Recombinant plasmid for expressing toxin and antitoxin as well as construction method and application of recombinant plasmid

Abstract

The invention provides a recombinant plasmid for expressing toxin and antitoxin as well as a construction method and application thereof in gene editing or biosynthesis, the toxin-antitoxin can maintain the plasmid to stably and efficiently express a target gene, and the recombinant plasmid plays an important role in research on halophilic microbial products.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a recombinant plasmid for expressing toxin and antitoxin, and a construction method and application thereof. Background Art

[0002] Halophiles are a type of microorganisms that grow in high-salt environments and are widely distributed in salt lakes, salt mines, saline-alkali soils, and high-salt products. Halophiles have long attracted research interest with their unique adaptation mechanisms. The environment in which halophiles live has a high salt concentration, and it is difficult for other microorganisms to survive and grow in these environments. If they are applied to the fermentation production of PHA, most of the contamination of bacteria can be avoided, the production links can be reduced, and they have strong cost advantages. Halophiles also have great advantages in high-salt wastewater treatment and ecological restoration. Because halophiles have a rich metabolic resource library, they can be found in high-salt wastewater and high-salt soil containing various nutrient sources. Some halophiles have strong tolerance to alkaline environments and heavy metal ions, and have strong bioremediation potential in environments rich in heavy metal ions such as high-salt organic wastewater and oilfield soil. Halophiles are gradually becoming an emerging force in new biochemical industries such as biodegradable materials, biofuels, enzyme preparations, and pharmaceuticals, as well as in pollution control and sewage treatment.

[0003] Halophilic microorganisms represented by Halomonas have great application potential as a platform for producing biodegradable bioplastics PHA and other high value-added chemicals. Researchers have developed a variety of corresponding genome editing technologies to achieve metabolic regulation of engineered bacteria. However, with the increasing demand for transformation, multiple editing often leads to decreased genome stability, which in turn affects the growth and production of engineered bacteria. Problems such as cumbersome editing process, long cycle and high cost are also becoming increasingly prominent.

[0004] Therefore, it is necessary to develop an extrachromosomal expression system suitable for halophilic microorganisms that can stably and efficiently express the target gene. Summary of the invention

[0005] The inventors of the present application, through years of research on the genomes of halophilic microorganisms, have elucidated that toxin-antitoxin derived from halophilic microorganisms is a key element in maintaining the stable propagation of endogenous plasmids, and have constructed a recombinant stable plasmid expressing toxin-antitoxin to carry the target gene, which can be stably and efficiently expressed in halophilic microorganisms.

[0006] In a first aspect, a recombinant plasmid is provided, wherein the recombinant plasmid expresses toxins and antitoxins, and the toxins and antitoxins are derived from halophilic microorganisms.

[0007] Preferably, the toxin and antitoxin are derived from halophilic microorganisms. Preferably, the toxin and antitoxin are derived from the same or different halophilic microorganisms.

[0008] Preferably, the toxin and antitoxin are derived from endogenous plasmids of halophilic microorganisms.

[0009] Preferably, the recombinant plasmid further comprises regulatory elements required for the expression of toxins and antitoxins. For example, a promoter, which can be any promoter disclosed in the prior art, as long as it can regulate the expression of toxins and antitoxins. Preferably, it can be a constitutive promoter or an inducible promoter.

[0010] In a specific embodiment of the present invention, the promoter may be derived from a halophilic microorganism.

[0011] In a specific embodiment, the promoter is hbpB / hbpC promoter.

[0012] The toxin and antitoxin are on the same operon and are regulated by the same promoter.

[0013] Preferably, the regulatory element also includes an RBS sequence.

[0014] Preferably, the promoter and RBS sequence regulate the initiation and extent of expression of toxin and antitoxin genes. Further preferably, the promoter is a promoter shared by toxin and antitoxin.

[0015] Preferably, the promoter and RBS sequences comprise SEQ ID NO: 5, 11, 30 or 35 or comprise a nucleotide sequence having more than 80% homology with SEQ ID NO: 5, 11, 30 or 35 and having the same or similar activity.

[0016] Preferably, the recombinant plasmid further comprises a replication initiation site and / or a replication initiation protein coding sequence, wherein the replication initiation site and / or the replication initiation protein can be any replication initiation site and / or replication initiation protein disclosed in the prior art, as long as the plasmid is guaranteed to have a certain copy number, preferably a high copy number, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70 or more copies. In a specific embodiment of the present invention, the replication initiation site and / or the replication initiation protein are derived from halophilic microorganisms, preferably from endogenous plasmids of halophilic microorganisms.

[0017] Preferably, the recombinant plasmid comprises one or more replication initiation sites.

[0018] In a specific embodiment, the replication origin site is pHbCP ori or pRO1600 oriV.

[0019] Preferably, the replication initiation site comprises SEQ ID NO: 6 or 12 or comprises a nucleotide sequence having more than 80% homology with SEQ ID NO: 6 or 12 and having the same or similar activity.

[0020] Preferably, the recombinant plasmid comprises one or more replication initiation protein coding sequences.

[0021] In a specific embodiment, the replication initiation protein is RepB protein.

[0022] Preferably, the repB gene comprises SEQ ID NO: 7 or comprises a nucleotide sequence having more than 80% homology with SEQ ID NO: 7 and having the same or similar activity.

[0023] In a specific embodiment, the replication initiation protein is pRO1600 Rep protein.

[0024] Preferably, the pRO1600Rep gene comprises SEQ ID NO: 13 or comprises a nucleotide sequence having more than 80% homology with SEQ ID NO: 13 and having the same or similar activity.

[0025] Preferably, the replication initiation protein pRO1600 Rep comprises SEQ ID NO: 24 or comprises an amino acid sequence having more than 80% homology with SEQ ID NO: 24 and having the same or similar activity.

[0026] Preferably, the replication initiation site and replication initiation protein coding sequence are essential elements for the plasmid to persist and replicate in halophilic microorganisms.

[0027] Preferably, the recombinant plasmid contains one or more toxin and antitoxin expression systems, and the toxin and antitoxin expression systems include toxin encoding genes, antitoxin encoding genes and regulatory elements required for expressing toxins and antitoxins.

[0028] Preferably, the recombinant plasmid may or may not express a resistance protein. Further preferably, the resistance protein comprises one or more of ampicillin, neomycin, chloramphenicol, tetracycline, kanamycin, apramycin or spectinomycin.

[0029] Preferably, the recombinant plasmid is a conjugative plasmid or a non-conjugative plasmid.

[0030] Preferably, the recombinant plasmid also includes elements required for conjugation and transformation, such as a replication initiation site and a plasmid transfer initiation site. The elements required for conjugation and transformation can be any element disclosed in the prior art, as long as conjugation and transformation can be performed. The elements required for conjugation and transformation can be used in Escherichia coli. Replication and / or transfer are preferred.

[0031] In a specific embodiment of the present invention, the elements required for conjugative transformation are derived from Escherichia coli.

[0032] Preferably, the conjugative transformation element is selected from the replication origin site and plasmid transfer origin site of pUC plasmid, pET plasmid, pGEX plasmid and pRO plasmid.

[0033] In a specific embodiment of the present invention, the elements required for conjugative transformation are derived from the optimized replication origin site pMB1 ori of the pUC series plasmids.

[0034] In a specific embodiment of the present invention, the elements required for conjugative transformation include the replication origin site pRO1600 oriV. Preferably, the replication origin site pRO1600 oriV is as shown in SEQ ID NO:12.

[0035] Preferably, the halophilic microorganism is a bacterium.

[0036] Preferably, the halophilic microorganisms include, but are not limited to, Chromohalobacter, Halomonas, Halovibrio, Vibrio, Cobetia, Kushneria, Salicola, Halotela or Carnimonas.

[0037] Preferably, the genus Chromohalobacter includes but is not limited to Chromohalobacter marismortui, Chromohalobacter salexigens or Chromohalobacter beijerinckii; the genus Halomonas includes but is not limited to Halomonas bluephagenesis, Halomonas campaniensis, Halomonasdesiderata, Halomonas venusta or Halomonas alkaliantarctica; the genus Halovibrio includes but is not limited to Halovibrio denitrificans, Halovibrio mesolongii or Halovibrio variabilis; the genus Vibrio includes but is not limited to Vibrio natriegens; the genus Salicola includes but is not limited to Salicola marasensis or Salicola salis; the genus Cobetia includes but is not limited to Cobetiamarina or Cobetia crustatorum; the genus Kushneria includes but is not limited to Kushneria aurantia or Kushneria indalinina; the genus Halotela includes but is not limited to Halotela alkalilenta; the genus Carnimonas includes but is not limited to Carnimonas nigrificans.

[0038] Preferably, the halophilic microorganisms include mild halophiles, moderate halophiles or extreme halophiles, and more preferably moderate halophiles.

[0039] Preferably, the halophilic microorganism is a halophilic microorganism that can grow under conditions containing a salt concentration of 0.5 mol / L-2.5 mol / L, preferably a halophilic microorganism that grows stably or grows best.

[0040] The amino acid sequence of the antitoxin comprises SEQ ID NO: 3, 28 or 33 or comprises an amino acid sequence having more than 80% homology and the same or similar activity as SEQ ID NO: 3, 28 or 33. Preferably, the antitoxin gene is hbpB.

[0041] The amino acid sequence of the toxin comprises SEQ ID NO: 4, 29 or 34 or comprises an amino acid sequence having more than 80% homology and the same or similar activity as SEQ ID NO: 4, 29 or 34. Preferably, the toxin gene is hbpC.

[0042] In a specific embodiment, the sequence of the hbpB gene comprises SEQ ID NO: 1, 26 or 31 or comprises a nucleotide sequence having more than 80% homology with SEQ ID NO: 1, 26 or 31 and having the same or similar activity.

[0043] In a specific embodiment, the sequence of the hbpC gene comprises SEQ ID NO: 2, 27 or 32 or comprises a nucleotide sequence having more than 80% homology with SEQ ID NO: 2, 27 or 32 and having the same or similar activity.

