Recombinant strain with elongated cell morphology, construction method therefor, and use thereof

Abstract

The present invention relates to the technical field of strain modification, and specifically relates to a recombinant strain with elongated cell morphology, a construction method therefor, and use thereof. The recombinant strain is constructed by means of subjecting a chassis cell to auto-induced expression of a cell morphology change gene minCD, so that the cell length of the recombinant strain is significantly increased; furthermore, the dry weight and the PHA yield of the cell are increased, and strain collection of the recombinant strain can be implemented at a lower centrifugal force, thus greatly simplifying the downstream processing process and reducing downstream processing costs.

Description

A recombinant strain with elongated cell morphology and its construction method and application Technical Field

[0001] The present invention relates to the technical field of bacterial strain transformation, and in particular to a recombinant bacterial strain with elongated cell morphology, a construction method and application thereof. Background Art

[0002] Polyhydroxybutyrate (PHA) is a naturally occurring intracellular polyester produced by a variety of bacteria. It is a natural way for bacteria to store carbon and energy when there is a nutritional or metabolic imbalance. It exists in the form of water-insoluble inclusion bodies in the bacteria (Shang, L., et al. Poly (3-Hydroxybutyrate) Synthesis in Fed-Batch Culture of Ralstonia eutropha with Phosphate Limitation under Different Glucose Concentrations. Biotechnol. Lett. 2003, 25 (17), 1415-1419.). PHA has material properties comparable to traditional petroleum-based plastics and has been used in packaging, food, medical treatment, cosmetics, agriculture, bioenergy and other fields. The market demand for PHA has huge potential, but PHA yield and production cost are the biggest obstacles restricting PHA from entering the market application.

[0003] Because PHA exists as intracellular inclusion bodies, when its content exceeds 50 wt%, especially when it exceeds 80 wt% in industrial production, cell volume becomes a significant factor influencing PHA yield and is also the determining factor for the upper limit of PHA content. One measure to increase PHA yield and reduce production costs is to increase cell volume. Modifying cell morphology can achieve both the goals of increasing yield and reducing costs. Increasing cell volume to accommodate more PHA not only helps increase PHA content, but also eases the difficulty of cell collection after fermentation, reducing product separation costs.

[0004] Although the prior art (Tan D, Wu Q, Chen JC, et al. Engineering Halomonas TD01 for the low-cost production of polyhydroxyalkanoates [J]. Metab Eng, 2014, 26: 34-47.) discloses that overexpressing minCD can cause cell elongation, thereby reducing downstream separation costs while increasing PHA production, this process requires the addition of the inducer IPTG, which increases production costs. The market demand for PHA has huge potential, but PHA production and production costs are the biggest obstacles to PHA's market application. The prior art (CN113999869A) discloses that overexpressing MinCD using the PhaP series promoter can self-induce cell elongation, but the original expression intensity is too high, which is not conducive to cell growth.

[0005] Summary of the Invention

[0006] The present invention introduces the minCD expression module, a deformation gene regulated by PHA synthesis, into chassis cells, and combines it with ribosome binding site (RBS) sequences of varying strengths to significantly increase the length of chassis cells. These cells, with increased length, can be separated and collected under low centrifugal force conditions, thereby increasing PHA production. Specifically:

[0007] In a first aspect of the present invention, a recombinant bacterium is provided, wherein the recombinant bacterium comprises an expression vector, wherein the expression vector comprises a minCD gene and a promoter;

[0008] The promoter contains a PhaR binding site;

[0009] The recombinant bacteria are recombinant bacteria that express PhaR protein.

[0010] Preferably, the nucleotide sequence of the minCD gene comprises the nucleotide sequence shown in SEQ ID NO: 1, or comprises a sequence having more than 80% homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0011] Preferably, the nucleotide sequence of the minCD gene is shown in SEQ ID NO: 1.

[0012] Preferably, the minCD gene is expressed on an expression vector and / or on a chromosome.

[0013] Preferably, the promoter includes a constitutive promoter and / or an inducible promoter.

[0014] Preferably, the expression of the minCD gene is regulated by the promoter.

