein with TF. ts A responsive promoter can operatively link to the target recombinant protein coding sequence; The target recombinant protein is different from TF. ts and TEV ts Furthermore, its expression is regulated by the coupled low-temperature response regulation module.

[0031] In a preferred embodiment, the endogenous plasmid has been knocked out in the recombinant engineered bacteria.

[0032] In another preferred embodiment, the recombinant engineered bacteria lacks the PHA synthase gene. phaC Genes to reduce competition for carbon sources and intracellular space in PHA synthesis.

[0033] In another preferred embodiment, the recombinant engineered bacteria are morphologically engineered bacteria, such as those in Δ sspB In the context of (Stringent starvation protein B), the MreB (actin-like gene) is fused with an SsrA (small stable RNA) degradation tag (e.g., SsrA16 or SsrA21) to transform rod-shaped cells into larger spherical cells. See also the applicant's earlier Chinese patent application No. 202311750846.2 entitled "Recombinant Halomonas strains that induce protein self-degradation and their construction method and application".

[0034] In some embodiments, the recombinant halomonas can be cultured under high-salt and / or alkaline conditions and is suitable for open or non-strictly aseptic fermentation.

[0035] A fourth aspect of the present invention provides the application of the aforementioned low-temperature response regulation module, recombinant expression vector, or recombinant Halomonas bacteria in the production of recombinant proteins that are difficult to express. By inhibiting the expression of the target recombinant protein during the bacterial growth stage and initiating or enhancing the expression of the target recombinant protein by lowering the temperature after accumulating a certain biomass, the present invention can achieve temporal decoupling between bacterial growth and target protein production.

[0036] In a preferred embodiment, the recombinant engineered bacteria is a deformed recombinant Halomonas bacterium, the low-temperature response regulation module and the recombinant protein are located on the endogenous plasmid pHbPBC vector, and / or the recombinant protein is selected from the suicidal proteins Cry1Ac and Vip3Aa16, and the production of the difficult-to-express recombinant protein is achieved by lowering the temperature. Preferably, the amino acid sequence and gene coding sequence of Cry1Ac are shown in SEQ ID NO: 4 and SEQ ID NO: 3, respectively, and the amino acid sequence and gene coding sequence of Vip3Aa16 are shown in SEQ ID NO: 6 and SEQ ID NO: 5, respectively.

[0037] In this invention, the cold-inactivated transcriptional regulatory protein TF is used... ts and heat-inactivated TEV protease TEV ts The coupled low-temperature response regulation module was introduced into Halomonas bacteria and utilized TF... tsBy identifying and inhibiting responsive promoters that regulate the expression of target recombinant proteins, recombinant engineered bacteria capable of dynamically regulating target protein production by altering culture temperature were obtained. The inventors unexpectedly discovered that for recombinant proteins whose continuous, premature, or high-level expression would impose a significant host burden on *Halomonas*, the above method allows for rapid initial biomass accumulation by the bacteria under conditions where the target protein is not expressed or expressed at low levels. Then, by lowering the culture temperature, the production of the target protein is concentrated, thereby simultaneously increasing both bacterial biomass and the yield of the target recombinant protein. Taking Cry1Ac and Vip3Aa16 as examples, using the described low-temperature dynamic expression system, their shake-flask yields increased from approximately 0.16 g / L and 0.38 g / L to approximately 0.50 g / L and 0.85 g / L, respectively, and reached approximately 2.03 g / L and 7.13 g / L, respectively, in 7 L open fed-batch fermentation. These results demonstrate that the present invention can effectively mitigate the adverse effects of continuous high-level recombinant protein expression on bacterial growth and protein production, and provides a new technical solution for the open, low-cost production of recombinant proteins by *Halomonas*. Attached Figure Description

[0038] Figure 1 Schematic diagram of the principle of dynamically regulating recombinant protein expression using low temperature response.

[0039] Figure 2 The expression of recombinant proteins Cry1Ac and Vip3Aa16 in Halomonas was dynamically regulated by low temperature response.

[0040] Figure 3 :different porin Screening results of promoter and expression of Cry1Ac and Vip3Aa16 at different culture temperatures.

[0041] Figure 4 The effects of constitutive promoters of different strengths on the expression of Cry1Ac and Vip3Aa16 on the growth of Halomonas host and the production of target proteins.

[0042] Figure 5 Comparison of cell dry weight and Cry1Ac / Vip3Aa16 yield between constitutive expression strains and low-temperature responsive expression strains in shake-flask culture.

[0043] Figure 6 Cell dry weight, yield, and fermentation time curves of Cry1Ac and Vip3Aa16 in 7L open non-sterile fed fermentation.

[0044] Figure 7 The insecticidal activity and specificity of Cry1Ac and Vip3Aa16 against fall armyworm and Asian corn borer were obtained.

[0045] Sequence List Description (Example sequences and sequences used in examples) SEQ ID NO: 1 – TF ts amino acid sequence MSISSRVKSKRIQLGLNQAELAQKVGTTQQSIEQLENGKTKRPRFLPELASALGVSVDWLLNGTSDSNVRENLYFQGFVGHVEPKGKYQLISMVRAGSWCEACEPYDIK DIDEWYDSDVNLLGNGFWLKVEGDSMTSPVGQSIPEGHMVLVDTGREPVNGSLVVAKLTDANEATFKKLVIDGGQKYLKGLNPSWPMTPINGNCKIIGVVVEARVKFV* SEQ ID NO: 2 – TEV ts amino acid sequence MGESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGSGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQT KSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMKN* SEQ ID NO: 3 – Cry1Ac nucleotide coding sequence SEQ ID NO: 4 – Cry1Ac amino acid sequence SEQ ID NO: 5 – Vip3Aa16 nucleotide coding sequence SEQ ID NO: 6 – Vip3Aa16 amino acid sequence MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDEILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVLNDVNNKLDAINTMLRVYLPKLTSMLSDVMKQNYALSLQIEYLSKQLQEISDKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDGSPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALKTASELITKENVKTSGSEVGNVYNFLIVLTALQAKAFLTLTTCRKLLGLADIDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVEAKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLLCPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEIDLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENSRLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLEPWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYTVKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILKSQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLYQGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYMSGAKDVSEMFTTKFEKDNFYIELSQGNNLYGGPIVHFYDVSIK* SEQ ID NO: 7 – TF ts Nucleotide coding sequence ATGAGTATTTCTTCCAGGGTAAAAAGCAAAAGAATCCAGCTTGGACTTAACCAGGCTGAACTTGCTCAAAAGGTGGGGACTACCCAGCAGTCTATAGAGCAGCTCGAAAACGGTAAAACTAAGCGACCACGCTTTTTACCAGAACTTGCGTCAGCTCTTGGCGTAAGTGTTGACTGGCTGCTCAATGGCACCTCTGATTCGAATGTTAGAGAGAACCTGTACTTCCAGGGCTTTGTTGGGCACGTTGAGCCCAAAGGGAAATATCAATTGATTAGCATGGTTAGAGCTGGTTCGTGGTGTGAAGCTTGTGAACCCTACGATATCAAGGACATTGATGAATGGTATGACAGTGACGTTAACTTATTAGGCAATGGATTCTGGCTGAAGGTTGAAGGTGATTCCATGACCTCACCTGTAGGTCAAAGCATCCCTGAAGGTCATATGGTGTTAGTAGATACTGGACGGGAGCCAGTGAATGGAAGCCTTGTTGTAGCCAAACTGACTGACGCGAACGAAGCAACATTCAAGAAACTGGTCATAGATGGCGGTCAGAAGTACCTGAAAGGCCTGAATCCTTCATGGCCTATGACTCCTATCAACGGAAACTGCAAGATTATCGGTGTTGTCGTGGAAGCGAGGGTAAAATTCGTATAA SEQ ID NO: 8 – TEV ts Nucleotide coding sequence ATGGGAGAAAGCTTGTTTAAGGGGCCGCGTGATTACAACCCGATATCGAGCACCATTTGTCATTTGACGAATGAATCTGATGGGCACACAACATCGTTGTATGGTATTGGATCTGGTCCCTTCATCATTACAAACAAGCACTTGTTTAGAAGAAATAATGGAACACTGTTGGTCCAATCACTACATGGTGTATTCAAGGTCAAGAACACCACGACTTTGCAACAACACCTCATTGATGGGAGGGACATGATAATTATTCGCATGCCTAAGGATTTCCCACCATTTCCTCAAAAGCTGAAATTTAGAGAGCCACAAAGGGAAGAGCGCATATGTCTTGTGACAACCAACTTCCAAACTAAGAGCATGTCTAGCATGGTGTCAGACACTAGTTGCACATTCCCTTCATCTGATGGCATATTCTGGAAGCATTGGATTCAAACCAAGGATGGGCAGTGTGGCAGTCCATTAGTATCAACTAGAGATGGGTTCATTGTTGGTATACACTCAGCATCGAATTTCACCAACACAAACAATTATTTCACAAGCGTGCCGAAAAACTTCATGGAATTGTTGACAAATCAGGAGGCGCAGCAGTGGGTTAGTGGTTGGCGATTAAATGCTGACTCAGTATTGTGGGGGGGCCATAAAGTTTTCATGGTGAAACCTGAAGAGCCTTTTCAGCCAGTTAAGGAAGCGACTCAACTCATGAAGAATTAG SEQ ID NO: 9 – P R Promoter sequence GATATTTACAAGAAAGTTTGTTAGATTTAACGTATGTACAAGAAAGTTTGTTGCATCGCGAACCAAAACC SEQ ID NO: 10 – RBS sequence AGAAAGAGGAGAAA SEQ ID NO: 11 – TEV protease recognition site GAGAACCTGTACTTCCAGGGC SEQ ID NO: 12 – P porin203 Promoter sequence ATGCCTCCACACCGCTCGTCACATCCTGTTGCGTTCACTGGAATCCCAGTATCCCTTTTGACCTGCGAGCAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAGT SEQ ID NO: 13 – P porin221 Promoter sequence ATGCCTCCACACCGCTCGTCACATCCTGTTGCGTTCACTGGAATCCCAGTATGCTATTTGACCTGCGAGCAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAGT SEQ ID NO: 14 – P porin278 Promoter sequence ATGCCTCCACACCGCTCGTCACATCCTGTTGCGTTCACTGGAATCCCAGTATTAAATTTGACCTGCGAGCAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAGT SEQ ID NO: 15 – P porin68 Promoter sequence ATGCCTCCACACCGCTCGTCACATCCTGTTGCGTTCACTGGAATCCCAGACTAGAGTTTGACCTGCGAGCAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAGT SEQ ID NO: 16 – P porin58 Promoter sequence ATGCTCCACACCGCTCGTCACATCCTGTTGCGTTCACTGGAATCCCAATATAGAGTTTGACCTGCGAGCAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAGT SEQ ID NO: 17 – P porin183 promoter sequence ATGCTCCACACCGCTCGTCACATCCTGTTGCGTTCACTGGAATCCCAGTATAGCATTTGACCTGCGAGCAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAGT SEQ ID NO: 18 – P porin140 promoter sequence ATGCTCCACACCGCTCGTCACATCCTGTTGCGTTCACTGGAATCCCAGTATAAGATTTGACCTGCGAGCAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAGT SEQ ID NO: 19 – P porin226 promoter sequence TTGCGTTCACTGGAATCCCAGTATAAAGTTTGACCTGCGAGCA Detailed Implementation