[0044] Preferably, the recombinant plasmid further comprises the target gene, and further preferably comprises regulatory elements and / or regulatory proteins required for replication, transcription and / or expression of the target gene, such as a promoter, terminator and RBS sequence.

[0045] Preferably, the recombinant plasmid comprises toxin and antitoxin expression systems, target gene expression systems and auxiliary sequences.

[0046] Preferably, the recombinant plasmid comprises a toxin and antitoxin expression system, a replication initiation site, a replication initiation protein coding sequence, a target gene expression system and an auxiliary sequence.

[0047] The target gene expression system includes the target gene and the regulatory elements and / or regulatory proteins required for the replication, transcription and / or expression of the target gene.

[0048] The toxin and antitoxin expression system is a system comprising toxins and antitoxins and regulatory elements required for expressing toxins and antitoxins.

[0049] In a specific embodiment of the present invention, the recombinant plasmid consists of a toxin and antitoxin expression system and a target gene expression system.

[0050] In a specific embodiment of the present invention, the recombinant plasmid consists of a toxin and antitoxin expression system, a replication initiation site, a replication initiation protein coding sequence and a target gene expression system.

[0051] In a specific embodiment of the present invention, the recombinant plasmid consists of a toxin and antitoxin expression system, a target gene expression system and an auxiliary sequence.

[0052] In a specific embodiment of the present invention, the recombinant plasmid consists of a toxin and antitoxin expression system, a replication initiation site, a replication initiation protein coding sequence, a target gene expression system and an auxiliary sequence.

[0053] The auxiliary sequence may be an element for ensuring or improving the self-replication function and / or expression function of the plasmid, or the auxiliary sequence may be a site required for connecting various elements.

[0054] For example, restriction sites; screening or marker genes; sequences that promote the replication, transcription and expression of target genes; sequences that promote the replication, transcription and expression of toxins and / or antitoxins; sequences that contribute to plasmid stability; spacer sequences; linker sequences; regulatory proteins; multiple cloning sites, etc.

[0055] Preferably, the recombinant plasmid is a linear plasmid or a circular plasmid.

[0056] Preferably, the recombinant plasmid is DNA or RNA.

[0057] In a specific embodiment, the recombinant plasmid is a circular plasmid, and the recombinant plasmid includes, in a clockwise direction, a toxin and antitoxin expression system, a replication initiation protein coding sequence and a replication initiation site, and a target gene expression system.

[0058] In a specific embodiment, the recombinant plasmid is a circular plasmid, which includes, in a clockwise direction, a toxin and antitoxin expression system, a replication initiation protein coding sequence and a replication initiation site, a target gene expression system, a starting site for conjugation transformation, and a starting site for plasmid transfer.

[0059] In a specific embodiment, the recombinant plasmid is a circular plasmid, which includes, in clockwise direction: a promoter shared by toxin and antitoxin, an antitoxin coding sequence, a toxin coding sequence, a replication initiation protein coding sequence and a replication initiation site, a target gene and its regulatory elements, a starting site for conjugation transformation, and a starting site for plasmid transfer.

[0060] In a second aspect, a toxin-antitoxin system is provided, wherein the toxin-antitoxin system is derived from halophilic microorganisms.

[0061] Preferably, the toxin and antitoxin are derived from the same or different halophilic microorganisms.

[0062] Preferably, the amino acid sequence of the antitoxin comprises SEQ ID NO: 3, 28 or 33 or comprises an amino acid sequence having more than 80% homology and the same or similar activity as SEQ ID NO: 3, 28 or 33. Preferably, the antitoxin gene is hbpB.

[0063] Preferably, the amino acid sequence of the toxin comprises SEQ ID NO: 4, 29 or 34 or comprises an amino acid sequence having more than 80% homology and the same or similar activity as SEQ ID NO: 4, 29 or 34. Preferably, the toxin gene is hbpC.

[0064] In a specific embodiment, the sequence of the hbpB gene comprises SEQ ID NO: 1, 26 or 31 or comprises a nucleotide sequence having more than 80% homology with SEQ ID NO: 1, 26 or 31 and having the same or similar activity.

[0065] In a specific embodiment, the sequence of the hbpC gene comprises SEQ ID NO: 2, 27 or 32 or comprises a nucleotide sequence having more than 80% homology with SEQ ID NO: 2, 27 or 32 and having the same or similar activity.

[0066] Preferably, the halophilic microorganism is a bacterium.

[0067] Preferably, the halophilic microorganisms include, but are not limited to, Chromohalobacter, Halomonas, Halovibrio, Vibrio, Cobetia, Kushneria, Salicola, Halotela or Carnimonas.

[0068] Preferably, the genus Chromohalobacter includes but is not limited to Chromohalobacter marismortui, Chromohalobacter salexigens or Chromohalobacter beijerinckii; the genus Halomonas includes but is not limited to Halomonas bluephagenesis, Halomonas campaniensis, Halomonasdesiderata, Halomonas venusta or Halomonas alkaliantarctica; the genus Halovibrio includes but is not limited to Halovibrio denitrificans, Halovibrio mesolongii or Halovibrio variabilis; the genus Vibrio includes but is not limited to Vibrio natriegens; the genus Salicola includes but is not limited to Salicola marasensis or Salicola salis; the genus Cobetia includes but is not limited to Cobetiamarina or Cobetia crustatorum; the genus Kushneria includes but is not limited to Kushneria aurantia or Kushneria indalinina; the genus Halotela includes but is not limited to Halotela alkalilenta; the genus Carnimonas includes but is not limited to Carnimonas nigrificans.

[0069] Preferably, the halophilic microorganisms include mild halophiles, moderate halophiles or extreme halophiles, and more preferably moderate halophiles.

[0070] Preferably, the halophilic microorganism is a halophilic microorganism that can grow under conditions containing a salt concentration of 0.5 mol / L-2.5 mol / L, preferably a halophilic microorganism that grows stably or grows best.

[0071] The toxin and antitoxin are on the same operon and are regulated by the same promoter.

[0072] Preferably, in the toxin-antitoxin system, hbpC is lethally toxic to cells, hbpB can inhibit the toxicity of hbpC and protect cells to maintain normal growth, and the toxin-antitoxin system can maintain the stability of the recombinant plasmid.

[0073] In a third aspect, a toxin-antitoxin system is provided for use in preparing a vector backbone, a recombinant plasmid and / or maintaining the stability of a recombinant plasmid in a halophilic microorganism.

[0074] Wherein, the toxin and antitoxin are derived from halophilic microorganisms. Preferably, the toxin and antitoxin are derived from the same or different halophilic microorganisms.

[0075] In a fourth aspect, a nucleic acid encoding the above-mentioned recombinant plasmid or the above-mentioned toxin-antitoxin system is provided.

[0076] In a fifth aspect, a method for constructing the above-mentioned recombinant plasmid is provided, and the construction method comprises connecting the toxin and antitoxin expression systems and the auxiliary sequences to obtain them.

[0077] Preferably, the method further comprises connecting to a target gene expression system.

[0078] Preferably, the method comprises the steps of connecting the replication initiation site and the replication initiation protein coding sequence.

[0079] Preferably, the method further comprises the step of connecting a resistance protein expression system.

[0080] Preferably, the resistance protein expression system comprises a gene encoding the resistance protein, and regulatory elements and / or regulatory proteins required for replication, transcription and / or expression of the gene encoding the resistance protein.

[0081] Preferably, the regulatory element includes a promoter that regulates the expression of the resistance protein.

[0082] Preferably, the resistance protein includes ampicillin, neomycin, chloramphenicol, tetracycline, kanamycin, apramycin or spectinomycin. The resistance protein is only used for plasmid screening and does not provide selection pressure for gene expression.

[0083] In a sixth aspect, a method for constructing a recombinant plasmid is provided, comprising connecting a toxin and an antitoxin expression system to a traditional plasmid.

[0084] The traditional plasmid is a plasmid that can replicate and express target genes in an organism, such as pUC plasmid, pET plasmid, pGEX plasmid, and pRO plasmid.

[0085] In a seventh aspect, a vector skeleton is provided, wherein the vector skeleton comprises a toxin and an antitoxin expression system.

[0086] The toxin and antitoxin expression system comprises a promoter, a toxin encoding gene and an antitoxin encoding gene, wherein the toxin and the antitoxin are on the same operon and are regulated by the same promoter.

[0087] The promoter may be any promoter disclosed in the prior art, as long as it can regulate the expression of toxins and antitoxins, and may preferably be a constitutive promoter or an inducible promoter.

[0088] In a specific embodiment of the present invention, the promoter may be derived from a halophilic microorganism.

[0089] In a specific embodiment, the promoter is hbpB / hbpC promoter.

[0090] Preferably, it also includes a replication initiation site and / or a replication initiation protein coding sequence, wherein the replication initiation site and / or the replication initiation protein can be any replication initiation site and / or replication initiation protein disclosed in the prior art, as long as it is ensured that the plasmid has a certain copy number, preferably a high copy number, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70 or more copies. In a specific embodiment of the present invention, the replication initiation site and / or the replication initiation protein are derived from halophilic microorganisms, preferably from endogenous plasmids of halophilic microorganisms.

[0091] Preferably, the elements required for transformation are also included.

[0092] Preferably, auxiliary sequences are also included.

[0093] In an eighth aspect, a method for expressing a target gene is provided, the expression method comprising using the above-mentioned recombinant plasmid.

[0094] Preferably, the expression method comprises:

[0095] 1) Knock out or knock down endogenous plasmids in microorganisms, or make endogenous plasmids non-functional;

[0096] 2) The recombinant plasmid is introduced into the microorganism obtained in 1) and cultured.

[0097] Preferably, the knockout or knockdown method comprises knocking out or knocking down hbpC while overexpressing hbpB in the microorganism.

[0098] Preferably, the microorganism obtained in step 1) does not contain endogenous plasmids and plasmids that may be introduced during the knockout or knockdown process.

