Toxin- and antitoxin-expressing recombinant plasmid, construction method therefor, and use thereof

Abstract

The present invention provides a toxin- and antitoxin-expressing recombinant plasmid, a construction method therefor, and a use thereof in gene editing or biosynthesis. A toxin-antitoxin can maintain plasmid stability, efficiently express a target gene, and play an important role in research on halophile products.

Description

A recombinant plasmid expressing toxin and antitoxin and its construction method and application 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 microorganism that thrives in high-salt environments and are widely distributed in salt lakes, salt mines, saline-alkali soils, and high-salt products. Their unique adaptive mechanisms have long fascinated research. Halophiles thrive in environments with high salt concentrations, making it difficult for other microorganisms to survive and grow. Their application in the fermentation production of PHAs can avoid contamination from most contaminants, reduce production steps, and offer significant cost advantages. Halophiles also offer significant advantages in high-salt wastewater treatment and ecological remediation. Due to their rich metabolic resource base, halophiles can be found in high-salt wastewater and soils containing a variety of nutrients. Some halophiles are highly tolerant to alkaline environments and heavy metal ions, demonstrating strong bioremediation potential in environments rich in heavy metal ions, such as high-salt organic wastewater and oilfield soils. Halophiles are becoming an emerging force in emerging biochemical industries, including biodegradable materials, biofuels, enzyme preparations, and pharmaceuticals, as well as in pollution control and wastewater treatment.

[0003] Halophilic microorganisms, such as Halomonas, also have great potential as platforms for producing biodegradable PHA bioplastics and other high-value-added chemicals. Researchers have developed a variety of genome editing technologies to achieve metabolic control of engineered bacteria. However, with the increasing demand for engineering, repeated editing often leads to decreased genome stability, which in turn affects the growth and production of engineered bacteria. Furthermore, the editing process is cumbersome, time-consuming, and costly.

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

[0005] Summary of the Invention

[0006] Through years of research on the genomes of halophilic microorganisms, the inventors of this application have clarified 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.

[0007] In a first aspect, a recombinant plasmid is provided, wherein the recombinant plasmid expresses a toxin and an antitoxin, and the toxin and the antitoxin are derived from halophilic microorganisms.

[0008] Preferably, the toxin and antitoxin are derived from halophilic microorganisms. Preferably, the toxin and antitoxin are derived from the same or different halophilic microorganisms.

[0009] Preferably, the toxin and antitoxin are derived from endogenous plasmids of halophilic microorganisms.

[0010] Preferably, the recombinant plasmid further comprises regulatory elements required for expression of the toxin and antitoxin. For example, a promoter can be any promoter disclosed in the prior art, as long as it can regulate the expression of the toxin and antitoxin. Preferably, it can be a constitutive promoter or an inducible promoter.

[0011] In a specific embodiment of the present invention, the promoter may be derived from a halophilic microorganism.

[0012] In a specific embodiment, the promoter is hbpB / hbpC promoter.

[0013] The toxin and antitoxin are on the same operon and regulated by the same promoter.

[0014] Preferably, the regulatory element further includes an RBS sequence.

[0015] Preferably, the promoter and RBS sequence regulate the initiation and degree of expression of toxin and antitoxin genes. Further preferably, the promoter is a promoter shared by toxin and antitoxin.

[0016] Preferably, the promoter and RBS sequence comprises SEQ ID NO: 5, 11, 30 or 35, or comprises a nucleotide sequence having more than 80% homology with SEQ ID NO: 5, 11, 30 or 35 and having the same or similar activity.

[0017] Preferably, the recombinant plasmid further comprises a replication origin site and / or a replication initiation protein coding sequence, wherein the replication origin site and / or replication initiation protein can be any replication origin site and / or replication initiation protein disclosed in the prior art, as long as 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 origin site and / or replication initiation protein are derived from a halophilic microorganism, preferably from an endogenous plasmid of a halophilic microorganism.

[0018] Preferably, the recombinant plasmid includes one or more replication origin sites.

[0019] In a specific embodiment, the replication origin is pHbCP ori or pRO1600 oriV.

[0020] Preferably, the replication initiation site comprises SEQ ID NO: 6 or 12 or comprises a nucleotide sequence having more than 80% homology to SEQ ID NO: 6 or 12 and having the same or similar activity.

[0021] Preferably, the recombinant plasmid comprises one or more replication initiation protein coding sequences.

[0022] In one embodiment, the replication initiation protein is RepB protein.

[0023] Preferably, the repB gene comprises SEQ ID NO: 7 or a nucleotide sequence having more than 80% homology to SEQ ID NO: 7 and having the same or similar activity.