[0046] Unless otherwise stated, the terms used herein have their general technical meanings as understood by those skilled in the art. For definitions, terms, and experimental procedures in this field, those skilled in the art are particularly recommended to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, New York (2012); and Ausubel et al., A Concise Guide to Molecular Biology Experiments (5th Edition), Science Press, 2008.

[0047] In this invention, the singular articles “a” and “the” cover a plurality of indicators unless the context clearly indicates otherwise. All references cited herein are incorporated herein by reference in their entirety.

[0048] In this invention, the terms "comprising" or "including" are open-ended expressions, referring to a specific component or step described, while not excluding other components or steps that do not substantially affect the meaning. When describing protein or nucleic acid sequences, the sequences may constitute the target molecule on their own, or may have additional amino acids or nucleotides added to one or both ends, or may be engineered to resemble proteins, while retaining the functional activity described in this invention.

[0049] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0050] In this invention, the term "halophiles" refers to a class of bacteria that live in high-salinity environments. In a preferred embodiment of the invention, the halophiles include, but are not limited to, *Halomonas* spp. Halomonas Preferably, the *Halomonas* genus includes, but is not limited to, those mentioned above. Halomonas bluephagenesis , Halomonas aydingkolgenesis , Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas sp. KM1 , Halomonas elongata and Halomonas smyrnensis Even better Halomonas bluephagenesis TD1.0 (See Zhao, H.) et al. (2017) Novel T7-1ikeexpression systems used for Halomonas Metabolic Engineering 39, 128-140, and disclosed in granted Chinese patent applications Nos. 2021108133976, 2022113288365 and 2021100113917; the public can obtain the bacterium from Tsinghua University. Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (CGMCC No. 19880) Halomonas campaniensisLS21 (CGMCC No. 6593) (All of the above strains have been deposited with the China General Microbiological Culture Collection Center (CGMCC) under the Budapest Treaty and have been disclosed in previous patent applications. For example, CGMCC No. 4353 has been disclosed in CN102120973A, CGMCC No. 19880 has been disclosed in CN111593006A, and CGMCC No. 6593 has been disclosed in CN102925382A).

[0051] In this invention, the term "cold-inactivated transcriptional regulatory protein" refers to a transcriptional regulatory protein whose DNA-binding and / or transcriptional regulatory activity is lower at relatively low temperatures than at relatively high temperatures. The term "heat-inactivated TEV protease" refers to a TEV protease whose cleavage activity is lower at relatively high temperatures than at relatively low temperatures.

[0052] In this invention, the core of the coupled cryogenic response switch is not simply cryogenic cultivation, but rather the use of TF... ts Mediated transcriptional repression and TEV ts Mediated TF ts The cleavage coupling allows the two temperature-responsive elements to jointly determine the expression state of the target protein. In one specific implementation, TEV... ts The expression module and the target recombinant protein expression module can be located in the same plasmid, or they can be distributed in two or more plasmids, or separately in the plasmid and the genome. Preferably, all three are located on the same stable expression vector to reduce the segregation of genetic elements and facilitate the maintenance of system integrity during open culture.

[0053] In this invention, the term "expression" can refer to "overexpression," which is defined as a gene expression level higher than the natural state, possibly achieved through increased transcription levels (producing more messenger mRNA) or improved translation efficiency (generating more functional proteins). In one specific embodiment, preferably, the expression or overexpression of a foreign gene can be achieved through insertion of the target gene into a non-translation site in the genome, or through plasmid overexpression. Preferably, gene insertion is performed using the CRISPR / Cas9 method (Qin Q, Ling C, Zhao Y, ...). et al. 2018. CRISPR / Cas9 editing genome of extremophile Halomonas(Spp. Metabolic Engineering 47: 219-229). Preferably, plasmid overexpression is introduced into the chassis strain via electroporation or conjugation transformation. When the host is *Halomonas*, the plasmid vector can be a pSEVA series vector (Martinez-Garcia E, Aparicio T, Goni-Moreno A, ...). et al. 2015. SEVA 2.0: an update of the Standard European Vector Architecture for de- / re-construction of bacterial functionalities. Nucleic Acids Research, 43: D1183-1189) or toxin-antitoxin endogenous plasmid pHbPBC vector (Ren K, Zhao YQ, Chen GQ, et al. 2023. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpC Toxin-Antitoxin System of Halomonas bluephagenesis . ACS Synthetic Biology, 13: 61-67), etc.

[0054] In this invention, the term "toxin-antitoxin system" (i.e., toxin-antitoxin, TA system) refers to a system that maintains plasmid stability through postsegregational killing (PSK). In a preferred embodiment, the recombinant halophilic bacteria of this invention comprises a suitable toxin-antitoxin system / plasmid, thereby enabling the plasmid to be naturally (i.e., without selective pressure such as antibiotics) stably maintained. Chinese Patent Application No. 2023112420281, entitled "A recombinant plasmid expressing toxins and antitoxins, a method for constructing the same and its application," discloses a TA system derived from halophilic microorganisms and a recombinant plasmid including the TA system, and discloses that the recombinant plasmid including the TA system can be naturally and stably maintained in halophilic microorganisms, etc.