[0099] Preferably, the introduction method includes chemical transformation, electroporation transformation and conjugation transformation.

[0100] In a specific embodiment, the introduction method is conjugation transformation.

[0101] Preferably, the halophilic microorganism is a bacterium.

[0102] Preferably, the halophilic microorganisms include, but are not limited to, Chromohalobacter, Halomonas, Halovibrio, Vibrio, Cobetia, Kushneria, Salicola, Halotela or Carnimonas.

[0103] Preferably, the genus Chromohalobacter includes but is not limited to Chromohalobacter marismortui, Chromohalobacter salexigens or Chromohalobacter beijerinckii; the genus Halomonas includes but is not limited to Halomonas bluephagenesis, Halomonas campaniensis, Halomonasdesiderata, Halomonas venusta or Halomonas alkaliantarctica; the genus Halovibrio includes but is not limited to Halovibrio denitrificans, Halovibrio mesolongii or Halovibrio variabilis; the genus Vibrio includes but is not limited to Vibrio natriegens; the genus Salicola includes but is not limited to Salicola marasensis or Salicola salis; the genus Cobetia includes but is not limited to Cobetiamarina or Cobetia crustatorum; the genus Kushneria includes but is not limited to Kushneria aurantia or Kushneria indalinina; the genus Halotela includes but is not limited to Halotela alkalilenta; the genus Carnimonas includes but is not limited to Carnimonas nigrificans.

[0104] Preferably, the halophilic microorganisms include mild halophiles, moderate halophiles or extreme halophiles, and more preferably moderate halophiles.

[0105] Preferably, the halophilic microorganism is a halophilic microorganism that can grow under conditions containing a salt concentration of 0.5 mol / L-2.5 mol / L, preferably a halophilic microorganism that grows stably or grows best.

[0106] Preferably, the expression method includes selection pressure such as adding antibiotics or not adding antibiotics.

[0107] In an eighth aspect, an engineered bacterium is provided, wherein the engineered bacterium comprises the above-mentioned recombinant plasmid, the above-mentioned toxin-antitoxin system or the above-mentioned nucleic acid.

[0108] Preferably, the endogenous plasmid is knocked out or knocked down in the engineered bacteria, or the endogenous plasmid is rendered non-functional.

[0109] Preferably, the knockout or knockdown method comprises overexpressing hbpB while knocking out or knocking down hbpC.

[0110] Preferably, the engineered bacteria include halophilic microorganisms;

[0111] Preferably, the halophilic microorganism is a bacterium.

[0112] Preferably, the halophilic microorganisms include, but are not limited to, Chromohalobacter, Halomonas, Halovibrio, Vibrio, Cobetia, Kushneria, Salicola, Halotela or Carnimonas.

[0113] Preferably, the genus Chromohalobacter includes but is not limited to Chromohalobacter marismortui, Chromohalobacter salexigens or Chromohalobacter beijerinckii; the genus Halomonas includes but is not limited to Halomonas bluephagenesis, Halomonas campaniensis, Halomonasdesiderata, Halomonas venusta or Halomonas alkaliantarctica; the genus Halovibrio includes but is not limited to Halovibrio denitrificans, Halovibrio mesolongii or Halovibrio variabilis; the genus Vibrio includes but is not limited to Vibrio natriegens; the genus Salicola includes but is not limited to Salicola marasensis or Salicola salis; the genus Cobetia includes but is not limited to Cobetiamarina or Cobetia crustatorum; the genus Kushneria includes but is not limited to Kushneria aurantia or Kushneria indalinina; the genus Halotela includes but is not limited to Halotela alkalilenta; the genus Carnimonas includes but is not limited to Carnimonas nigrificans.

[0114] Preferably, the halophilic microorganisms include mild halophiles, moderate halophiles or extreme halophiles, and more preferably moderate halophiles.

[0115] Preferably, the halophilic microorganism is a halophilic microorganism that can grow under conditions containing a salt concentration of 0.5 mol / L-2.5 mol / L, preferably a halophilic microorganism that grows stably or grows best.

[0116] In a ninth aspect, a method for preparing the above-mentioned engineered bacteria is provided, the preparation method comprising introducing a recombinant plasmid into the engineered bacteria.

[0117] Preferably, the introduction is chemical transformation, electroporation transformation or conjugation transformation.

[0118] In the tenth aspect, a method is provided for using the above-mentioned recombinant plasmid, the above-mentioned vector backbone, the above-mentioned toxin-antitoxin system, the above-mentioned nucleic acid or the above-mentioned engineered bacteria in gene editing or biosynthesis.

[0119] The gene editing is halophilic microorganism gene editing.

[0120] Preferably, the biosynthetic products include but are not limited to PHA, ectoine, amino acids, inositol, 1,3-propanediol, 3-hydroxypropionic acid, 1,5-pentanediamine, 5-aminolevulinic acid, lipase, amylase, saccharification enzyme, protease, cellulase, esterase, glucanase, pectinase, phospholipase, gelatinase, nuclease, microtubule protein FtsZ, cytoskeletal protein MreB, cell division regulatory factor MinCD, lactose operon repressor protein LacI, tetracycline operon repressor protein TetR, sgRNA used in CRISPRi / CRISPRa or one or more of lycopene.

[0121] Preferably, the PHA is selected from 3-hydroxybutyric acid homopolymer PHB, 3-hydroxybutyric acid and 4-hydroxybutyric acid copolymer P3HB4HB, 3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvaleric acid terpolymer PHBV4HB, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid copolymer PHBHHx, homopolymer or copolymer of 3-hydroxypropionic acid, the homopolymer of 3-hydroxypropionic acid is P3HP, and the copolymer of 3-hydroxypropionic acid is P(3HB-co-3HP) or PHBHP.

[0122] In the eleventh aspect, a fermentation method is provided, which comprises fermenting and culturing the above-mentioned engineered bacteria.

[0123] In a twelfth aspect, a method for product biosynthesis is provided, wherein the method comprises culturing the above-mentioned engineered bacteria.

[0124] No antibiotics were added to the cultures.

[0125] Through the above technical solution, the present invention has the following advantages:

[0126] This achievement, through the discovery and mechanism research of the endogenous toxin-antitoxin system of halophilic microorganisms, established an extrachromosomal expression system suitable for halophilic microorganisms, and achieved stable and efficient expression of exogenous target genes without the need for selection pressure such as external antibiotics.

[0127] The terms “comprising” or “including” described in the present invention are open-ended terms. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0128] The "homology" mentioned in the present invention refers to that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39 %, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity.

[0129] The "halophilic microorganism" of the present invention is a microorganism whose optimal salt concentration for growth is greater than 0.2 mol / L. According to the need of microorganisms for salt, they can be divided into non-halophilic microorganisms, weak halophilic microorganisms, moderate halophilic microorganisms, and extreme halophilic microorganisms.

[0130] The "mild halophilic bacteria" of the present invention can grow under the condition of 0.2 mol / L-0.5 mol / L salt concentration, preferably stably grow or grow best, when the culture conditions other than salt concentration of the bacteria are kept consistent;

[0131] "Moderately halophilic bacteria" refers to bacteria that can grow under conditions of 0.5 mol / L to 2.5 mol / L salt concentration, preferably with stable growth or the best growth, when other culture conditions except salt concentration of the bacteria are kept the same;

[0132] "Extreme halophiles" are those that can grow under conditions of 2.5 mol / L-5.2 mol / L salt concentration, preferably with stable growth or the best growth, when other culture conditions except salt concentration of the bacteria are kept the same.

[0133] Wherein, the "salt" may be a salt commonly used in the prior art for microbial culture, and the salt is an inorganic salt, and the inorganic salt is used to maintain the osmotic pressure required by the microorganism. For example, potassium salt and / or sodium salt. The potassium salt includes, but is not limited to, one or more of potassium chloride, potassium sulfate, potassium phosphate, potassium citrate, potassium acetate, potassium gluconate, potassium nitrate, potassium carbonate, potassium bicarbonate or potassium dihydrogen phosphate. The sodium salt includes, but is not limited to, one or more of sodium chloride, sodium sulfate, sodium phosphate, sodium citrate, sodium acetate, sodium gluconate, sodium nitrate, sodium carbonate, sodium bicarbonate or dihydrogen phosphate. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] Figure 1 :Effects of orfF2 and orfF3 overexpression on the growth of Halomonas.

[0135] Figure 2 : Toxin encoding gene hbpC knockout system, in which, Figure 2 A: pSEVA321 plasmid expresses Cas9, Figure 2 B: pSEVA341 plasmid expresses gRNA and homology arms, Figure 2 C: gRNA is designed on the toxin encoding gene hbpC, oriT: conjugation transfer initiation site; HR (Homologous arm): homologous arm; CmR: chloramphenicol resistance protein; KanR: kanamycin resistance protein; SpeR: spectinomycin resistance protein.

[0136] Figure 3 : Knockout results of the toxin encoding gene hbpC using CRISPR / Cas9 editing technology, Figure 3 A directly knocks out hbpC, Figure 3 B. Overexpress hbpB and knock out hbpC.

[0137] Figure 4 : Knocking out the stable endogenous plasmid pHbCP does not affect the growth rate of Halomonas.

[0138] Figure 5 : Expression of toxin-antitoxin system in Halomonas H.campaniensis.

[0139] Figure 6 : The toxin-antitoxin system hbpB / hbpC is the key stabilizing element of pHbPBC.

[0140] Figure 7 :Applicable to the plasmid replication initiation site and replication initiation protein of Halomonas and Escherichia coli, Figure 7 A is the replication origin site and replication initiation protein from the endogenous plasmid pRO1600 of Pseudomonas aeruginosa; Figure 7B The replication origin site and replication initiation protein from the endogenous plasmid pHbCP of Halomonas bluephagenesis; Figure 7 C is suitable for the replication origin of Escherichia coli S17-1 strain; Figure 7 D junction transfer initiation site.