[0015] Preferably, the promoter can be a conventional promoter in the prior art.

[0016] Preferably, the constitutive promoter is selected from but not limited to Sp6 promoter or its variants or porin promoter or its mutants.

[0017] Among them, the mutant of the porin promoter is selected from any one of porin-203, porin-221, porin-194, porin-278, porin-68, porin-58 or porin-42 (Ye et.al.Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas.Metabolic Engineering.57(2020)85-95.).

[0018] Preferably, the inducible promoter is selected from but not limited to luc promoter or variants thereof, lac promoter or variants thereof, trp promoter or variants thereof, tac promoter or variants thereof, phage promoter or variants thereof, araBAD promoter or variants thereof or phaP promoter or variants thereof.

[0019] Wherein, the phage promoter includes but is not limited to a λ phage promoter or a T7 phage promoter.

[0020] Preferably, the phaP promoter includes but is not limited to phaP1 promoter, phaP2 promoter or phaP3 promoter.

[0021] In a specific embodiment of the present invention, the promoter is phaP1, and the phaP1 promoter is located at base 109 upstream of the open reading frame of the phaP1 gene.

[0022] Preferably, the phaP1 promoter comprises the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence having more than 80% homology thereto and having the same or similar activity.

[0023] In a specific embodiment of the present invention, the phaP1 promoter is shown as SEQ ID NO: 2.

[0024] Preferably, the recombinant bacteria can be recombinant bacteria that can express PhaR protein itself, or the recombinant bacteria can be modified to express PhaR protein (for example, an expression vector containing a gene encoding PhaR protein is introduced into the recombinant bacteria).

[0025] Preferably, when the PhaR protein expressed by the recombinant bacteria binds to the PhaR binding site on the promoter, it inhibits the expression of the promoter. When the PHA synthesized in the system binds to PhaR, PhaR is detached from the promoter, the expression of the promoter is restored, and the expression of the promoter is regulated by PHA synthesis.

[0026] Preferably, the promoter itself contains a PhaR binding site, or the promoter can be modified, for example, by adding a PhaR binding site to the promoter. The promoter with the added PhaR binding site still has the function of a promoter, and the expression of the promoter can be regulated by PHA synthesis.

[0027] Preferably, the expression vector further comprises an RBS sequence. In order to regulate the phaP1 promoter and meet the expression requirements of different strengths, the core region of the original phaP1 RBS was mutated to obtain a library of 1024 RBS with different strengths.

[0028] Preferably, the sequence of the RBS library is shown in SEQ ID NO: 3 (wherein, N is a mutation site, which can be any one of A, T, C, G or U).

[0029] Preferably, the nucleotide sequence of the RBS is selected from an RBS library, and the nucleotide sequence of the RBS includes SEQ ID NO: 4-13 or includes a nucleotide sequence having more than 90% homology with SEQ ID NO: 4-13, or a nucleotide sequence as shown in SEQ ID NO: 4-13.

[0030] In a specific embodiment of the present invention, the nucleotide sequence of the RBS includes SEQ ID NO: 7-13 or a nucleotide sequence having more than 90% homology with SEQ ID NO: 7-13, or a nucleotide sequence as shown in SEQ ID NO: 7-13.

[0031] In a specific embodiment of the present invention, the promoter, minCD gene and RBS sequence contained in the expression vector are in the order of promoter, RBS sequence, minCD gene.

[0032] Preferably, in the expression vector, the RBS is adjacent to the start codon of the minCD gene.

[0033] Preferably, the expression vector is capable of replication, transcription and translation in the host cell, and therefore, it may further comprise other conventional expression elements in expression vectors, such as terminators, enzyme cleavage sites, and the like.

[0034] In a specific embodiment of the present invention, the expression vector comprises a promoter, a minCD gene, an RBS sequence and a terminator, and the order of the expression elements and genes in the expression vector is promoter, RBS sequence, minCD gene and terminator.

[0035] Preferably, the expression vector can be a prokaryotic expression vector or a eukaryotic expression vector, and more preferably a prokaryotic expression vector.

[0036] In a specific embodiment of the present invention, the expression vector is a plasmid vector.