[0055] In this invention, the term "expression module" refers to a functionally independent and structurally complete combination of DNA elements, typically including basic elements such as promoters, RBSs, genes, and terminators to express the expression of one or more target genes.

[0056] In this invention, the term "operable link" refers to a functional DNA element linking relationship, that is, linking a regulatory sequence (such as a promoter) to a target gene sequence, so that the regulatory sequence can effectively control the expression of the target gene.

[0057] Addressing the shortcomings of existing technologies, this invention utilizes a coupled low-temperature response dynamic regulation strategy. By adjusting the expression timing of the target recombinant protein, it maintains the recombinant protein, which is difficult to express, at a low level during the bacterial growth stage. After the bacterial cell accumulates a certain biomass, the expression is initiated or enhanced by lowering the culture temperature. This alleviates the host growth inhibition and decreased production capacity caused by continuous, premature, or high-level expression of the target protein, achieving dynamic decoupling between bacterial growth and target protein production. In one specific embodiment of this invention, *Haloxylon ammodendron* (*Haloxylon spp.*) is used. Halomonas bluephagenesis As a platform for the production of difficult-to-express recombinant proteins, it utilizes the cold-inactivated transcriptional regulatory protein TF. ts and heat-inactivated TEV protease TEV ts The coupled low-temperature response control module produced Cry1Ac and Vip3Aa16, respectively. In shake-flask culture, the yields of Cry1Ac and Vip3Aa16 were increased to approximately 0.50 g / L and 0.85 g / L, respectively. Furthermore, in an open, non-strictly sterile 7 L bioreactor, gram-level production was achieved, with the highest yields reaching approximately 2.03 g / L and 7.13 g / L, respectively.

[0058] A first aspect of the present invention provides a method for dynamically regulating the expression of recombinant proteins at low temperatures in *Halomonas*. The method is characterized by employing a cold-inactivated transcriptional regulatory protein TF. ts and heat-inactivated TEV protease TEV ts The coupled low-temperature response control module constitutes the TF ts The target gene is maintained at no or low expression levels at higher temperatures, and its expression is initiated or enhanced after the culture temperature is lowered. Preferably, TF ts Expression driven by a constitutive promoter, TEV ts From being able to be TF ts Identification and suppression of responsive promoter regulation; TF after lowering the culture temperature ts The transcriptional repression activity of TEV is reduced, while TEV ts Increased activity and further cleavage of inactivated TF ts This further relieves the inhibition of target gene expression.

[0059] Preferably, TF ts Depend on porin promoter (i.e., P) porin (Promoter) or its variants drive the expression. porinFor promoters or their variants, see (Shen R, Yin J, Ye JW, Xiang RJ, Ning ZY, Huang WZ, Chen GQ. 2018. Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis. ACS Synthetic Biology 7(8): 1897-1906. doi:10.1021 / acssynbio.8b00102. Epub 2018 Jul 31. PMID: 30024739) porin promoters or their variants, for example porin , porin226, porin58 mutant porin42 mutant porin68 mutant porin140 mutant porin278 mutant porin183 mutant porin221 mutant porin203, mutant porin3 One or more of the following.

[0060] A second aspect of the present invention provides a recombinant *Haloxylon ammodendron* strain with cryogenic dynamic gene expression regulation capabilities. The recombinant *Haloxylon ammodendron* strain comprises a cryogenically inactivated transcriptional regulatory protein TF. ts and heat-inactivated TEV protease TEV ts The coupled low-temperature response control module is constructed, and it can be controlled by TF ts A responsive promoter is identified and inhibited; the responsive promoter is used to regulate the expression of a foreign target gene, so that the foreign target gene can switch its expression state by changing the culture temperature.

[0061] Preferably, the TF ts Derived from phage 434 CI transcriptional repressor protein or a functional variant thereof; the responsive promoter is P. R promoter, P L Promoters or their functional variants.

[0062] Preferably, the higher temperature is 35-39°C, and the lowered temperature is 14-30°C; more preferably, the target gene is expressed first by culturing at 37°C and then lowered to 16°C.

[0063] The genus *Haloxymonas* includes Halomonas bluephagenesis or its derivatives Halomonas campaniensis or its derivatives or Halomonas aydingkolgenesisOr one or more of its derivatives.

[0064] A third aspect of the present invention provides a method for constructing the above-mentioned low-temperature dynamic regulation recombinant halomonas, wherein the method includes introducing a low-temperature dynamic regulation module into the genome or plasmid of a wild-type strain to obtain low-temperature dynamic regulation recombinant halomonas.

[0065] The importation can be electroconversion, bonding conversion, chemical conversion, etc.

[0066] In one specific embodiment of the present invention, the low-temperature dynamic regulation module and the recombinant protein expression module are jointly constructed onto the endogenous plasmid pHbPBC vector and introduced into Halomonas bacteria through conjugation transformation.

[0067] In a fourth aspect, the present invention provides a method for producing recombinant proteins that are difficult to express using Halomonas bacteria, characterized in that the expression of the target recombinant protein is dynamically regulated by a coupled low-temperature response regulation method, so that the target recombinant protein is not expressed or expressed at a low level during the bacterial growth stage, and the expression is initiated or enhanced by lowering the culture temperature after the bacterial cells have accumulated a certain biomass.

[0068] The method includes: culturing the recombinant Halomonas bacteria at a higher first temperature than the first temperature, so that TF ts Maintaining strong transcriptional repression activity and inhibiting the expression of target recombinant proteins to promote cell growth and biomass accumulation; subsequently, the culture temperature was lowered to a second temperature, allowing TF... ts The transcriptional repression effect is reduced, while TEV is reduced. ts Increased protease activity, TEV ts Further cutting and inactivating TF ts This allows the inhibition of the target recombinant protein expression to be lifted and the protein production stage to begin.

[0069] The first temperature is any value between 35 and 39°C, and the second temperature is any value between 14 and 30°C; more preferably, the recombinant halometazoa is first cultured at 37°C, and then the temperature is lowered to 16°C for the production of the target recombinant protein.

[0070] The culture time can be 5-48 h at a first temperature, preferably 8-20 h, or 5-48 h at a second temperature, preferably 10-40 h.

[0071] Preferably, the target recombinant protein is a difficult-to-express recombinant protein whose continuous expression, premature expression, or high-level expression will adversely affect the growth, survival, protein production capacity, or genetic stability of the host cell; in a specific embodiment of the present invention, the target recombinant protein is Cry1Ac or Vip3Aa16.

[0072] The Cry1Ac or Vip3Aa16 genes mentioned above are derived from Bacillus thuringiensis.

[0073] Preferably, the nucleotide coding sequence of the Cry1Ac insecticidal protein comprises SEQ ID NO: 3 or has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more homology with SEQ ID NO: 3. The amino acid sequence of the Cry1Ac insecticidal protein comprises the sequence shown in SEQ ID NO: 4, or has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more homology with SEQ ID NO: 4.

[0074] Preferably, the nucleotide coding sequence of the Vip3Aa16 insecticidal protein comprises SEQ ID NO: 5 or has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more homology with SEQ ID NO: 5. The amino acid sequence of the Vip3Aa16 insecticidal protein comprises the sequence shown in SEQ ID NO: 6, or has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more homology with SEQ ID NO: 6.

[0075] More preferably, the insecticidal protein includes Cry family proteins, Cyt family proteins, Vip family proteins, or truncated, modified, or mutant forms thereof; even more preferably, the Cry family protein is a Cry1 class insecticidal protein, and the Vip family protein is a Vip3 class insecticidal protein.