[0141] Figure 8 : A stable recombinant plasmid vector pHbPBC with the exogenous target protein sfGFP encoding gene inserted, PromoterHbpBC-HbpB-HbpC: toxin-antitoxin stable element expression system; pMB1 ori: replication origin site suitable for Escherichia coli S17-1 strain; pHbCP ori: replication origin site suitable for Halomonas; pHbCP RepB: replication initiation protein; oriT: conjugative transfer origin site; rrnB T1 terminator-J23119 promoter-RBS-sfGFP-lambdat0 terminator: sfGFP expression system; KanR: kanamycin resistance protein; SpeR: spectinomycin resistance protein.

[0142] Fig. 9 :sfGFP encoding gene is integrated into the genome, Fig. 9 A: pSEVA321 plasmid expressing Cas9; Fig. 9 B: pSEVA341 plasmid expresses Grna, oriT: conjugative transfer initiation site; HR-L / R: upstream and downstream homology arms of IR 00227-00228 site; rrnB T1 terminator-J23119 promoter-RBS-sfGFP-lambda t0 terminator: sfGFP expression system; CmR: chloramphenicol resistance protein; KanR: kanamycin resistance protein; SpeR: spectinomycin resistance protein.

[0143] Fig.10 : sfGFP encoding gene was inserted into the vector plasmid pSEVA341, pMB1 ori: replication origin site suitable for Escherichia coli S17-1 strain; pRO1600 oriⅤ: replication origin site suitable for Halomonas; pRO1600 RepB: replication initiation protein; oriT: conjugative transfer origin site; rrnB T1 terminator-J23119 promoter-RBS-sfGFP-lambda t0 terminator: sfGFP expression system; KanR: kanamycin resistance protein; SpeR: spectinomycin resistance protein.

[0144] Fig.11:The target gene can be integrated into the genome and remain stable without loss. The square icon indicates the average proportion of fluorescent cells in the H.bluephagenesis sfGFP strain; the circular icon indicates the average proportion of fluorescent cells in the H.bluephagenesis LCP strain.

[0145] Fig.12 : The stability of pSEVA341 depends on the selection pressure of antibiotics. The diamond icon indicates the average proportion of fluorescent cells in the pSEVA341-sfGFP group under antibiotic-free culture conditions; the triangle icon indicates the average proportion of fluorescent cells in the pSEVA341-sfGFP group under antibiotic culture conditions.

[0146] Fig.13 : The stability of pHbPBC does not depend on the selection pressure of antibiotics. The circular icon indicates the average proportion of fluorescent cells in the pHbPBC-sfGFP group under antibiotic-free culture conditions; the square icon indicates the average proportion of fluorescent cells in the pHbPBC-sfGFP group under antibiotic culture conditions.

[0147] Fig.14 : Comparison of the stability of target gene expression strategies. The square icon indicates the average proportion of fluorescent cells in the genome integration group under antibiotic-free culture conditions; the circular icon indicates the average proportion of fluorescent cells in the pHbPBC-sfGFP group under antibiotic-free culture conditions; the triangular icon indicates the average proportion of fluorescent cells in the pSEVA341-sfGFP group under antibiotic culture conditions.

[0148] Fig.15 :High-level expression of the target protein can be achieved using the pHbPBC stable vector. The square icon indicates the average fluorescence intensity of single cells in the genome integration group under antibiotic-free culture conditions; the triangle icon indicates the average fluorescence intensity of single cells in the pSEVA341-sfGFP group under antibiotic culture conditions; the circle icon indicates the average fluorescence intensity of single cells in the pHbPBC-sfGFP group under antibiotic-free culture conditions.

[0149] Fig.16 :The addition of antibiotics has a negative impact on the expression level of the target protein. The circular icon indicates the average fluorescence intensity of single cells in the pHbPBC-sfGFP group under antibiotic-free culture conditions; the square icon indicates the average fluorescence intensity of single cells in the pHbPBC-sfGFP group under antibiotic culture conditions.

[0150] Fig.17 : Expression of toxin-antitoxin system in V. natriegens.

[0151] Fig.18: Expression of toxin-antitoxin systems from H. venusta and H. alkaliantarctica.

[0152] Fig.19 : Expression of toxin-antitoxin system in moderately halophilic environment. DETAILED DESCRIPTION

[0153] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0154] Table 1 Sources of bacteria used in the examples

[0155]

[0156] Example 1: Functional verification of the toxin-antitoxin system in halophilic microorganisms

[0157] 1. In the toxin-antitoxin system, hbpC is lethally toxic to cells, and hbpB can inhibit the toxicity of hbpC and protect cells to maintain normal growth.

[0158] The bacterial culture was carried out in a 96-well plate with a 200uL system in an ELISA instrument, and the absorbance value (OD) of the bacterial solution at a wavelength of 600nm was monitored in real time. 600 ) to characterize the growth of bacteria. Taking Halomonas as an example among halophilic microorganisms, sfGFP, orfF2 (i.e., hbpB) sequence, orfF3 (i.e., hbpC) sequence, and orfF2+3 (i.e., hbpB+C) full-length sequence encoding green fluorescent protein were overexpressed in Halomonas H.bluephagenesis TD01. Among them, the sequence of hbpB is shown in SEQ ID NO: 1, the sequence of hbpC is shown in SEQ ID NO: 2, the amino acid sequence of the corresponding HbpB protein is shown in SEQ ID NO: 3, and the amino acid sequence of the HbpC protein is shown in SEQ ID NO: 4.

[0159] SEQ ID NO: 1

[0160] atggccatccgtaaacttagcgacctcaaacccgtgtttagcagtgagcgtgtcaccgagtggcagcccgtacccctggggccgcg

[0161] ctaccgttatgagcgagatcgggctgcggtcgggcaggaaatcacacctggcagtgagcagtacgaatggtacgtgttggcgaaa

[0162] aacgacctggctaacgccaagcgcaaagtctttgcgttgatcacagaggaatatctgtaa

[0163] SEQ ID NO: 2

[0164] atggcgcgcacgcgatccgtacaggccgctgaggcgcatttgtggctagaggtgttgcttacgtatgcgtttggtacttcgcctgttca

[0165] gcgggccgcacggcttgatttgttaggtgttgcccatgatgccactgcgtatccagacgacataccgcattggcgattggctcaactg

[0166] ctgctggcctgggtagagaaatatgtgtcaccggaggattggaggaggcttcaggcgcgagttaggaagcggcgcagccaatagSEQ ID NO: 3

[0167] MAIRKLSDLKPVFSSERVTEWQPVPLGPRYRYERDRAAVGQEITPGSEQYEWYVLA

[0168] KNDLANAKRKVFALITEEYL

[0169] SEQ ID NO: 4

[0170] MARTRSVQAAEAHLWLEVLLTYAFGTSPVQRAARLDLLGVAHDATAYPDDIPHWR

[0171] LAQLLLAWVEKYVSPEDWRRLQARVRKRRSQ

[0172] The results are as Figure 1As shown, compared with the strain overexpressing green fluorescent protein, the strain overexpressing the orfF2 sequence maintained a normal growth rate, without growth slowdown or death. At the same time, it may be because the orfF2 sequence is short in length, only 231bp, so the molecular weight of the encoded product is also small. Compared with the full-length 238aa sfGFP (protein as shown in SEQ ID NO: 9, encoding sequence as shown in SEQ ID NO: 10), the metabolic pressure on the bacteria is small, so it has a relatively slightly faster growth rate than the control group. On the contrary, the expression of the orfF3 sequence directly caused the death of bacteria, that is, the orfF3 expression product was lethal to Halomonas H. bluephagenesis TD01. It is worth noting that overexpression of the orfF2 / orfF3 system does not cause lethal consequences to bacteria. When orfF2 and orfF3 are located on the same operon, regulated by the same promoter, and overexpressed in bacteria at the same time, orfF2 can inhibit the damage of orfF3 to cells, allowing bacteria to grow and reproduce normally. In other words, orfF2 / orfF3 conforms to the action mode of the toxin-antitoxin system. In the toxin-antitoxin system, hbpC is lethal to cells, and hbpB can inhibit the toxicity of hbpC, protecting cells from normal growth.

[0173] SEQ ID NO: 9

[0174] MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVP

[0175] WPTLVTTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAE

[0176] VKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIR

[0177] HNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK*

[0178] SEQ ID NO: 10

[0179] atgcgtaaaggcgaagagctgttcactggtgtcgtccctattctggtggaactggatggtgatgtcaacggtcataagttttccgtgcg

[0180] tggcgagggtgaaggtgacgcaactaatggtaaactgacgctgaagttcatctgtactactggtaaactgccggtaccttggccgac

[0181] tctggtaacgacgctgacttatggtgttcagtgctttgctcgttatccggaccatatgaagcagcatgacttcttcaagtccgccatgcc

[0182] ggaaggctatgtgcaggaacgcacgatttcctttaaggatgacggcacgtacaaaacgcgtgcggaagtgaaatttgaaggcgat

[0183] accctggtaaaccgcattgagctgaaaggcattgactttaaagaagacggcaatatcctgggccataagctggaatacaattttaac

[0184] agccacaatgtttacatcaccgccgataaacaaaaaaatggcattaaagcgaattttaaaattcgccacaacgtggaggatggcagc

[0185] gtgcagctggctgatcactaccagcaaaacactccaatcggtgatggtcctgttctgctgccagacaatcactatctgagcacgcaa

[0186] agcgttctgtctaaagatccgaacgagaaacgcgatcatatggttctgctggagttcgtaaccgcagcgggcatcacgcatggtatg

[0187] gatgaactgtacaaataa

[0188] 2. The toxin - antitoxin system hbpB / hbpC is a key element for maintaining the stability of endogenous plasmids.

[0189] Halophilic microorganisms contain an endogenous plasmid, pHbCP, which can be stably replicated and propagated as the bacteria grow and reproduce without the need for additional selection pressure. pHbCP has a toxin-antitoxin system, hbpB / hbpC. In order to explore the role of the toxin-antitoxin system, hbpB / hbpC, on pHbCP, a knockout experiment of the toxin encoding gene hbpC was conducted.