[0037] In a specific embodiment of the present invention, the plasmid vector is an Escherichia coli expression vector.

[0038] Preferably, the expression vector integrates the minCD gene into the genome of the recombinant bacteria, or the expression vector is free in the recombinant bacteria.

[0039] Preferably, the expression vector also integrates the promoter, RBS sequence and / or terminator into the genome of the recombinant bacteria.

[0040] Preferably, the recombinant bacteria are recombinant bacteria that synthesize PHA or monomers that constitute PHA.

[0041] Preferably, the recombinant bacteria can be recombinant bacteria that naturally synthesize PHA or monomers that constitute PHA, or recombinant bacteria that can synthesize PHA or monomers that constitute PHA after being modified (for example, by introducing PHA synthesis-related genes into the recombinant bacteria).

[0042] Preferably, the expression vector may also contain genes encoding product synthesis pathways.

[0043] Preferably, the product comprises monomers constituting PHA or PHA.

[0044] Preferably, the monomers constituting PHA include but are not limited to one or more of 2-hydroxypropionic acid, 3-hydroxypropionic acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 3-hydroxyvaleric acid, 5-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, and 3-hydroxydodecanoic acid;

[0045] Preferably, the PHA includes but is not limited to one or more of P(HB-LA), P3HP, PHB, P4HB, PHV, PHO, PHN, PHD, PHBV, P34HB, PHBHHp, PHBHHx, P3HB4HB3HV, P3HB4HB5HV or other medium- and long-chain hydroxy fatty acids.

[0046] Preferably, the recombinant bacteria is selected from the genus Escherichia, Ralstonia, Alcaligenes, Pseudomonas, Aeromonas or Halomonas.

[0047] Preferably, the genus Escherichia includes but is not limited to Escherichia coli or its derivatives.

[0048] Preferably, the genus Ralstonia includes but is not limited to Ralstonia eutropha (also known as Alcaligenes eutrophus, Cupriavidus necator) or its derivatives.

[0049] Preferably, the genus Alcaligenes includes but is not limited to Alcaligenes latus or its derivatives.

[0050] Preferably, the genus Pseudomonas includes but is not limited to Pseudomonas putida or its derivatives.

[0051] Preferably, the genus Aeromonas includes but is not limited to Aeromonas hydrophila or its derivatives.

[0052] Preferably, the genus Halomonas includes but is not limited to Halomonas bluephagenesis or its derivatives, Halomonas campaniensis or its derivatives, or Halomonas aydingkolgenesis or its derivatives.

[0053] In a specific embodiment of the present invention, the recombinant bacteria are Halomonas, and the recombinant bacteria include but are not limited to Halomonas bluephagenesis TD01 CGMCC.No.4353, Halomonas campaniensis LS21 CGMCC No.6593, Halomonas aydingkolgenesis M1 CGMCC NO.19880, Halomonas bluephagenesis WZY254 or Halomonas bluephagenesis WZY278.

[0054] The second aspect of the present invention provides a method for preparing the recombinant bacteria described in the first aspect, characterized in that the preparation method comprises introducing the expression vector into the recombinant bacteria.

[0055] Preferably, the expression vector integrates the minCD gene into the genome of the recombinant bacteria, or the expression vector is free in the recombinant bacteria.

[0056] Preferably, the expression vector also integrates the promoter, RBS sequence and / or terminator into the genome of the recombinant bacteria.

[0057] Preferably, the relevant limitations on expression vector, promoter, RBS sequence, and recombinant bacteria are the same as those in the first aspect of the present invention.

[0058] The third aspect of the present invention provides a recombinant bacterium obtained by the preparation method described in the second aspect.

[0059] The fourth aspect of the present invention provides an expression vector.

[0060] The relevant limitations on expression vectors are the same as those in the first aspect of the present invention.

[0061] The fifth aspect of the present invention provides a fermentation method.

[0062] The fermentation method comprises fermenting and culturing the recombinant bacteria described in the first aspect.

[0063] In a sixth aspect, the present invention provides a method for producing PHA.