[0076] In one specific embodiment of the present invention, the method includes: 1) Construct an endogenous plasmid containing a low-temperature response module and a recombinant protein sequence; 2) The plasmid constructed in 1) was introduced into Escherichia coli S17-1; 3) Plasmids were introduced into Halomonas bacteria using conjugation transformation; 4) Use a urea concentration of 1-10 g / L (60LB or 60MM) medium, add 30-50 g / L of glucose as needed, and culture the recombinant bacteria at 37℃ for 8-36 h, then cool to 16℃ and culture for 12-36 h to harvest the recombinant protein.

[0077] In one specific embodiment of the method of the present invention, preferably for the deformed recombinant halomonas, a 60MM medium with 10 g / L urea and the optimal culture conditions are selected: culture at 37°C for 12 h, followed by cooling to 16°C and culture for 36 h, which yields the best production of recombinant protein Cry1Ac or Vip3Aa16. Shaking flasks can yield 0.5 g / L Cry1Ac or 0.85 g / L Vip3Aa16.

[0078] A fifth aspect of the invention provides a recombinant halomonas bacterium for producing recombinant proteins that are difficult to express. The recombinant halomonas bacterium contains the cold-inactivated transcriptional regulatory protein TF. ts and heat-inactivated TEV protease TEV ts The coupled low-temperature response control module is constructed, and it is independent of TF. ts and TEV ts The target recombinant protein expression unit is a coding sequence; the target recombinant protein coding sequence is a TFBoy that can be expressed by TF. ts The responsive promoter that is identified and inhibited is operablely linked, thereby allowing the expression of the target recombinant protein to be controlled by the coupled cryo-responsive regulatory module.

[0079] The genus *Haloxymonas* includes Halomonas bluephagenesis or its derivatives Halomonas campaniensis or its derivatives or Halomonas aydingkolgenesis Or one or more of its derivatives.

[0080] In a sixth aspect, the present invention provides the application of the above-described low-temperature dynamic regulation method, recombinant Halomonas bacteria, and their related expression systems in the production of recombinant proteins that are difficult to express at stable high levels. Preferably, the difficult-to-express recombinant protein is a protein whose continuous expression, premature expression, or high-level expression would adversely affect the growth, survival, protein production capacity, or genetic stability of the host cell; more preferably, it is an insecticidal protein, including Cry, Cyt, or Vip family proteins; more preferably, it is Cry1Ac or Vip3Aa16. The application can be used in shake-flask culture, fed-batch fermentation, or open fermentation production.

[0081] In a seventh aspect, the present invention provides a recombinant protein Cry1Ac produced by the above method.

[0082] In an eighth aspect, the present invention provides a recombinant protein Vip3Aa16 produced by the above method.

[0083] In a ninth aspect, the present invention provides a deformed recombinant bacterium that produces the above-mentioned high-yield, difficult-to-express recombinant protein.

[0084] The recombinant bacteria may not express or may express at least one recombinant protein.

[0085] Compared with the continuous or constitutive expression of the target recombinant protein, the present invention has at least the following advantages: (1) Reduce the expression level of the target recombinant protein during the bacterial growth stage to reduce the impact of the target protein on host growth and biomass accumulation; (2) By lowering the culture temperature to initiate or enhance the expression of the target recombinant protein, there is no need to add exogenous chemical inducers such as IPTG to initiate the expression of the target gene, which helps to simplify fermentation operations and reduce the cost of large-scale production. (3) By dynamically decoupling the bacterial growth stage and the protein production stage, the production capacity of recombinant proteins that are difficult to express is improved, and the genetic stability of the expression system is also improved. (4) In a specific embodiment, after adopting the low-temperature response dynamic expression system, the yield of Cry1Ac in shake flask culture was significantly increased from about 0.16 g / L to about 0.50 g / L, and the yield of Vip3Aa16 was significantly increased from about 0.38 g / L to about 0.85 g / L; in 7 L open fed fermentation, the yields of Cry1Ac and Vip3Aa16 reached about 2.03 g / L and about 7.13 g / L, respectively.

[0086] The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. All chemicals used in the following reactions are commercially available products unless otherwise specified.

[0087] The strains used in the embodiments of this application are: Used in the examples Halomonas bluephagenesis TD01 (CGMCC No. 4353) is a moderately halophilic bacterium screened from Ayding Lake in Xinjiang. Its optimal growth salt concentration is 5-6% (w / v) and its optimal pH is 9.0. It has been published in CN102120973A.

[0088] The Escherichia coli S17-1 used in the examples can be obtained from Tsinghua University or purchased commercially.

[0089] The culture medium formulation used in the examples is as follows: 10LB medium: containing 10 g / L NaCl, 10 g / L peptone (purchased from OXIOD, UK, catalog number LP0042), 5 g / L yeast extract (purchased from OXIOD, UK, catalog number LP0021), with the remainder being water, autoclaved.

[0090] 20LB medium: except that the NaCl concentration is 20 g / L, the other components are the same as those of 10LB.

[0091] 60LB medium: Except for the NaCl concentration of 60 g / L, the other components are the same as those of 10LB.

[0092] 60MM medium: 1 g / L yeast extract (OXID, catalog number LP0021), 60 g / L sodium chloride, 2 w / v% component I, 2 w / v% component II, 2 w / v% component III + 2 w / v% component IV, the remainder being water, pH adjusted with NaOH.

[0093] Component I contains: 10 g / L magnesium sulfate, the remainder being water.

[0094] Component II contains: 191.25 g / L disodium hydrogen phosphate, 75 g / L potassium dihydrogen phosphate, and the remainder is water.

[0095] Component III contains: 5 g / L ferric ammonium citrate, 2 g / L calcium chloride dihydrate, and the remainder is water.

[0096] Component IV contains: 0.03 g / L sodium molybdate dihydrate, 0.03 g / L manganese chloride tetrahydrate, 0.01 g / L copper sulfate pentahydrate, 0.02 g / L nickel chloride hexahydrate, 0.2 g / L cobalt chloride hexahydrate, 0.1 g / L zinc sulfate heptahydrate, 0.3 g / L boric acid, and the remainder is water.

[0097] In actual culture, a certain concentration of antibiotics can be added to the culture medium to maintain the stability of the plasmid. Commonly used antibiotics include chloramphenicol stock solution (25 mg / ml).

[0098] If a solid culture medium is required, add 1.5% agar to the liquid culture medium described above, sterilize, cool to 60°C, add an appropriate amount of antibiotic as needed, mix well, and pour into plates.

[0099] The detection method used in the examples: Cell dry weight (CDW) determination method: Take 35 mL of culture medium, centrifuge at 5000×g for 10 min to collect bacterial cells, and discard the supernatant. Wash the obtained bacterial cell pellet once with 15 mL of distilled water, and centrifuge again under the same conditions. Freeze-dry the washed bacterial cell pellet for 24 h and weigh it. Cell dry weight (CDW, g / L) is calculated as the ratio of dried bacterial cell mass to the corresponding culture medium volume.

[0100] Protein concentration detection method (BCA assay): Prepare BSA standard systems with final concentrations of 2000, 1500, 1000, 750, 500, 250, 125, 25, and 0 mg / L; Prepare BCA working solution: Add 1 volume of BCA reagent B to 50 volumes of BCA reagent A (A:B=50:1) and mix thoroughly. Add 25 μL of standard and sample to each well of the microplate. Add 200 μL of BCA working solution to each well and shake for 30 s to mix thoroughly. Cover the microplate and incubate at 37°C for 30 min. Cool to room temperature and measure absorbance in the wavelength range of 540-595 nm using a microplate reader, with 562 nm being the optimal wavelength. Plot a standard curve based on the absorbance of the BSA standard (subtract the OD value of the blank well in the standard to obtain the final reading). (X - protein concentration μg / mL; Y - final OD value) 562nm The protein concentration of the sample was calculated based on the standard curve and the sample dilution factor.

[0101] Recombinant protein content detection method (SDS-PAGE grayscale quantification method): Cells were collected from a certain volume of fermentation broth, lysed, and sonicated to obtain cell lysate. 40 μL of the lysate was mixed with 10 μL of 5×SDS loading buffer, heated at 100℃ for 10 min, and then electrophoresed on a 12% SDS-PAGE gel at 120 V for 70–80 min. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 and then destained. The grayscale value of the target protein band was measured using ImageJ software, and a standard curve was established between grayscale value and protein mass using different known loading amounts of BSA as standard proteins. The target protein mass was calculated based on the grayscale value of the target band in the sample, and the target protein yield was calculated in g / L, taking into account the sample volume and dilution factor.