[0190] First, taking H. bluephagenesis TD01 as an example, CRISPR / Cas9 editing technology was used to select hbpC as the target sequence on the endogenous plasmid pHbCP to design gRNA (SEQ ID NO: 25) and directly knock it out ( Figure 2 A, B). The results showed that this strategy could not successfully knock out the toxin encoding gene hbpC, and the experiment failed to obtain an effective single clone ( Figure 3 A). Secondly, the CRISPR / Cas9 editing technology was also used, and the same gRNA was used as for direct knockout, but hbpB was overexpressed in Halomonas at the same time during the knockout ( Figure 2 A, C). The experiment proved that this knockout strategy was effective, and a large number of strains with normal growth were obtained. However, colony PCR and sequencing confirmed that the toxin encoding gene hbpC on the stable plasmid pHbCP was not knocked out in these strains, but the endogenous stable plasmid pHbCP was lost ( Figure 3 B). It is speculated that this phenomenon is due to the fact that CRISPR / Cas9 editing itself targets the toxin encoding gene hbpC, destroying the stabilizing elements of the endogenous stable plasmid pHbCP. Therefore, when Cas9 cuts pHbCP at the corresponding position, the homologous recombination step based on the homology arm fails to complete, and the linearized pHbCP is directly lost during the bacterial growth and reproduction process. Therefore, the knockout of the toxin encoding gene hbpC in this experiment is actually the knockout of the stable plasmid pHbCP.

[0191] SEQ ID NO: 25 (gF3)

[0192] atccagacgacataccgcat

[0193] This result is consistent with the classic "postsegregational killing (PSK)" model. Under appropriate growth conditions, the toxin is relatively stable in the cell, while the antitoxin is relatively unstable. When the plasmid is lost, the antitoxin is first degraded, and the toxin loses its corresponding inhibition, leading to cell death. In other words, cells that fail to inherit the plasmid copy will be killed by the toxin-antitoxin module (Gerdes et al., 1986; Ogura and Hiraga, 1983; Unterholzner et al., 2013). In the knockout experiment conducted in Halomonas, CRISPR / Cas9 editing caused a double-strand break gap on the endogenous plasmid pHbCP. pHbCP could not replicate and propagate normally as the bacteria grew and reproduced. The hbpB expression product on it was first degraded, and the concentration of the free hbpC expression product increased, exerting a toxic effect and causing bacterial death. Therefore, when pHbCP was directly knocked out using CRISPR / Cas9 editing technology, all edited bacteria died, so no effective single clone was obtained. When hbpB was continuously overexpressed while pHbCP was knocked out, the endogenous hbpB expression products that were first degraded were effectively supplemented, and these exogenous overexpression products continued to play a role in inhibiting the toxicity of endogenous hbpC. Therefore, the endogenous plasmid pHbCP was successfully knocked out, and the strain with pHbCP knocked out grew normally in large quantities on the plate.

[0194] Therefore, the toxin-antitoxin system hbpB / hbpC maintains the stability of the endogenous plasmid pHbCP in halophilic microorganisms and is the key element for the stable replication and propagation of pHbCP as the bacteria grow and reproduce.

[0195] Example 2: Construction of a recombinant stable plasmid vector based on the toxin-antitoxin system hbpB / hbpC

[0196] 1. Construction of the LCP of the plasmid-free Halomonas bluephagenes

[0197] This achievement uses the toxin-antitoxin system hbpB / hbpC to construct a recombinant stable plasmid vector suitable for halophilic microorganisms as an extrachromosomal expression system to enrich the editing and transformation tools available for halophilic microorganisms. Taking Halomonas H.bluephagenesis as an example, in order to facilitate the construction and improve its stability, the endogenous plasmid pHbCP that adopted the same stabilization strategy in the original strain was first knocked out. The specific knockout sequences used are shown in Table 2. After the knockout was successful, the strain was cultured for 48 hours without adding antibiotics to remove the two exogenous plasmids introduced during the CRISPR / Cas9 editing process. At this point, a strain free of plasmids (including the stable endogenous plasmid pHbCP, the exogenous plasmid pSEVA321 used for editing in Example 1 ( Figure 2 A) and pSEVA341( Figure 2 C), which will be named Halomonas bluephagenesis LCP (H. bluephagenesis LCP) hereinafter.

[0198] Table 2 gRNA sequences used for knockout

[0199]

[0200]

[0201] The growth rate of H. bluephagenesis LCP was measured using a medium containing 1.03 mol / L NaCl. The growth curve showed that knocking out the stable endogenous plasmid pHbCP had no significant effect on the growth rate of H. bluephagenesis ( Figure 4 ), H.bluephagenesis LCP maintained normal growth under suitable culture conditions.

[0202] In summary, it can be considered that under normal suitable culture conditions, knocking out the stable endogenous plasmid pHbCP has no significant effect on the growth and metabolism of halophilic microorganisms. Halomonas H. bluephagenesis LCP is a chassis strain with good growth conditions and can be used for subsequent detection of stable recombinant plasmid vectors and production experiments of related products.

[0203] 2. Construction of recombinant stable plasmid vector based on toxin-antitoxin system hbpB / hbpC

[0204] After identifying the key stabilizing elements and constructing a suitable chassis bacteria, this work selected other important elements to construct a recombinant stable plasmid vector based on the toxin-antitoxin system hbpB / hbpC, which is named pHbPBC below, including the replication start site, replication start protein, conjugation transfer start site, and construction of screening markers, etc.

[0205] 3. Toxin-antitoxin system hbpB / hbpC is a key stabilizing element

[0206] This study discovered a new toxin-antitoxin system hbpB / hbpC from the endogenous plasmids of halophilic microorganisms. This genetic module has the function of maintaining the stable propagation of plasmids without the addition of antibiotics and other selection pressures. Therefore, the toxin-antitoxin system hbpB / hbpC was used as the key stabilizing element for constructing the recombinant plasmid vector pHbPBC. Since hbpB and hbpC are located in the same operon, and the toxin-antitoxin complex often interacts directly with the shared promoter, the original promoter was retained when using the hbpB / hbpC module in this study. Similarly, due to the lack of relevant data and databases on halophilic microorganisms, it is difficult to directly determine the promoter sequence of the toxin-antitoxin module. Therefore, this study combined the prokaryotic promoter computational models reported in many literatures (Cassiano and Silva-Rocha, 2020; Mishra et al., 2020; Rahman et al., 2019; Solovyev and Salamov, 2013) to predict the promoter sequence of hbpB / hbpC. Based on the prediction results of various models, this study selected the 250bp upstream of hbpB / hbpC as the promoter region ( Figure 6 ), wherein the sequences of the promoter and RBS region are as shown in SEQ ID NO: 5. In addition, other promoter and RBS region regulatory sequences that increase the expression level of hbpB / hbpC can also be selected, such as the sequence shown in SEQ ID NO: 11, wherein the partial sequence of the promoter is SEQ ID NO: 22, and the regulatory sequence of the RBS region is SEQ ID NO: 23.

[0207] SEQ ID NO: 5

[0208] aattgcccctcaacagacgtaggtatccgtacctacattctgactctctaagggacagcaatctttctaattgataggtttgctgattccg

[0209] agacaagggcctgctctcctatggagacaggcccttttctttttggggaatttaaaaactcaagataatgaggataatcttgagttttgg

[0210] gccataagcgtgaaaacttcacgcttatgatccaagataagaggactcacatcaatcagggcaccctcc

[0211] SEQ ID NO: 11

[0212] ttgacagctagctcagtcctaggtataatactagtaataattttgtttaactttaagaaggaga

[0213] SEQ ID NO: 22

[0214] ttgacagctagctcagtcctaggtataatactagt

[0215] SEQ ID NO: 23

[0216] tttgtttaactttaagaaggaga

[0217] 4. Replication initiation site and replication initiation protein of recombinant stable plasmid vector

[0218] To improve stability, we first screened the replication origin site and replication origin protein coding region of the endogenous plasmid pHbCP, and used them as retention elements on the stable recombinant plasmid vector pHbPBC to ensure replication and subculture in H. bluephagenesis LCP.

[0219] The 861 bp repB gene (SEQ ID NO: 7) is the only sequence with known function on the endogenous plasmid pHbCP, except for the newly discovered toxin-antitoxin system hbpB / hbpC in this study. It encodes a replication initiation protein RepB (SEQ ID NO: 8), which is also found in other halophilic microorganisms.