[0024] In one embodiment, the replication initiation protein is pRO1600Rep protein.

[0025] Preferably, the pRO1600Rep gene comprises SEQ ID NO: 13 or a nucleotide sequence having more than 80% homology to SEQ ID NO: 13 and having the same or similar activity.

[0026] Preferably, the replication initiation protein pRO1600Rep comprises SEQ ID NO: 24 or an amino acid sequence having more than 80% homology to SEQ ID NO: 24 and having the same or similar activity.

[0027] Preferably, the replication initiation site and replication initiation protein coding sequence are essential elements for the plasmid to persist and replicate in halophilic microorganisms.

[0028] 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.

[0029] 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.

[0030] Preferably, the recombinant plasmid is a conjugative plasmid or a non-conjugative plasmid.

[0031] Preferably, the recombinant plasmid further comprises elements required for conjugative transformation, such as an origin of replication and an initiation site for plasmid transfer. The elements required for conjugative transformation may be any element disclosed in the prior art, as long as conjugative transformation is possible. The elements required for conjugative transformation are capable of being used in Escherichia coli. Replication and / or transfer are preferred.

[0032] In one embodiment of the present invention, the elements required for conjugative transformation are derived from Escherichia coli.

[0033] Preferably, the conjugative transformation element is selected from the replication origin and plasmid transfer origin of pUC plasmid, pET plasmid, pGEX plasmid, and pRO plasmid.

[0034] In a specific embodiment of the present invention, the elements required for conjugative transformation are derived from the optimized replication origin pMB1ori of the pUC series plasmids.

[0035] 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 shown in SEQ ID NO: 12.

[0036] Preferably, the halophilic microorganism is a bacterium.

[0037] Preferably, the halophilic microorganisms include but are not limited to Chromohalobacter, Halomonas, Halovibrio, Vibrio, Cobetia, Kushneria, Salicola, Halotela, or Carnimonas.

[0038] Preferably, the genus Chromohalobacterium 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, Halomonas desiderata, 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 Cobetia marina 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.

[0039] Preferably, the halophilic microorganisms include mild halophiles, moderate halophiles or extreme halophiles, and more preferably moderate halophiles.

[0040] Preferably, the halophilic microorganism is a halophilic microorganism that can grow under conditions containing a salt concentration of 0.5 mol / L to 2.5 mol / L, preferably a halophilic microorganism that grows stably or grows best.

[0041] 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.

[0042] 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.

[0043] 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 to SEQ ID NO: 1, 26 or 31 and having the same or similar activity.

[0044] 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 to SEQ ID NO: 2, 27 or 32 and having the same or similar activity.

[0045] Preferably, the recombinant plasmid further includes the target gene, and further preferably includes 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.

[0046] Preferably, the recombinant plasmid includes toxin and antitoxin expression systems, target gene expression system and auxiliary sequences.

[0047] Preferably, the recombinant plasmid includes toxin and antitoxin expression systems, a replication initiation site, a replication initiation protein coding sequence, a target gene expression system and auxiliary sequences.

[0048] The target gene expression system includes the target gene and regulatory elements and / or regulatory proteins required for the replication, transcription and / or expression of the target gene.

[0049] The toxin and antitoxin expression system is a system comprising toxins and antitoxins and regulatory elements required for the expression of toxins and antitoxins.

[0050] 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.

[0051] In a specific embodiment of the present invention, the recombinant plasmid consists of a toxin and antitoxin expression system, a replication origin site, a replication initiation protein coding sequence and a target gene expression system.

[0052] 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 auxiliary sequences.

[0053] 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.

[0054] The auxiliary sequence may be an element that ensures or improves the self-replication function and / or expression function of the plasmid, or the auxiliary sequence may be a site required for connecting various elements.

[0055] For example, enzyme cutting 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.

[0056] Preferably, the recombinant plasmid is a linear plasmid or a circular plasmid.

[0057] Preferably, the recombinant plasmid is DNA or RNA.

[0058] 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.

[0059] 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, an initiation site for conjugation transformation, and an initiation site for plasmid transfer.

[0060] In a specific embodiment, the recombinant plasmid is a circular plasmid, which includes, in clockwise direction: a promoter shared by the 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, an initiation site for conjugation transformation, and an initiation site for plasmid transfer.

[0061] In a second aspect, a toxin-antitoxin system is provided, wherein the toxin-antitoxin system is derived from halophilic microorganisms.

[0062] Preferably, the toxin and antitoxin are derived from the same or different halophilic microorganisms.

[0063] 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.

[0064] 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.

[0065] 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 to SEQ ID NO: 1, 26 or 31 and having the same or similar activity.