[0102] Methods for detecting the toxicity of insecticidal proteins: The bioactivity of recombinant insecticidal proteins was evaluated using a surface treatment method for artificial feed. First, bacterial cell lysates containing the target insecticidal protein were prepared, and the actual concentration of the target protein in the lysates was determined before toxicity assays. The lysates containing the target insecticidal protein were then serially diluted with lysis buffer and applied to the surface of artificial feed to obtain different treatment doses of the target protein.

[0103] Newly hatched larvae of the insects to be tested were transferred to treated artificial feed, with one larva placed in each well, and cultured for 7 days under suitable insect culture conditions. After the culture was completed, the number of larval deaths and the developmental status of each treatment group were recorded.

[0104] Larval mortality was calculated as the proportion of dead larvae to the total number of treated larvae. Effective inhibition rate was calculated as the proportion of the sum of dead larvae and larvae still in the first instar to the total number of treated larvae, reflecting both the lethal effect of the insecticidal protein and its inhibitory effect on larval development. Dose-response curves were plotted based on mortality or effective inhibition rate at different treatment doses, and the median lethal concentration (LC50) was calculated using Probit analysis. 50 and / or half-maximal inhibitory concentration (EC50) 50 and its 95% confidence interval.

[0105] Example 1: Construction of a gene cryogenic response switch The control module used in this embodiment includes TF ts Expression unit, TEV ts Expression units and TF ts Downstream gene expression units regulated. See the sequence listing description section for specific sequences.

[0106] TF ts This is a temperature-sensitive variant of the phage 434 CI transcriptional repressor protein. The phage 434 CI transcriptional repressor protein includes an N-terminal DNA-binding domain and a C-terminal dimerization domain, with a cleavage site recognized by the TEV protease located in the linker region between the two domains. The TEV protease recognition site is ENLYFQG and is located between amino acids 69 and 70 of CI434.

[0107] TF ts Constituent porin Promoter-driven expression. TEV ts The encoded sequence is located in a region that can be TF-1 ts The downstream of the responsive promoter is identified and inhibited. The responsive promoter is a P-type promoter derived from the phage 434 regulatory system. R Promoters and their corresponding operator sequences. Downstream genes to be regulated are also located in TF-mediated regions. ts Suppressed responsive promoter P R Downstream.

[0108] Therefore, the coupled cryogenic response system includes at least the following three expression units: (1) Constitutive type porin226 Promoter-TF ts Encoded sequence - terminator; (2) Response promoter P R -TEV ts Encoded sequence - terminator; (3) Response promoter P R -Downstream target gene coding sequence-Terminator.

[0109] The three expression units can be located in the same recombinant vector, or they can be set in two or more vectors, or they can be fully or partially integrated into the host genome.

[0110] In the specific construct used in this embodiment, TF ts For TF ts -2, TEV ts For TEV ts -6, P porin226 Promoter, TF ts TEV ts P R The nucleotide sequences of the promoter, RBS, TEV protease recognition site, and other regulatory elements are shown in SEQ ID NOs: 7-11 and 19, respectively. See the schematic diagram for the construction. Figure 1 .

[0111] The specific implementation process is as follows: Based on the above structure, the functional elements were sequentially assembled into the endogenous plasmid expression vector pHbPBC, which can be stably maintained in *Halomonas*, using methods such as PCR amplification, DNA synthesis, and Gibson assembly and / or restriction endonuclease ligation (see Ren K, Zhao YQ, Chen GQ, ...). et al. 2023. Construction of a StableExpression System Based on the Endogenous hbpB / hbpC Toxin-Antitoxin System of Halomonas bluephagenesis In ACS Synthetic Biology 13: 61-67, the coupled low-temperature response expression vector pG01 was obtained.

[0112] After the constructed plasmid was verified to be correct by sequencing, it was first introduced into Escherichia coli S17-1, and then transformed into Halomonas through conjugation. Halomonas bluephagenesis .

[0113] Specifically, *E. coli* S17-1 carrying the target plasmid and *Halomonas*, the recipient bacterium, were cultured at 37°C and 200 rpm for approximately 12 h. After collecting the bacterial cells, the donor and recipient bacteria were mixed at approximately a 1:1 ratio and cultured on 20 LB solid medium without antibiotics at 37°C for 6–8 h for conjugation transfer. Subsequently, the bacterial cells were spread on 60 LB solid medium containing the corresponding screening antibiotic and cultured at 37°C for 36–48 h to obtain recombinant *Halomonas* containing the coupled low-temperature response expression vector. These conjugation transformation conditions are consistent with the method used in the experiments of this application.

[0114] After verification by colony PCR and / or sequencing, engineered strains were obtained for subsequent low-temperature response expression experiments.

[0115] A schematic diagram of the constructed low-temperature response expression system is shown below. Figure 1 As shown.

[0116] Example 2: Functional verification of the coupled low-temperature response regulation system in Halomonas. To verify whether the coupled cryo-response regulatory system constructed in Example 1 can achieve temperature-dependent regulation in *Halomonas*, Cry1Ac and Vip3Aa16 were used as downstream reporter target proteins. See the sequence listing section for specific sequences.

[0117] Place the Cry1Ac encoded sequence or the Vip3Aa16 encoded sequence in TF ts Regulated responsive promoter P R Downstream, and with TF ts Expression units and TEV ts The expression units together constitute the low-temperature response expression vector.

[0118] The specific implementation process is as follows: I. Construction and Reorganization Halomonas bluephagenesis ZY-Cry-01 / 21 and ZY-Vip-01 / 21 express Cry1Ac and Vip3Aa16 strains at low temperatures. Based on the above structure, the functional elements were sequentially assembled into the endogenous plasmid expression vector pHbPBC, which can be stably maintained in Halomonas, through PCR amplification, DNA synthesis, and Gibson Assembly and / or restriction endonuclease ligation, to obtain expression vectors pG02 and pG03 that couple the low-temperature response expression of recombinant proteins Cry1Ac and Vip3Aa16.

[0119] After the constructed plasmid was verified to be correct by sequencing, it was first introduced into Escherichia coli S17-1, and then transformed into wild-type Halomonas through conjugation. Halomonas bluephagenesis TD01 and deformed recombinant halomonas Halomonas bluephagenesis TD01- mreB -21 (See the applicant's earlier Chinese patent application CN120700080A entitled "A strain for efficient production of microbial single-cell proteins and recombinant proteins and a method for constructing the same"), which yielded recombinant strains respectively. Halomonas bluephagenesis ZY-Cry-01 / 21 (i.e.) Halomonas bluephagenesis ZY-Cry-01 and Halomonas bluephagenesis ZY-Cry-21 and Halomonas bluephagenesis ZY-Vip-01 / 21 (i.e.) Halomonas bluephagenesis ZY-Vip-01 andHalomonas bluephagenesis ZY-Vip-21).

[0120] II. Cultivation and Recombination Halomonas bluephagenesis ZY-Cry-01 / 21 and ZY-Vip-01 / 21 produce Cry1Ac and Vip3Aa16. 1. Shaking flask experiment 1) Inoculation with primary seed culture: Pick bacterial cells from the plate and inoculate them into 60LB medium, and incubate at 37℃ and 200 rpm for 12h; 2) Inoculation with secondary seed culture: Transfer the primary seed culture to fresh 60 LB medium at a ratio of 1% (v / v) and incubate at 37℃ and 200 rpm for 8-10 h. At this time, OD 600 =3~5; 3) Shake-flask fermentation: Transfer the secondary seed culture to 60LB or 60MM medium at a ratio of 5% (v / v). Add glucose 30-50 g / L and urea 0-10 g / L as needed. Culture at 200 rpm. The culture conditions are divided into three groups to verify the function of the low-temperature promoter in Halomonas: a) 37℃ for 48 h, b) 30℃ for 48 h, c) 37℃ for 12 h, then cooled to 16℃ for 36 h.