[0220] SEQ ID NO: 7

[0221] atgcacgatgcccaaatatataaagccaatgcactgatcgaagcgtcttatcgtctcagtatttatgagcaacgcgtcattctggcatgtatttctaaggtgcgtagggatgagcccttgaccgaccagaaactctaccgtgttagcgcccaggaaattgctgacatgagtggcacccagatcggaaccgcttaccaaaatcttaaggccgcatctgagcgtttatttgaacgccgtgtttctctacacgcctctcctaatgggggcagcaaggtcaaggtgcgtctgactcgctgggtgcaaacaatcgaatatgacgaaggaggcggggctgtctcactgcgctttggcaccgacatggtgccgtatttatcgcagctaaccgaacaattcactcgttacgccttggcagacattgccaaaatgaccagttctcacgcggttcgtctctatgagctgctggctcaatggcgaggtacaggggaacgatcagtttccatcgagtggttgcgtgaggcattccagcttgaggatcgttactccaatattcgtgactttaaacggtgggtaattgaaccagccgttaaccaagtcaacgagtacagccccctatggtgcaaatggagtcaacggaagacggggcggcgagtatctcatctaactttcacctttggtgaaaaagctaaaactatccctcctaaggcccgccaagcaaaaactaagggcaaagcaggggaagtctctcagaacagctcaggggcgctctatgggattcctcgcgccgtcatcgagagtaaagcacgccctggcgagggctatgaagataccgcgttgcggttgctgagcgaagctaagggctag

[0222] SEQ ID NO:8

[0223] MHDAQIYKANALIEASYRLSIYEQRVILACISKVRRDEPLTDQKLYRVSAQEIADMSGTQIGTAYQNLKAASERLFERRVSLHASPNGGSKVKVRLTRWVQTIEYDEGGGAVSLRFGTDMVPYLSQLTEQFTRYALADIAKMT SSHAVRLYELLAQWRGTGERSVSIEWLREAFQLEDRYSNIRDFKRWVIEPAVNQVNEYSPLWCKWSQRKTGRRVSHLTFTFGEKAKTIPPKARQAKTKGKAGEVSQNSSGALYGIPRAVIESKARPGEGYEDTALRLLSEAKG*

[0224] However, due to the incomplete study of the genetic background of Halomonas H.bluephagenesis TD01, the replication initiation site sequence of its endogenous plasmid is still unknown, and attempts to compare the current replication initiation site public database and use the prediction method reported in the literature have failed to determine the replication initiation site region on pHbCP. Therefore, pHbCP was connected to the pSEVA341 backbone, but the original replication initiation site sequence suitable for Halomonas was deleted from the pSEVA341 vector, the broad-host replication initiation site pRO1600 oriⅤ (SEQ ID NO: 12) from the endogenous plasmid of Pseudomonas aeruginosa, and the corresponding replication initiation protein pRO1600 Rep coding region (SEQ ID NO: 13). The corresponding expressed replication initiation protein pRO1600 Rep sequence is shown in SEQ ID NO: 24 ( Figure 7 A); then the pHbCP sequence was segmented and deleted, first with 500 bp as a segment, and the segment area was gradually reduced. It was found that the approximately 360 bp sequence upstream of the gene encoding the replication initiation protein RepB was essential for the retention and replication of the plasmid in Halomonas. Therefore, this fragment sequence was used as the replication initiation site pHbCP ori (SEQ ID NO: 6) and retained to construct a stable recombinant plasmid vector pHbPBC ( Figure 7 B).

[0225] SEQ ID NO: 6

[0226] tttagcgaccagctaacgccgtaggtggggccagccgcgaagcggctcgacagccccacctactcgacttgtcgaggcatttttaggtttaggcctcgcgaagcgaggggcggagggaggaggaacgacgaccggagggtaggcgagcgcgtacgcgctcttttatgtacttttcccttttcccttttaagctttataagatgctgaattttaagtattaataacgttaaaaggggagagattgatggttaaaagaggagggattgatggttaaaagaggagggattgatggttgtaagggagagggattgatggttaagtgtgagccaaataaatcactcacattatagggtaggcaaatccat

[0227] SEQ ID NO:12

[0228] tccgcctcgataccctgattactcgcttcctgcgccctctcaggcggcgataggggactggtaaaacggggattgcccagacgcctcccccgccccttcaggggcacaaatgcggccccaacggggccacgtagtggtgcgttttttgcgtttccacccttttcttccttttcccttttaaaccttttaggacgtctacaggccacgtaatccgtggcctCtagagtttaaaaagggacggatttgttgccattaagggacggatttgttgttaagaagggacggatttgttgttgtaaagggacggatttgttgtattgtgggacgcagatacagtgtccccttatacaca

[0229] SEQ ID NO:13

[0230] gtggcctcacccccaatggtttacaaaagcaatgccctggtcgaggccgcgtatcgcctcagtgttcaggaacagcggatcgttctggcctgtattagccaggtgaagaggagcgagcctgtcaccgatgaagtgatgtattcagtgacggcggaggacatagcgacgatggcgggtgtccctatcgaatcttcctacaaccagctcaaagaagcggccctgcgcctgaaacggcgggaagtccggttaacccaagagcccaatggcaaggggaaaagaccgagtgtgatgattaccggctgggtgcaaacaatcatctaccgggagggtgagggccgtgtagaactcaggttcaccaaagacatgctgccgtacctgacggaactcaccaaacagttcaccaaatacgccttggctgacgtggccaagatggacagcacccacgcgatcaggctttacgagctgctcatgcaatgggacagcatcggccagcgcgaaatagaaattgaccagctgcgaaagtggtttcaactggaaggccggtatccctcgatcaaggacttcaagttgcgagtgcttgatccagccgtgacgcagatcaacgagcacagcccgctacaggtggagtgggcgcagcgaaagaccgggcgcaaggtcacacatctgttgttcagttttggaccgaagaagcccgccaaggcggtgggtaaggccccagcgaagcgcaaggccgggaagatttcagatgctgagatcgcgaaacaggctcgccctggtgagacatgggaagcggcccgcgctcgactaacccagatgccgctggatctggcctag

[0231] SEQ ID NO:24

[0232] MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEVMYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQTIIYREGEGRVELRFTKDMLPYLTELTKQFTKYAL ADVAKMDSTHAIRLYELLMQWDSIGQREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHLLFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA

[0233] In addition, in order to transform the plasmid into Halomonas, chemical transformation, electroporation transformation and conjugation transformation are currently the three most commonly used methods for transferring exogenous DNA into bacteria. This embodiment uses conjugation as an example to transform the plasmid into Halomonas. Conjugation transformation also requires the addition of other replication initiation sites on pHbPBC.

[0234] In order to construct a shuttle plasmid that is adaptable to multiple hosts, it can be replicated and propagated in both Halomonas and the E. coli S17-1 strain used for conjugation transformation. Therefore, the optimized replication origin pMB1 ori from the pUC series plasmids was selected as the replication origin of the stable recombinant plasmid vector pHbPBC in the E. coli S17-1 strain, and pMB1 ori relies on the replication initiation protein encoded by the host bacterial genome, and no additional addition is required ( Figure 7 C). oriT is the starting point of plasmid transfer during conjugation and is also an essential sequence on pHbPBC ( Figure 7 D).

[0235] In addition, other high copy number regulatory element sequences may also be selected as needed, such as the broad-host replication origin site pRO1600 oriV (SEQ ID NO: 12), and the corresponding replication initiation protein pRO1600 Rep coding region (SEQ ID NO: 13).

[0236] 5. Other sequences

[0237] In order to facilitate the subsequent insertion of exogenous target genes into the stable recombinant plasmid vector pHbPBC to achieve the expression of the target product, the kanamycin resistance gene kan was added to pHbPBC. R and spectinomycin resistance gene spe R, respectively, for positive clone screening on Escherichia coli S17-1 strain and Halomonas H.bluephagenesis LCP strain plates. This resistance screening marker is only used in the plasmid construction and conjugation transformation stage. No antibiotics need to be added in the subsequent exogenous target gene expression and production stage. pHbPBC with the target gene fragment inserted will rely on its own toxin-antitoxin system hbpB / hbpC to maintain stable replication and propagation in the host cell.

[0238] In addition, in order to facilitate the subsequent stability test and expression level detection of the stable recombinant plasmid vector pHbPBC, the sfGFP gene encoding green fluorescent protein was inserted into the stable vector as the target gene fragment.

[0239] Thus, this study successfully constructed a stable recombinant plasmid vector pHbPBC suitable for halophilic microorganisms (the stable recombinant plasmid vector for inserting the target gene and resistance gene is shown in Figure 8 ).

[0240] Example 3: Stability Characterization of Recombinant Stable Plasmid Vector pHbPBC

[0241] 1. Strategy for expression of exogenous target genes

[0242] In order to characterize the stability of the recombinant plasmid vector pHbPBC in halophilic microorganisms and the expression level after the insertion of exogenous target genes, this study adopted three exogenous target gene expression strategies for comparison.

[0243] Strategy 1( Fig. 9 ): Integrate the exogenous target gene into the genome of the H. bluephagenesis LCP strain, culture it under antibiotic-free conditions, and measure the target cell ratio and target protein expression level respectively. This is the most commonly used strategy for engineering bacteria. The transformation has good stability and does not require additional selection pressure. However, the transformation cycle is long, the operation is cumbersome, and multiple operations are likely to interfere with the stability of the genome;

[0244] Strategy 2 Fig.10 ): Insert the exogenous target gene into a general plasmid expression vector, introduce it into the Halomonas H. bluephagenesis LCP strain through conjugation transformation, culture it with and without antibiotics, and measure the plasmid retention ratio and target protein expression level. This strategy is also used in the transformation and actual production of engineered bacteria. It is simple and flexible to operate and does not destroy the integrity of the genome. However, since it requires additional selection pressure such as antibiotics to maintain the stable existence of the expression vector in the bacteria, it increases the production cost and is harmful to the ecological environment;

[0245] Strategy 3 Figure 8): The exogenous target gene was inserted into the recombinant plasmid vector pHbPBC constructed in this study, and introduced into the Halomonas H. bluephagenesis LCP strain by conjugation transformation. The strain was cultured with and without antibiotics, and the plasmid retention ratio and target protein expression level were determined.

[0246] 2. Stable replication of the recombinant plasmid vector pHbPBC in H. bluephagenesis LCP

[0247] This achievement uses flow cytometry to detect whether there is fluorescence in a single cell and to measure the average fluorescence intensity of a single cell. In the three exogenous target gene expression strategies described above, the green fluorescent protein sfGFP encoding gene of strategy 1 is integrated into the bacterial genome, and the green fluorescent protein sfGFP encoding gene of strategies 2 and 3 is located on the plasmid, so the cells carrying the plasmid will emit fluorescence, that is, the ability to detect fluorescence is used to characterize that the plasmid in the cell is still stably retained.

[0248] The H.bluephagenesis LCP strains modified by three different strategies were subcultured for 24 h × 7 days using a culture medium containing 1.03 mol / L NaCl, i.e. the bacterial culture was transferred at a ratio of 1:1000 every 24 hours for a total of 7 days; the proportion of cells emitting fluorescence and the average fluorescence intensity of individual cells were recorded by flow cytometry every 24 hours; each group had three biological parallels, and 30,000 cell data were recorded for each sample each time.