[0064] The method comprises fermenting and culturing the recombinant bacteria described in the first aspect.

[0065] A seventh aspect of the present invention provides a method for promoting cell elongation.

[0066] The method comprises introducing an expression vector into a recombinant bacterium, wherein the expression vector comprises a minCD gene and a promoter; the promoter comprises a PhaR binding site; and the recombinant bacterium is a recombinant bacterium expressing a PhaR protein.

[0067] Preferably, the nucleotide sequence of the minCD gene comprises the nucleotide sequence shown in SEQ ID NO: 1, or comprises a sequence having more than 80% homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0068] Preferably, the nucleotide sequence of the minCD gene is shown in SEQ ID NO: 1.

[0069] Preferably, the relevant limitations on expression vectors and recombinant bacteria are the same as those in the first aspect of the present invention.

[0070] In an eighth aspect, the present invention provides a method for isolating recombinant bacteria, wherein the method comprises centrifuging the recombinant bacteria.

[0071] Preferably, the centrifugation can be performed at any centrifugal force.

[0072] Preferably, the centrifugal force is greater than 300g.

[0073] Preferably, the centrifugal force of the centrifugation is any value in the range of 300-15000g, for example 300, 500, 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000 or 15000g.

[0074] Preferably, the separation time is more than 3 minutes, such as 3, 4, 5, 6, 7, 8, 9, 10, or longer.

[0075] Due to the elongation of the cells of the recombinant bacteria described in the present application, bacterial cell collection can be completed in a short time, for example, within 10 minutes, under low centrifugal force, thereby simplifying the downstream processing process and reducing downstream processing costs. Beneficial effects:

[0076] In PHA production strains, the PhaP protein acts as a PHA particle-binding protein, and its expression level is positively correlated with the percentage of PHA content. By placing the cell deformation-related gene minCD downstream of the PhaP promoter, the phaP promoter regulates minCD expression, enabling auto-induced expression of MinCD in PHA production without the need for inducers. During fermentation, cell length increases to nearly ten times its original length, while simultaneously increasing dry weight and PHA content. Furthermore, the lengthened cells can be separated and collected under low centrifugal force conditions, thereby reducing downstream processing costs for PHA. This will help PHA gain wider market applications and has applications in molecular biology, genetic engineering, metabolic engineering, synthetic biology, fermentation engineering, and downstream separation and purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1: Recombinant plasmid pSEVA321-P p1 -RBS-minCD structure diagram, minCD gene is regulated by phaP1 promoter, where oriT represents the transfer origin of bacterial conjugative plasmid, Cm R represents chloramphenicol resistance, T represents terminator, and the semicircle in the figure represents the ribosome binding site RBS.

[0078] Figure 2: Strain WZY278 (pSEVA321-P p1 ) and WZY278(pSEVA321-P p1 -RBS3-minCD) cell morphology at the end point of the shake flask fermentation experiment.

[0079] Figure 3: Strain WZY278 (pSEVA321-P p1 ) and WZY278(pSEVA321-P p1 -RBS3-minCD) shake flask fermentation broth was centrifuged at 500g for 3min, 6min, and 10min respectively; ① was the control strain WZY278 (pSEVA321-P p1 ), ② is the deformed strain WZY278 (pSEVA321-P p1 -RBS3-minCD). DETAILED DESCRIPTION

[0080] The present invention is further described below with reference to the Examples, sequence listing, and accompanying drawings, but is not intended to be limiting. Experimental methods used in the Examples, unless otherwise specified, were based on conventional methods, conventional procedures, or the conditions described in the instructions. Reagents or drugs not otherwise specified are commonly available commercially.

[0081] Escherichia coli was grown in LB medium containing 10 g / L sodium chloride, 10 g / L peptone, and 5 g / L yeast extract.

[0082] Halomonas bluephagenes WZY278 and its derivatives were cultured in LB50 or MM30 medium. LB50 medium is identical to LB except that the sodium chloride concentration is increased to 50 g / L. MM30 medium consists of 30 g / L sodium chloride, 1 g / L yeast extract, 30 g / L glucose, 0.5 g / L urea, and other trace elements.