[0121] 2. Target protein identification and purification 1) Collect 10 mL of the bacterial culture obtained from the shake flask and centrifuge at 10,000 rpm for 10 minutes; 2) The obtained cell pellet was resuspended in 2 mL of lysis buffer and placed on ice for 30 minutes. Then, the cells were disrupted by sonication at 200 W power, with a sonication on time of 2 s and a sonication off time of 3 s, for a total working time of 4 minutes. 3) Centrifuge the obtained bacterial lysate at 4°C, 12000 rpm, for 25 minutes to obtain the supernatant and precipitate. 4) SDS-PAGE was used to detect the target protein in the supernatant and precipitate. The voltage was 80 V and the gel was run for 2 hours. The protein gel was then stained with Coomassie Brilliant Blue. After staining for 1 hour, the target band was observed by development. 5) Verify the target protein using mass spectrometry sequencing; 3. Preliminary results Figure 2 The results showed that the low-temperature response regulation system successfully induced the expression of the target protein in Halomonas. The highest yield of the target protein was achieved when cultured at 37°C for 12 h and then at 16°C for 36 h, and the deformed strain still maintained its yield advantage.

[0122] Example 3: Host burden caused by constitutive high-level expression of Cry1Ac and Vip3Aa16 in Halomonas To evaluate the effects of high-level expression of the target recombinant protein on the host, an endogenous plasmid pHbPBC was used as a vector, and a series of constitutive strains with different expression intensities were employed. porin The promoter controls the expression of Cry1Ac and Vip3Aa16. A series of constitutively expressed expression vectors for the recombinant proteins Cry1Ac and Vip3Aa16 were obtained. After the constructed plasmids were verified to be correct by sequencing, they were first introduced into *E. coli* S17-1, and then transformed into wild-type *Halomonas* TD01 via conjugation, resulting in a series of recombinant expression strains. Halomonas bluephagenesis Cry1Ac-203 / 221 / 278 / 68 / 58 / 183 / 140 and Halomonas bluephagenesis Vip3Aa16-203 / 221 / 278 / 68 / 58. Promoter strength ranking is: P porin203 <P porin221 < P porin278 < P porin68 < P porin58 < P porin183 < P porin140 The promoter sequence corresponds to SEQ ID NO: 12-18.

[0123] Constitutive expression of the target protein in Halomonas was verified using the same shake-flask conditions: a) 37℃ for 48 h, b) 30℃ for 48 h, c) 37℃ for 12 h, then cooled to 16℃ and incubated for 36 h.

[0124] The bacterial culture obtained from the shake flasks was collected using the same method. The expression level of the target protein was detected by SDS-PAGE, and the cell dry weight of the recombinant Halomonas was also detected.

[0125] Figure 3 and Figure 4 The results showed that for Cry1Ac, the accumulation of Cry1Ac protein increased within a certain range as the promoter strength increased; however, the dry weight of bacterial cells decreased as the expression level of Cry1Ac increased. Cry1Ac expression became undetectable after exceeding a certain promoter strength.

[0126] For Vip3Aa16, the expression level of Vip3Aa16 protein first increases with the increase of promoter strength; when using a stronger promoter to further increase the expression level, it is impossible to construct the correct expression vector, and some high expression pressure constructs have mutations such as stop codons that inactivate translation.

[0127] The above results indicate that sustained high-level expression of both Cry1Ac and Vip3Aa16 can create a host burden on Halomonas, but their specific manifestations differ: Cry1Ac mainly affects bacterial growth or biomass, while the strong sustained expression pressure of Vip3Aa16 can lead to selection for expression-inactivating mutants.

[0128] Example 4: Low-temperature dynamic expression improves shake-flask production levels of Cry1Ac and Vip3Aa16 To verify the benefits of cryogenic promoters for the production of burdensome recombinant proteins, the constitutive expression vector with the highest expression levels of Cry1Ac and Vip3Aa16 in Example 3, namely P... porin68 Cry1Ac expression vector driven and P porin58 The Vip3Aa16 expression vector was driven and conjugated to *Haloxylon ammodendron*. Halomonas bluephagenesis TD01- mreB -21, to obtain recombinant strain Halomonas bluephagenesis Cry-21 and Vip-21, along with the low-temperature responsive engineered bacteria in Example 2 Halomonas bluephagenesis ZY-Cry-21 and ZY-Vip-21 are produced simultaneously using shake-flask production.

[0129] The cells were cultured at 37℃ and 200 rpm for 12 h to allow for cell growth. The culture temperature was then lowered to 16℃ and cultured for another 36 h. After the culture was completed, the yields of CDW, Cry1Ac, and Vip3Aa16 proteins were measured.

[0130] Figure 5 The results showed that the constitutive expression strains Halomonas bluephagenesis The dry weight of Cry-21 cells is approximately 1–2 g / L, while those expressed dynamically at low temperatures... Halomonas bluephagenesis The dry weight of ZY-Cry-21 cells increased to approximately 5–6 g / L.

[0131] Under constitutive expression conditions, the Cry1Ac yield was approximately 0.16 g / L; after using a low-temperature dynamic expression system, the Cry1Ac yield increased to approximately 0.50 g / L, which is about 3.1 times that of the constitutive expression system.

[0132] Constitutive expression strains Halomonas bluephagenesis The cell dry weight of Vip-21 is approximately 4–5 g / L, while the cell dry weight of the cryogenically expressed strain is increased to approximately 7–8 g / L.

[0133] In the constitutive expression system, the yield of Vip3Aa16 was approximately 0.38 g / L; after adopting the low-temperature response dynamic expression system, the yield of Vip3Aa16 increased to approximately 0.85 g / L, which is about 2.2 times that of the constitutive expression system.

[0134] The above results indicate that by inhibiting the expression of Cry1Ac and Vip3Aa16 during the bacterial growth stage and initiating protein production after cooling, the limitation of continuous expression on host biomass accumulation can be effectively alleviated, and the final production level of Cry1Ac and Vip3Aa16 can be improved.

[0135] Example 5: Production of Cry1Ac and Vip3Aa16 in a 7 L fermenter using a low-temperature response control system Recombinant bacteria Halomonas bluephagenesis ZY-Cry-01 / 21 and ZY-Vip-01 / 21 were inoculated into 20 mL of 60 LB medium and cultured for 12-16 h. Then, they were transferred to new 60 LB medium at a volume ratio of 1% and cultured for another 8-12 h. 300 mL of seed solution was prepared as inoculum for a 7 L bioreactor (NBS Bioflo3000).

[0136] Prepare 2.7 L of substrate culture medium containing sodium chloride (107.8 g), sodium sulfate (15 g), glucose (60 g), urea (15 g), magnesium sulfate (2 g), potassium dihydrogen phosphate (14.6 g), 60 ml / L trace element solution I, and 6 ml / L trace element solution II. Trace element solution I consisted of 5 g / L ferric ammonium citrate and 2 g / L CaCl2, prepared with 1 M HCl. Trace element solution II consisted of 100 mg / L ZnSO4•7H2O, 30 mg / L MnCl2•4H2O, 300 mg / L H3BO3, 200 mg / L CoCl2•6H2O, 10 mg / L CuSO4•5H2O, 20 mg / L NiCl2•6H2O, and 30 mg / L NaMoO4•2H2O, prepared with 1 M HCl. The final pH of the culture medium was adjusted to 8-9 using 5 M NaOH solution. Fermentation was carried out using an open, non-strictly aseptic culture method. Oxygen levels were regulated during fermentation through agitation and aeration. The glucose content in the fermentation medium was monitored hourly by measuring residual glucose with a glucometer. Feeding was initiated when the base sugar content reached approximately 10 g / L, continuing until the OD (Oxygen Demand) reached 90%. 600 To stop growth, the principle of feeding is to control residual sugar at around 10 g / L.

[0137] For Cry1Ac production, the engineered bacteria were first cultured at 37°C for 16 h to accumulate biomass, and then the culture temperature was lowered to 16°C to initiate Cry1Ac expression. The total fermentation time was 44 h.

[0138] Figure 6 The results showed that low-temperature responsive, morphologically engineered bacteria Halomonas bluephagenesisZY-Cry-21 achieved a CDW of approximately 17 g / L at the fermentation endpoint, with a Cry1Ac yield of approximately 2.03 g / L.