[0249] First, for the sample group using strategy 1, the unmodified H. bluephagenesis LCP without the green fluorescent protein sfGFP encoding gene was used as a control, and basically no fluorescent cells were detected ( Fig.11 ); in the H.bluephagenesis sfGFP group, in which the green fluorescent protein sfGFP coding gene was integrated into the genome, the average percentage of fluorescent cells remained above 95% after 7 consecutive days of transfer culture. This result was in line with expectations, that is, the target gene integrated into the genome can remain stable without loss ( Fig.11 , Table 3).

[0250] Table 3 The target gene can be integrated into the genome and remain stable without loss

[0251]

[0252]

[0253] For strategy 2, pSEVA341-sfGFP was introduced into H. bluephagenesis LCP by conjugation transformation, and the cells were transferred and subcultured for 7 days with and without antibiotics. The results of flow cytometry analysis showed that for the antibiotic group, the average percentage of fluorescent cells decreased slightly within 7 days, from 99.4% on Day 1 to 91.9% on Day 7, and remained at a high level of more than 90% overall, but slightly lower than the 95.8% on Day 7 of the H. bluephagenesis sfGFP group in strategy 1; in the group without antibiotics, the average percentage of fluorescent cells dropped sharply from 99.5% on Day 1 to 25.6% on Day 7, and nearly three-quarters of the cells could no longer detect fluorescence, that is, the recombinant plasmid pSEVA341-sfGFP had been lost ( Fig.12 , Table 4).

[0254] Table 4 The stability of pSEVA341 depends on the selective pressure of antibiotics

[0255]

[0256] This proves that for general expression plasmid vectors without special stabilizing elements, such as pSEVA341, their stability in host cells mainly depends on additional selection pressure. For example, the corresponding antibiotics provide a survival advantage for cells retaining the plasmid, and this level of stability is not as good as genome integration. Under culture conditions without additional selection pressure, general expression plasmid vectors cannot maintain stability in cells and will gradually be lost as the bacteria grow and multiply.

[0257] For strategy 3, the newly constructed pHbPBC-sfGFP in this study was introduced into the Halomonas H. bluephagenesis LCP by conjugation transformation, and the culture was transferred and subcultured for 7 days with and without the addition of antibiotics. The results of flow cytometry analysis showed that for the antibiotic group, the average proportion of fluorescent cells decreased slightly within 7 days, from 99.4% on Day 1 to 93.3% on Day 7; in the group without the addition of antibiotics, the average proportion of fluorescent cells also decreased slightly within 7 days, from 99.5% on Day 1 to 95.5% on Day 7. However, both groups remained at a high level as a whole, and compared with the general plasmid vector of strategy 2, the stability was closer to the genome integration of strategy 1 ( Fig.13 , Table 5).

[0258] Table 5 The stability of pHbPBC is not dependent on the selective pressure of antibiotics

[0259]

[0260] The stability of two commonly used strategies for engineering bacteria transformation, namely, integrating the target gene into the genome and culturing under antibiotic-free conditions (i.e., strategy 1 without antibiotics), and inserting the target gene into a general expression vector to form a recombinant plasmid and adding antibiotics to culture (i.e., strategy 2 with antibiotics), was compared with the newly developed expression strategy in this study, namely, inserting the target gene into the stable expression vector pHbPBC based on the toxin-antitoxin system hbpB / hbpC to form a stable recombinant plasmid and culturing under antibiotic-free conditions (i.e., strategy 3 without antibiotics). Fig.14 ). The results show that the toxin-antitoxin system hbpB / hbpC found in the endogenous plasmid pHbCP of Halomonas H.bluephagenesis TD01 in this study has the effect of maintaining the stable existence of the plasmid without loss. The recombinant plasmid expression vector pHbPBC with this as the key stabilizing element can stably replicate and propagate with the growth and reproduction of host cells without the addition of antibiotics, and has good stability similar to genome integration.

[0261] 3. High-level expression of recombinant plasmid vector pHbPBC in H. bluephagenesis LCP

[0262] The medium containing 1.03 mol / L NaCl was also used, and flow cytometry was used to record the average fluorescence intensity of individual cells in each group of samples during the 7-day transfer and subculture experiment to characterize the expression level of the target protein green fluorescent protein sfGFP. The results showed that the stable recombinant plasmid pHbPBC-sfGFP showed a relatively high level of target protein expression, which was 3.5 to 5 times that of the genome integration strategy under the same conditions, and 1.5 to 2 times that of the pSEVA341-sfGFP group ( Fig.15 ).

[0263] Since the expression of genes on the intracellular plasmid is directly related to the copy number of the plasmid in the cell, this study measured the copy number of the stable recombinant plasmid vector pHbPBC in the Halomonas H. bluephagenesis LCP. According to the real-time quantitative PCR experiment, the copy number of pHbPBC was only 3, which is a low-copy number plasmid. As a high-copy plasmid, pSEVA341 was measured to have a copy number of more than 300 in the cell, but compared with the genome with a copy number of 1, the expression level of the target gene inserted into it was only 2 to 2.5 times that of the genome integration, and was lower than the recombinant plasmid vector pHbPBC with a lower copy number ( Fig.15This may be due to the fact that the antibiotics added as a selection pressure during the culture process caused negative interference with the growth and metabolic processes of the cells. For this reason, the newly constructed pHbPBC-sfGFP strategy was used as an example to measure the expression level of the green fluorescent protein sfGFP under the conditions of adding and not adding antibiotics.

[0264] The statistical results show that in the group without additional antibiotics, the average fluorescence intensity of single cells is 1 to 1.5 times that of the group with antibiotics, which means that adding antibiotics during the culture process will indeed lead to a decrease in the expression level of the target protein ( Fig.16 ).

[0265] 4. The recombinant plasmid vectors pHvPBC and pHaPBC composed of hbpB / hbpC from Halomonas venusta and Halomonas alkaliantarctica are stably replicated in H. bluephagenesis LCP

[0266] The hbpB / hbpC genes and their upstream promoter regions from H. venusta and H. alkaliantarctica were used to replace the corresponding elements in the plasmid pHbPBC-sfGFP, respectively, to obtain the plasmids pHvPBC-sfGFP and pHaPBC-sfGFP. Flow cytometry was used to detect whether there was fluorescence in individual cells. The Halomonas H. bluephagenesis LCP carrying the plasmids pHvPBC-sfGFP and pHaPBC-sfGFP was subcultured for 7 days in a culture medium containing 1.03 mol / L NaCl in the absence of antibiotics and with the addition of antibiotics. The bacterial solution was transferred and cultured at a ratio of 1:1000 every 24 hours for a total of 7 days; the proportion of cells that emitted fluorescence was recorded by flow cytometry every 24 hours. The results are as follows: Fig.18 As shown, the proportion of cells emitting fluorescence in the four groups remained at a high level. This shows that in the Halomonas H. bluephagenesis LCP, the toxin-antitoxin system hbpB / hbpC from H. venusta and H. alkaliantarctica also has the effect of maintaining the stable existence of plasmids.

[0267] Among them, the sequence of hbpB from H. venusta is shown in SEQ ID NO: 26, the sequence of hbpC is shown in SEQ ID NO: 27, the amino acid sequence of the corresponding HbpB protein is shown in SEQ ID NO: 28, and the amino acid sequence of the HbpC protein is shown in SEQ ID NO: 29. In this study, the upstream 250 bp of hbpB / hbpC was selected as the promoter region, and the sequence of the promoter and RBS region was SEQ ID NO: 30.

[0268] SEQ ID NO: 26

[0269] atggctcttcgcaaaatcagcgacctcaaacccgtgtttagcagtgagcgtgtcaccgagtggcagcccgtgcccctggggccacg

[0270] ataccgttatgagcgagatcgggccgcggtcgggcaggaagtcacgcctggcagtgagcagtatgagtggcacgtcttggcgaa

[0271] caatgacctggcccatgccaagcgcaaagtgtttgcgctgatcaccgaggagtacctgtaa

[0272] SEQ ID NO: 27

[0273] atggcgcgcacgcgggccatacagcgctgaggcgcctttatggctagaggtgctgttagcgtacgcgtttggctctgagcctgc

[0274] tcagcgagctgcacagctggatttgctgggggtggcctatgatgccactgcatatcccaatgatatccccgatgctcgattggccga

[0275] gctgctgctggcctgggctgagcagtatgtgcctggggaggattggcaacggttgcaggcccgaatcaggcaacggcgctcaca

[0276] gttgcgatag

[0277] SEQ ID NO: 28

[0278] MALRKISDLKPVFSSERVTEWQPVPLGPRYRYERDRAAVGQEVTPGSEQYEWHVL

[0279] ANNDL AHAKRKVFAL ITEEYL

[0280] SEQ ID NO: 29MARTRAIQSAEAPLWLEVLLAYAFGSEPAQRAAQLDLLGVAYDATAYPNDIPDARLAELLLAWAEQYVPGEDWQRLQARIRQRRSQLR

[0281] SEQ ID NO: 30

[0282] aaatctccttagctagggtttaggtacgttaagcaatggacgctttgcgcaaggacgaccgagcacggaacgcttgatgatctcatggacgtatgagcaggacgctcaacagagttcgctaggaa gcgatgtaagaattctgcccgacccgaaaaggtcgggcttttctttgtctgtcgttatttaaacgttatattttttaacgtttattggtctacacttgtagacacctctccccttgagtgaacagcc

[0283] Among them, the sequence of hbpB from H. alkaliantarctica is shown in SEQ ID NO: 31, the sequence of hbpC is shown in SEQ ID NO: 32, the amino acid sequence of the corresponding HbpB protein is shown in SEQ ID NO: 33, and the amino acid sequence of the HbpC protein is shown in SEQ ID NO: 34. In this study, the upstream 250 bp of hbpB / hbpC was selected as the promoter region, and the sequence of the promoter and RBS region was SEQ ID NO: 35.