[0083] The shaking growth conditions for Escherichia coli, H. bluephagenesis and their derivatives were 37°C and 200 rpm.

[0084] Example: Construction of a recombinant strain with elongated cell morphology

[0085] First, to regulate the phaP1 promoter and adapt to different expression strengths, we performed saturation mutagenesis on the core region of the original phaP1 RBS, generating a library of 1024 RBSs with varying strengths. The sequence of this RBS library is shown in SEQ ID NO: 3 (where N represents the saturation mutation site).

[0086] Ten RBSs of varying strengths were screened (see Table 1, where RBS0 is the natural RBS and RBS1-RBS9 are modified RBSs. The expression intensity of RBS0 is too high, which is detrimental to cell growth) to construct a recombinant expression plasmid for minCD. The minCD gene was placed downstream of the phaP1 promoter, and the RBS was located upstream of minCD, immediately adjacent to the start codon of minCD. RBS1-RBS9 were derived from the RBS library calculator constructed by Salis Lab. phaP1 RBS X Library (https: / / salislab.net / software / design_rbs_library_calculator). This library is based on RBS0 (i.e. P phaP1The natural RBS) homologous sequence CTCACTTAXXXXXATTATGTG (where X is any base among A, T, C, G or U) was used as a template, minCD was used as the expression gene, and the five X sites were randomly mutated. Each RBS corresponds to its own relative strength. Then, the strain H. bluephagenesis WZY278 (H. bluephagenesis WZY278 strain: the 4HB-CoA transferase encoding gene was integrated into the recombinant H. bluephagenesis TD1.0 genome, ΔphaP1, ΔlpxL, ΔlpxM, and the P expressing phaCAB was used. Mmp1 The promoter was replaced by P porin The promoter was used to overexpress an additional phaCAB linked to the essential gene ompW (for details, see Ji, M., et al. PHB production from food waste hydrolysates by Halomonas blue phagenesis Harboring PHB operon linked with an essential gene. Metabolic Engineering, 2023, 77, 12-20). The recombinant expression plasmid was incorporated into the host to generate the corresponding engineered strain. Shake flask fermentation experiments were performed on each strain, and analysis of changes in cell morphology, dry weight, and PHB percentage revealed significant increases in cell length for each strain, and one strain showed both increased PHB yield and CDW.

[0087] Table 1: Nucleic acid sequences and relative intensities of RBS0-RBS9

[0088] 1. Construction of the autoinducible minCD recombinant expression plasmid

[0089] First, the H. bluephagenesis TD01 genome was selected as a template and minCD was obtained by PCR. Then, the pSEVA321 plasmid was used as a vector and the phaP1 promoter (P p1 ) is the promoter of gene expression, and the 10 RBSs with different strengths in Table 1 were used to construct recombinant expression plasmids with different minCD expression strengths, namely pSEVA321-P p1 -RBS0-minCD、pSEVA321-P p1 -RBS1-minCD, pSEVA321-P p1 -RBS2-minCD, pSEVA321-P p1 -RBS3-minCD, pSEVA321-Pp1 -RBS4-minCD, pSEVA321-P p1 -RBS5-minCD, pSEVA321-P p1 -RBS6-minCD, pSEVA321-P p1 -RBS7-minCD, pSEVA321-P p1 -RBS8-minCD, pSEVA321-P p1 -RBS9-minCD. Among them, the plasmid pSEVA321-P without minCD was used. p1 Each plasmid was transformed into competent E. coli S17-1 cells. Single clones were selected for PCR identification and sequencing. Correctly sequenced single clones were selected to obtain 10 auto-inducing recombinant expression plasmids with different minCD expression intensities (plasmid structures are shown in Figure 1) and a control plasmid.