[0139] For Vip3Aa16 production, the engineered bacteria were first cultured at 37°C for 14 h to accumulate biomass, and then the culture temperature was lowered to 16°C to initiate Vip3Aa16 expression. The total fermentation time was 44 h.

[0140] Figure 6 The results showed that low-temperature responsive strains using morphologically engineered hosts... Halomonas bluephagenesis ZY-Vip-21 ultimately achieved a Vip3Aa16 yield of approximately 7.13 g / L.

[0141] The above results indicate that the low-temperature response dynamic expression system can be applied to open fed-batch fermentation of Halomonas bacteria and can be used in conjunction with host chassis modification such as morphological engineering to achieve gram-level production of Cry1Ac and Vip3Aa16 per liter.

[0142] Example 6: Validation of the bioactivity of recombinant Cry1Ac and Vip3Aa16 To verify whether Cry1Ac and Vip3Aa16 produced by the low-temperature dynamic expression system of this invention retain insecticidal biological activity, insect toxicity was determined using lysates of Halomonas containing Cry1Ac and Vip3Aa16, respectively.

[0143] 1. Preparation of the sample to be tested Recombinant halomonas bacteria expressing Cry1Ac and Vip3Aa16 under low temperature dynamics were collected, and the bacterial cells were collected by centrifugation and cell disruption to obtain bacterial lysates containing Cry1Ac or Vip3Aa16.

[0144] The actual protein concentrations of Cry1Ac and Vip3Aa16 in the lysate were determined by SDS-PAGE grayscale quantification. The lysate was then diluted accordingly based on the measured target protein concentrations to achieve the predetermined insecticidal protein application doses for different treatment groups.

[0145] Lysis buffer was used as a negative control. In the high-dose specificity experiment, a wild-type sample was also included. Halomonas bluephagenesis The bacterial lysate was used as a host-derived component control to eliminate the influence of Halomonas cellular components on the tested insects. The above control setup is consistent with the method used in this application.

[0146] 2. Verification of high-dose insecticidal activity and specificity The insecticidal activity of Cry1Ac and Vip3Aa16 was evaluated using an artificial feed surface treatment method.

[0147] Bacterial lysates containing either Cry1Ac or Vip3Aa16 were uniformly applied to the surface of artificial feed (Multiple Species Diet, Southland Products, USA) in 24-well plates, respectively, to achieve a final application dose of 2000 ng / cm² for the target insecticidal protein. 2 .

[0148] Fall armyworms (Mortises fasciatus) were continuously reared in the laboratory. Spodoptera frugiperda ) and Asian corn borer ( Ostrinia furnacalis Newly hatched larvae were used as test insects. One newly hatched larva was placed in each well and cultured under suitable conditions for 7 days. Larval mortality and developmental status were recorded.

[0149] Figure 7 The results showed that at 2000 ng / cm 2 Under high-dose conditions, Vip3Aa16 showed significant insecticidal activity against fall armyworm, causing complete death of the tested fall armyworm larvae; in contrast, Vip3Aa16 had lower insecticidal activity against Asian corn borer.

[0150] Cry1Ac exhibited a different insect activity spectrum. At 2000 ng / cm³... 2 Under certain conditions, Cry1Ac exhibits strong insecticidal activity against the Asian corn borer, causing near-total mortality, while its insecticidal effect against the fall armyworm is relatively weak.

[0151] The above results indicate that Cry1Ac and Vip3Aa16 produced by this invention maintain their respective insecticidal biological activities and exhibit target insect activity spectra corresponding to each insecticidal protein.

[0152] 3. Dose-response relationship determination To further quantitatively evaluate the insecticidal activity of the produced Cry1Ac and Vip3Aa16, the bacterial cell lysate containing the target insecticidal protein was serially diluted with lysis buffer to obtain a series of test samples with different target protein concentrations: 400, 200, 100, 50, 25, 12.5, 6.25, and 3.125 ng / cm³. 2 And set 0 ng / cm 2 Negative control. Samples of each gradient were uniformly applied to the surface of the artificial feed in a 24-well plate to achieve the corresponding dosage per unit area for Cry1Ac or Vip3Aa16.

[0153] Cry1Ac was mainly evaluated for dose-response using newly hatched larvae of the Asian corn borer; Vip3Aa16 was mainly evaluated for dose-response using newly hatched larvae of the fall armyworm.

[0154] One newly hatched larva was placed in each well. After 7 days of cultivation, the number of dead larvae and the developmental stage of each larva were recorded.

[0155] The mortality rate is calculated using the following formula: Mortality rate (%) = Number of dead larvae / Total number of tested larvae × 100%.

[0156] For larvae that showed significant growth inhibition but had not yet died, the effective inhibition rate was also calculated. The effective inhibition rate was calculated as the ratio of the sum of the number of dead larvae and the number of larvae still in the first instar stage to the total number of tested larvae, thereby comprehensively evaluating the lethal effect and severe developmental inhibition effect of the insecticidal protein.

[0157] Based on the dose-response relationship between different target protein doses and larval mortality, Probit analysis was used to calculate the median lethal dose (LC50). 50 And its 95% confidence interval; based on the effective inhibition rate, EC can be further calculated. 50 .

[0158] 4. Results of dose-response experiments Cry1Ac exhibited a significant dose-dependent insecticidal effect against the Asian corn borer. Probit analysis was performed based on larval mortality rates at different dosages to determine the LC50 of Cry1Ac against the Asian corn borer. 50 Approximately 48 ng / cm 2 .

[0159] Vip3Aa16 also showed a significant dose-dependent insecticidal effect against fall armyworm, with its LC50 against fall armyworm being [missing information]. 50 Approximately 59 ng / cm 2 (Table 1)

[0160]

[0161] Note: LC 50 This indicates the insecticidal protein dose that causes 50% mortality in the tested larvae; the 95% confidence interval represents the LC. 50 or EC 50 95% confidence interval; EC 50 This indicates the dosage of insecticidal protein that induces 50% effective inhibition, where effective inhibition is calculated based on a combination of larval mortality and developmental arrest.

[0162] The high-dose specificity experiment and dose-response experiment described above together demonstrate that Cry1Ac and Vip3Aa16 produced by the low-temperature responsive recombinant Halomonas strain of this invention not only achieve high levels of protein accumulation, but also maintain the corresponding biological activity of the obtained proteins. Among them, Cry1Ac mainly exhibits strong insecticidal activity against the Asian corn borer, while Vip3Aa16 mainly exhibits strong insecticidal activity against the fall armyworm.

[0163] Those skilled in the art should understand that although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught in this invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the invention, and the equivalent embodiments obtained therefrom are all within the scope of this invention.

Claims

1. A method utilizing Halomonas ( Halomonas A method for producing the target recombinant protein, comprising: (1) Constructing a recombinant halometabolite, wherein the recombinant halometabolite comprises: a) Transcription regulatory protein expression module, which includes the cold-inactivated transcription regulatory protein TF. ts The encoding gene of the TF ts It contains a TEV protease (tobacco erosion virus protease) recognition site; b) TEV protease expression module, which contains heat-inactivated TEV protease TEV. ts The encoding gene of the TEV ts The encoding gene and the TF that can be used ts Suppressed first-response promoters are operable; as well as c) A target recombinant protein expression module, comprising a gene encoding the target recombinant protein, wherein the gene encoding the target recombinant protein is expressed by the TF. ts The suppressed second-response promoter is operable; (2) The recombinant Halomonas bacteria were cultured at a first temperature to allow the TF to... ts Maintaining transcriptional repression activity, while the TEV ts The cutting activity is relatively low, thus making the TEV ts The target recombinant protein is not expressed or is expressed at a low level, and the bacterial cells accumulate biomass. (3) Lower the culture temperature to a second temperature lower than the first temperature, so that the TF ts Reduced transcriptional repression activity and decreased TEV activity ts The cutting activity of the TEV is improved. ts Cut the TF ts Furthermore, by releasing the inhibition of the first and second responsive promoters, the expression of the target recombinant protein is induced or enhanced; and (4) Recover bacterial cells, bacterial cell lysates and / or the target recombinant protein containing the target recombinant protein.