[0284] SEQ ID NO: 31

[0285] atgactcttcgcaaaatcagcgaccttaaacccgtgtttagcagtgagcgtgtcaccgagtggcagctcgcccctatgggaccacggtaccgttatgagcgagatcgggccgcggtcgggcaggaaataacgcctggcagtgagcagtatgagtggcacgttctggccaacaacgacctgacccacgccaaacgcaaggtgtttgcgctaatcaccgaggactatctgtaa

[0286] SEQ ID NO:32

[0287] atggcccgcgtgcgggccatacaggccgctgaggcgtctttatggctagaggtgttactaacctacgcatttggccctgcgcctgctcagcaggctgcgcagctggatttagtggggattgcccacgacgccaccgcgtatcccgacgacataccgaatgggcgattggctcagctgctgcttgcctgggctgagcgttacgttgaggcgggagattggcagcggttacaggccagaatcaggcagcggcgctcacagtcgggatag

[0288] SEQ ID NO:33

[0289] MTLRKISDLKPVFSSERVTEWQLAPMGPRYRYERDRAAVGQEITPGSEQYEWHVLANNDLTHAKRKVFALITEDYL

[0290] SEQ ID NO:34

[0291] MARVRAIQAAEASLWLEVLLTYAFGPAPAQQAAQLDLVGIAHDATAYPDDIPNGRLAQLLLAWAERYVEAGDWQRLQARIRQRRSQSG

[0292] SEQ ID NO:35

[0293] aatctccctagctagggtgtaggtacgttaagcaacggacgctttgcgtaaggacgaccgagcacggaacgcttgaggatctcatggacgtatgagcaggacgctcaacagagtttcgctaggaa gcggcacaagaattttgcccgacccgaaagggtcgggcttttctttgtctgtcgttatttaaacgtgattttttcacgtttattagtctacacttgtagacatctctccccttgagtgaacagcc

[0294] 5. Stable replication of the recombinant plasmid vector pHbPBC in H. bluephagenesis LCP under moderately halophilic environment

[0295] Flow cytometry was used to detect whether there was fluorescence in individual cells. The H. bluephagenesis LCP carrying the plasmid pHbPBC-sfGFP was subcultured for 7 days in a medium containing 0.51 mol / L and 2.50 mol / L NaCl, respectively, without antibiotics or with antibiotics. The bacterial solution was transferred and cultured at a ratio of 1:1000 every 24 hours for a total of 7 days; the proportion of cells that emitted fluorescence was recorded by flow cytometry every 24 hours. The results are shown in the figure below. Fig.19 As shown, the proportion of cells emitting fluorescence in the four groups remained at a high level.

[0296] In addition, H. bluephagenesis has difficulty growing in culture media with a salt-loving environment beyond a moderate level. When the salt concentration is too high or too low, the bacteria grow slowly or cannot grow.

[0297] Further combined with the results obtained using a culture medium containing 1.03 mol / L NaCl in Example 2, it can be seen that in Halomonas H. bluephagenesis LCP, under a moderately halophilic environment, the toxin-antitoxin system hbpB / hbpC has the effect of maintaining the stable existence of the plasmid.

[0298] 6. Stable replication of the recombinant plasmid vector pHbPBC in Halomonas campaniensis

[0299] This study used flow cytometry to detect fluorescence in individual cells. The culture medium containing 0.68 mol / L NaCl was used to subculture the H. campaniensis carrying the plasmid pHbPBC-sfGFP for 7 days without or with antibiotics. The bacterial culture was transferred at a ratio of 1:1000 every 24 hours for a total of 7 days. The proportion of cells that emitted fluorescence was recorded by flow cytometry every 24 hours. The results are shown in Figure 2. Figure 5 As shown, the proportion of cells emitting fluorescence in both groups remained at a high level. This shows that in H. campaniensis, the toxin-antitoxin system hbpB / hbpC also has the effect of maintaining the stable existence of plasmids.

[0300] 7. The replication stability of recombinant plasmid vectors carrying toxin-antitoxin system is increased in Vibrio natriegens

[0301] The pUC replicon was used to replace the replicon of the plasmid pHbPBC-sfGFP to obtain the plasmid pUC-HbPBC-sfGFP; the plasmid pUC-sfGFP was obtained by deleting the toxin-antitoxin hbpB / hbpC genes in pUC-HbPBC-sfGFP. This result uses flow cytometry analysis technology to detect whether there is fluorescence in individual cells. In the absence of antibiotics, the culture medium containing 0.51 mol / LNaCl was used to subculture V. natriegens carrying the plasmids pUC-HbPBC-sfGFP and pUC-sfGFP for 7 days. The bacterial solution was transferred and cultured at a ratio of 1:1000 every 24 hours for a total of 7 days; the proportion of cells that emit fluorescence was recorded by flow cytometry every 24 hours. The results are as follows Fig.17 As shown, the proportion of cells emitting fluorescence in the group with the toxin-antitoxin system remained at a higher level. This shows that in V. natriegens, the toxin-antitoxin system hbpB / hbpC also has the effect of maintaining the stable existence of plasmids.

[0302] In summary, based on the newly discovered toxin-antitoxin system hbpB / hbpC on the endogenous plasmid of halophilic microorganisms, this achievement constructed an extrachromosomal expression system suitable for halophilic microorganisms - stable recombinant plasmid vectors such as pHbPBC, pHvPBC and pHaPBC, and achieved high-level expression of exogenous target proteins, enriching the editing and transformation tools available for halophilic microorganisms, and providing more possibilities for halophilic microorganisms to use as chassis cells to produce a variety of PHAs and high-value-added chemicals.

[0303] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A recombinant plasmid, characterized in that: The recombinant plasmid expresses toxin and antitoxin, and the toxin and antitoxin are derived from halophilic microorganisms.

2. The recombinant plasmid according to claim 1, characterized in that The recombinant plasmid also includes regulatory elements required for expressing toxins and antitoxins; Preferably, a promoter is included; Further preferably, the recombinant plasmid further comprises a replication initiation site and / or a replication initiation protein coding sequence.

3. The recombinant plasmid according to claim 1 or 2, characterized in that The toxin and antitoxin are on the same operon and are regulated by the same promoter.

4. The recombinant plasmid according to any one of claims 1 to 3, characterized in that The recombinant plasmid is a conjugative plasmid or a non-conjugative plasmid.

5. The recombinant plasmid according to any one of claims 1 to 4, characterized in that: The halophilic microorganisms include moderate halophilic bacteria; Preferably, the moderately halophilic bacteria can grow under the condition of salt concentration of 0.5 mol / L-2.5 mol / L.

6. The recombinant plasmid according to any one of claims 1 to 5, characterized in that: The halophilic microorganisms include Chromohalobacter, Halomonas, Halovibrio, Vibrio, Cobetia, Kushneria, Salicola, Halotela or Carnimonas; Preferably, the Halomonas genus includes Halomonas bluephagenesis, Halomonas campaniensis, Halomonas desiderata, Halomonas venusta or Halomonas alkaliantarctica.

7. The recombinant plasmid according to any one of claims 1 to 6, characterized in that: The amino acid sequence of the antitoxin comprises SEQ ID NO: 3, 28 or 33 or comprises an amino acid sequence having more than 80% homology with SEQ ID NO: 3, 28 or 33 and having the same or similar activity; The amino acid sequence of the toxin comprises SEQ ID NO: 4, 29 or 34 or comprises an amino acid sequence having more than 80% homology with SEQ ID NO: 4, 29 or 34 and having the same or similar activity.

8. The recombinant plasmid according to any one of claims 1 to 7, characterized in that: The recombinant plasmid also includes the target gene, and preferably also includes regulatory elements and / or regulatory proteins required for the replication, transcription and / or expression of the target gene.

9. The recombinant plasmid according to any one of claims 1 to 8, characterized in that: The recombinant plasmid includes toxin and antitoxin expression systems, target gene expression systems and auxiliary sequences; Preferably, the auxiliary sequence is an element that ensures or improves the self-replication function and / or transcription and / or expression function of the plasmid, or the auxiliary sequence is a site required for connecting various elements.

10. The recombinant plasmid according to any one of claims 1 to 9, characterized in that: The recombinant plasmid is a linear plasmid or a circular plasmid.

11. A method for constructing a recombinant plasmid according to any one of claims 1 to 10, characterized in that: The method comprises connecting toxin and antitoxin expression systems and auxiliary sequences; Preferably, the method further comprises connecting to a target gene expression system.

12. An engineered bacterium, characterized in that: The engineered bacteria comprises the recombinant plasmid described in any one of claims 1-10.

13. The engineered bacteria according to claim 12, characterized in that: The endogenous plasmid is knocked out or knocked down in the engineered bacteria, or the endogenous plasmid is rendered non-functional.

14. The engineered bacteria according to claim 12 or 13, characterized in that: The engineered bacteria include halophilic microorganisms; Preferably, the halophilic microorganisms include moderate halophilic bacteria; further preferably, the moderate halophilic bacteria can grow under the condition of 0.5 mol / L-2.5 mol / L salt concentration; Preferably, the halophilic microorganisms include Chromohalobacter, Halomonas, Halovibrio, Vibrio, Cobetia, Kushneria, Salicola, Halotela or Carnimonas; Further preferably, the Halomonas genus includes Halomonas bluephagenesis, Halomonas campaniensis, Halomonas desiderata, Halomonas venusta or Halomonas alkaliantarctica.

15. A method for preparing the engineered bacteria according to any one of claims 12 to 14, characterized in that: The preparation method comprises introducing a recombinant plasmid into an engineering bacterium; Preferably, the introduction is chemical transformation, electroporation transformation or conjugation transformation.

16. Use of the recombinant plasmid according to any one of claims 1 to 10 or the engineered bacteria according to any one of claims 12 to 14 in gene editing or biosynthesis.

17. Use of a toxin-antitoxin system in preparing a vector backbone, a recombinant plasmid and / or maintaining the stability of a recombinant plasmid in a halophilic microorganism, characterized in that: The toxin and antitoxin are derived from halophilic microorganisms.

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