[0090] 2. Construction of strains with elongated cell morphology

[0091] Using H. bluephagenesis WZY278 as the host cell, the 10 autoinducing recombinant expression plasmids and the control plasmid obtained above were respectively transferred into H. bluephagenesis WZY278 (for specific methods, see Zhao, H., et al. Novel T7-like expression systems used for Halomonas. Metabolic Engineering, 2017, 39, 128-140.), and WZY278 (pSEVA321-P p1 -RBS0-minCD), WZY278(pSEVA321-P p1 -RBS1-minCD), WZY278(pSEVA321-P p1 -RBS2-minCD), WZY278(pSEVA321-P p1 -RBS3-minCD), WZY278(pSEVA321-P p1 -RBS4-minCD), WZY278(pSEVA321-P p1 -RBS5-minCD), WZY278(pSEVA321-P p1 -RBS6-minCD), WZY278(pSEVA321-P p1 -RBS7-minCD), WZY278(pSEVA321-P p1 -RBS8-minCD), WZY278(pSEVA321-Pp1 -RBS9-minCD) and the control strain WZY278 (pSEVA321-P p1 ).

[0092] 3. Determination of CDW and PHB% of Strain

[0093] First, shake flask fermentation must be carried out. The 11 engineered strains obtained above were inoculated into 20 mL of LB50 medium containing 34 mg / L chloramphenicol and cultured for 12-16 hours, and then transferred to a new 20 mL of LB50 medium containing 34 mg / L chloramphenicol at a volume ratio of 1% and continued to be cultured for 8-10 hours to obtain a shake flask seed solution. 2.5 mL of seed bacterial solution was inoculated into a 500 mL conical flask containing 50 mL of MM30 medium containing 34 mg / L chloramphenicol (inoculation ratio 1: 20), and fermented for 48 hours at 37 ° C and 200 rpm. After the fermentation was completed, the samples were collected and the samples were analyzed for CDW and PHB% (for specific experimental methods, see Ye, J., et al. Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas. Metabolic Engineering, 2020, 57, 85-95). The results are shown in Table 2. As can be seen from Table 2, compared with the control, the dry weight and PHB% of some strains containing the auto-inducing minCD recombinant expression plasmid were increased, and the effect varied with the strength of their RBS. Among them, RBS7 had the best effect, and WZY278 (pSEVA321-P p1 -RBS7-minCD) increased by about 15.5% in dry weight and about 6.7% in PHB%.

[0094] Table 2: phaP1 promoter drives PHB synthesis gene to improve PHB production

[0095] 4. Analysis of bacterial morphology and centrifugation of bacterial liquid

[0096] At the end of the shake flask fermentation experiment, WZY278 (pSEVA321-P p1 )、WZY278(pSEVA321-P p1 -RBS7-minCD) were used to observe the cell morphology under a microscope and analyze the centrifugation of the bacterial solution. The cell morphology of the two strains is shown in Figure 2. As can be seen from Figure 2, WZY278 (pSEVA321-P p1 -RBS7-minCD) cells increased significantly in length, reaching 10 μm, which was approximately 10 times the length of the control strain cells.

[0097] Regarding the analysis of the centrifugation of the bacterial solution, 35 ml of the bacterial solution was first taken into 50 ml EP tubes and centrifuged at 500 g for 3 min, 6 min, and 10 min, respectively. After each centrifugation, the opacity of the bacterial solution in the two tubes was observed. The results are shown in Figure 3, where ① is the control strain WZY278 (pSEVA321-P p1 ), ② is the deformed strain WZY 278 (pSEVA321-P p1 As shown in Figure 3, under a centrifugal force of 500 g, the control strain WZY278 (pSEVA321-P p1 ) was separated for 10 min, the supernatant was still turbid, while the strain WZY278 (pSEVA321-P p1 -RBS7-minCD) after 10 min of separation, the supernatant was significantly clearer than that of the control strain group, that is, the deformed strain can achieve faster centrifugal separation, which helps to improve the efficiency of downstream separation processing.

Claims

1. A recombinant bacterium, characterized in that: The recombinant bacteria contains an expression vector, and the expression vector contains a minCD gene and a promoter; The promoter contains a PhaR binding site; The recombinant bacteria are recombinant bacteria expressing PhaR protein.

2. The recombinant bacterium according to claim 1, characterized in that The nucleotide sequence of the minCD gene comprises the nucleotide sequence shown in SEQ ID NO: 1, or comprises a nucleotide sequence having more than 80% homology with the nucleotide sequence shown in SEQ ID NO:

1.