2. The method according to claim 1, wherein, The TF ts Derived from CI434 transcriptional repressor protein (phage 434CI transcriptional repressor protein) or its functional variants, and comprising a DNA-binding domain, a dimerization domain, and a linker region between the two; the TEV protease recognition site is disposed in the linker region, preferably, the TEV protease recognition site includes ENLYFQG; and / or the first responsive promoter and the second responsive promoter are respectively selected from P containing the CI434 operator sequence. R promoter, P L Promoters or their functional variants.

3. The method according to claim 1 or 2, wherein, The TF ts By not being subject to the TF ts Repressive promoter-driven expression, preferably constitutive promoter-driven expression; the first responsive promoter and the second responsive promoter are the same or different; and / or the transcriptional regulatory protein expression module, the TEV ts The expression module and the target recombinant protein expression module are located on the same expression vector, on different expression vectors, or on the genome of the recombinant halometa.

4. The method according to any one of claims 1 to 3, wherein: 1) By regulating the expression of the TF ts promoter strength, the TEV ts The strength of the ribosome binding site, the strength of the ribosome binding site of the target recombinant protein, and / or the operation sequence of the first or second responsive promoter, to reduce leakage expression at the first temperature and increase induced expression at the second temperature; and / or 2) The target recombinant protein is a difficult-to-express recombinant protein. The difficult-to-express recombinant protein refers to a target recombinant protein that, relative to a control that does not express or expresses the target recombinant protein at a low level, causes at least one of the following detectable changes in the host due to its continuous expression, premature expression, or high-level expression: decreased growth rate, prolonged lag phase, decreased final biomass, decreased cell viability, decreased plasmid retention rate, increased frequency of expression module mutation or inactivation, or decreased yield of the target recombinant protein during continuous culture.

5. The method according to claim 4, wherein: 1) The difficult-to-express recombinant protein is selected from suicide-inducing proteins, antimicrobial proteins or antimicrobial peptides, cell lysis-related proteins, membrane proteins, proteases, nucleases, phospholipases, proteins that interfere with host metabolism or cell division, and industrial enzymes, food proteins, feed proteins, cytokines, therapeutic proteins, or biomaterial proteins whose continuous expression, premature expression, or high-level expression reduces host growth, productivity, or genetic stability; or 2) The recombinant protein that is difficult to express is an insecticidal protein of Bacillus thuringiensis, selected from Cry family proteins, Cyt family proteins, Vip family proteins and their truncated, fused or variants that retain the main insecticidal activity; preferably Cry1Ac or Vip3Aa16.

6. The method according to any one of claims 1 to 5, wherein: 1) The first temperature is 30-42°C, the second temperature is 10-30°C, and the second temperature is lower than the first temperature; preferably, the first temperature is 35-39°C, and the second temperature is 14-30°C; more preferably, the first temperature is 37°C, and the second temperature is 16°C. 2) The expression conversion of the target recombinant protein is triggered solely by lowering the culture temperature, and IPTG (isopropyl-β-D-1-thiogalactopyranoside), arabinose, formaldehyde, AHL (N-acylhomoserine lactone), oleic acid, or other exogenous chemical inducers for initiating the expression of the target recombinant protein are not added in step (3); and / or 3) The low-temperature response regulation module and the target recombinant protein expression module are set on the genome of Halomonas, on a plasmid, or on an expression vector that can be stably maintained in Halomonas, preferably on the endogenous plasmid pHbPBC of Halomonas or its derivative vector; more preferably, the endogenous plasmid in the recombinant Halomonas that has the same or incompatible replication maintenance system as the expression vector is knocked out, removed or inactivated.

7. The method according to any one of claims 1 to 6, wherein: 1) The Halomonas species include Halomonas bluephagenesis , Halomonas aydingkolgenesis Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas sp. KM1、 Halomonas elongata and Halomonas smyrnensis Preferred Halomonas bluephagenesis TD1.0 Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (CGMCC No. 19880) and Halomonas campaniensis LS21 (CGMCC No. 6593) or its derivative strains; 2) The recombinant halometazobacter also has one or more of the following modifications: phaC (PHA synthase gene) gene deletion or inactivation; sspB Gene deletion; C-terminal fusion of the MreB protein with an SsrA degradation tag; and / or morphological engineering to increase cell volume relative to unmodified strains; and / or 3) The culture is carried out in an open and / or non-sterile manner in a culture medium containing 20-100 g / L NaCl and pH 7.0-10.0; preferably, the culture is a fed-batch culture, the initial culture medium contains 10-40 g / L glucose and 1-10 g / L urea, and the glucose concentration is maintained at 5-15 g / L by feeding during the culture.

8. A recombinant halomonas strain for producing a target recombinant protein, comprising: a) Transcription regulatory protein expression module, which includes the cold-inactivated transcription regulatory protein TF. ts The encoding gene of the TF ts It contains a TEV protease (tobacco erosion virus protease) recognition site; b) TEV protease expression module, which contains heat-inactivated TEV protease TEV. ts The encoding gene of the TEV ts The encoding gene and the TF that can be used ts Suppressed first-response promoter operable connection; and c) A target recombinant protein expression module, comprising a gene encoding the target recombinant protein, wherein the gene encoding the target recombinant protein is expressed by the TF. ts The suppressed second-response promoter is operable.

9. The recombinant halomonas according to claim 8, wherein: 1) The TF ts Derived from CI434 transcriptional repressor protein (phage 434 CI transcriptional repressor protein) or its functional variants, and comprising a DNA-binding domain, a dimerization domain, and a linker region between the two; the TEV protease recognition site is disposed in the linker region, preferably, the TEV protease recognition site includes ENLYFQG; and / or the first responsive promoter and the second responsive promoter are respectively selected from P containing the CI434 operator sequence. R promoter, P L Promoters or their functional variants; 2) The TF ts By not being subject to the TF ts The expression is driven by a repressive promoter, preferably by a constitutive promoter; the first responsive promoter and the second responsive promoter may be the same or different; the transcriptional regulatory protein expression module, the TEV ts The expression module and the target recombinant protein expression module are located on the same expression vector, on different expression vectors, or on the genome of the recombinant Halomonas bacterium; 3) The target recombinant protein is a difficult-to-express recombinant protein, which is selected from insecticidal proteins, antimicrobial proteins or antimicrobial peptides, cell lysis-related proteins, membrane proteins, proteases, nucleases, phospholipases, proteins that interfere with host metabolism or cell division, as well as industrial enzymes, food proteins, feed proteins, cytokines, therapeutic proteins or biomaterial proteins whose continuous expression, premature expression or high-level expression will reduce host growth, productivity or genetic stability. Preferably, the difficult-to-express recombinant protein is a Bacillus thuringiensis insecticidal protein, selected from Cry family proteins, Cyt family proteins, Vip family proteins and their truncated, fused or variants that maintain the main insecticidal activity; more preferably, it is Cry1Ac or Vip3Aa16. 4) The Halomonas include Halomonas bluephagenesis , Halomonas aydingkolgenesis Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas sp. KM1、 Halomonas elongata and Halomonas smyrnensis Preferred Halomonas bluephagenesis TD1.0 Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (CGMCC No. 19880) and Halomonas campaniensis LS21 (CGMCC No. 6593) or its derivatives; and / or 5) The recombinant halomonas bacteria also has one or more of the following modifications: phaC (PHA synthase gene) gene deletion or inactivation; sspB Gene deletion; C-terminal fusion of the MreB protein with an SsrA degradation tag; and / or Morphological engineering to increase cell volume relative to unmodified strains.

10. A temperature-responsive recombinant expression vector for *Halomonas*, comprising: Genetic elements used for replication and / or stable maintenance of the vector in Halomonas bacteria; TF, a cold-inactivating transcriptional regulatory protein containing a TEV protease recognition site ts The expression module; and the TF that can be expressed by the TF ts Inhibitory responsive promoters can be operatively linked to heat-inactivated TEV proteases. ts Encoded sequences; and sequences for use with the TF ts The identified responsive promoter can be operated to connect to the target recombinant protein insertion site or the target recombinant protein coding sequence.

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