3. The recombinant bacterium according to claim 1, characterized in that The minCD gene is expressed on an expression vector and / or on a chromosome.

4. The recombinant bacterium according to any one of claims 1 to 3, characterized in that The promoter includes a constitutive promoter and / or an inducible promoter. Preferably, the constitutive promoter is selected from Sp6 promoter or its variant or porin promoter or its variant; Preferably, the inducible promoter is selected from luc promoter or variants thereof, lac promoter or variants thereof, trp promoter or variants thereof, tac promoter or variants thereof, phage promoter or variants thereof, araBAD promoter or variants thereof or phaP promoter or variants thereof.

5. The recombinant bacterium according to any one of claims 1 to 4, characterized in that The phaP promoter includes phaP1 promoter, phaP2 promoter or phaP3 promoter.

6. The recombinant bacterium according to claim 5, characterized in that The phaP1 promoter includes SEQ ID NO: 2 or a nucleotide sequence having more than 80% homology with the nucleotide sequence shown in SEQ ID NO:

2.

7. The recombinant bacterium according to any one of claims 1 to 6, characterized in that The expression vector further comprises an RBS sequence; Preferably, the promoter, minCD gene and RBS sequence contained in the expression vector are in the order of promoter, RBS sequence and minCD gene.

8. The recombinant bacterium according to claim 7, characterized in that In the expression vector, the RBS is adjacent to the start codon of the minCD gene.

9. The recombinant bacterium according to claim 7 or 8, characterized in that The RBS sequence is as follows GCGTGTCGAATCGCTAGCTCACTTANNNNNATTATGTG (SEQ ID NO: 3) is shown, Wherein, N is selected from any one of A, T, C, G or U.

10. The recombinant bacterium according to any one of claims 7 to 9, characterized in that: The RBS sequence includes SEQ ID NO: 4-13 or a nucleotide sequence having more than 90% homology with SEQ ID NO: 4-13.

11. The recombinant bacterium according to claim 10, characterized in that The RBS sequence includes SEQ ID NO: 7-13 or a nucleotide sequence having more than 90% homology with SEQ ID NO: 7-13.

12. The recombinant bacterium according to any one of claims 1 to 11, characterized in that: The recombinant bacteria are recombinant bacteria that synthesize PHA or constitute monomers of PHA.

13. The recombinant bacterium according to any one of claims 1 to 12, characterized in that: The recombinant bacteria is selected from Escherichia or its derivatives, Ralstonia or its derivatives, Alcaligenes or its derivatives, Pseudomonas or its derivatives, Aeromonas or its derivatives, or Halomonas or its derivatives; The Halomonas genus preferably includes Halomonas bluephagenesis or its derivatives, Halomonas campaniensis or its derivatives or Halomonas aydingkolgenesis or its derivatives.

14. A method for preparing the recombinant bacteria according to any one of claims 1 to 13, characterized in that: The preparation method comprises introducing the expression vector into the recombinant bacteria.

15. A method for promoting cell fibrillation, characterized in that: The method comprises introducing an expression vector into a recombinant bacterium; the expression vector comprises a minCD gene and a promoter; The promoter contains a PhaR binding site; The recombinant bacteria are recombinant bacteria expressing PhaR protein.

16. The method according to claim 15, characterized in that The nucleotide sequence of the minCD gene comprises the nucleotide sequence shown in SEQ ID NO: 1, or comprises a nucleotide sequence having more than 80% homology with the nucleotide sequence shown in SEQ ID NO:

1.

17. A method for isolating recombinant bacteria, characterized in that: The separation method comprises centrifugation to separate the recombinant bacteria according to any one of claims 1 to 13; Preferably, the centrifugal force of the centrifugation is greater than 300g.

18. A fermentation method, characterized in that: The fermentation method comprises fermenting and culturing the recombinant bacteria according to any one of claims 1 to 13.

19. A method for producing PHA, characterized in that: The method comprises fermenting and culturing the recombinant bacteria according to any one of claims 1 to 13.

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