[0066] 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 to SEQ ID NO: 2, 27 or 32 and having the same or similar activity.

[0067] Preferably, the halophilic microorganism is a bacterium.

[0068] Preferably, the halophilic microorganisms include but are not limited to Chromohalobacter, Halomonas, Halovibrio, Vibrio, Cobetia, Kushneria, Salicola, Halotela, or Carnimonas.

[0069] Preferably, the genus Chromohalobacterium 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, Halomonas desiderata, 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 Cobetia marina 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.

[0070] Preferably, the halophilic microorganisms include mild halophiles, moderate halophiles or extreme halophiles, and more preferably moderate halophiles.

[0071] Preferably, the halophilic microorganism is a halophilic microorganism that can grow under conditions containing a salt concentration of 0.5 mol / L to 2.5 mol / L, preferably a halophilic microorganism that grows stably or grows best.

[0072] The toxin and antitoxin are on the same operon and regulated by the same promoter.

[0073] Preferably, in the toxin-antitoxin system, hbpC is lethally toxic to cells, hbpB can inhibit the toxicity of hbpC, protect cells and maintain normal growth, and the toxin-antitoxin system can maintain the stability of the recombinant plasmid.

[0074] 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.

[0075] Wherein, the toxin and antitoxin are derived from halophilic microorganisms. Preferably, the toxin and antitoxin are derived from the same or different halophilic microorganisms.

[0076] In a fourth aspect, a nucleic acid encoding the above-mentioned recombinant plasmid or the above-mentioned toxin-antitoxin system is provided.

[0077] In a fifth aspect, a method for constructing the above-mentioned recombinant plasmid is provided, which comprises connecting the toxin and antitoxin expression systems and auxiliary sequences.

[0078] Preferably, the method further comprises connecting to a target gene expression system.

[0079] Preferably, the method includes the steps of connecting the replication origin site and the replication initiation protein coding sequence.

[0080] Preferably, the method further comprises the step of connecting a resistance protein expression system.

[0081] Preferably, the resistance protein expression system includes a gene encoding the resistance protein, and regulatory elements and / or regulatory proteins required for the replication, transcription and / or expression of the gene encoding the resistance protein.

[0082] Preferably, the regulatory element includes a promoter that regulates the expression of the resistance protein.

[0083] 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.

[0084] In a sixth aspect, a method for constructing a recombinant plasmid is provided, comprising connecting a toxin and antitoxin expression system to a traditional plasmid.

[0085] The traditional plasmids are plasmids that can replicate and express target genes in organisms, such as pUC plasmids, pET plasmids, pGEX plasmids, and pRO plasmids.

[0086] In a seventh aspect, a vector skeleton is provided, wherein the vector skeleton comprises a toxin and an antitoxin expression system.

[0087] The toxin and antitoxin expression system comprises a promoter, a toxin encoding gene and an antitoxin encoding gene, wherein the toxin and antitoxin are on the same operon and regulated by the same promoter.

[0088] The promoter can be any promoter disclosed in the prior art, as long as it can regulate the expression of toxins and antitoxins, and preferably can be a constitutive promoter or an inducible promoter.

[0089] In a specific embodiment of the present invention, the promoter may be derived from a halophilic microorganism.

[0090] In a specific embodiment, the promoter is hbpB / hbpC promoter.

[0091] Preferably, the 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 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 a halophilic microorganism, preferably from an endogenous plasmid of a halophilic microorganism.

[0092] Preferably, the elements required for transformation are also included.

[0093] Preferably, auxiliary sequences are also included.

[0094] In an eighth aspect, a method for expressing a target gene is provided, wherein the expression method comprises using the above-mentioned recombinant plasmid.

[0095] Preferably, the expression method comprises:

[0096] 1) Knockout or knockdown of endogenous plasmids in microorganisms, or rendering endogenous plasmids non-functional;

[0097] 2) The recombinant plasmid is introduced into the microorganism obtained in 1) and cultured.

[0098] Preferably, the knockout or knockdown method comprises knocking out or knocking down hbpC while overexpressing hbpB in the microorganism.

[0099] Preferably, the microorganism obtained in step 1) does not contain endogenous plasmids and plasmids that may be introduced during the knockout or knockdown process.

[0100] Preferably, the introduction method includes chemical transformation, electroporation transformation and conjugation transformation.

[0101] In one embodiment, the introduction method is conjugation transformation.

[0102] Preferably, the halophilic microorganism is a bacterium.

[0103] Preferably, the halophilic microorganisms include but are not limited to Chromohalobacter, Halomonas, Halovibrio, Vibrio, Cobetia, Kushneria, Salicola, Halotela, or Carnimonas.

[0104] Preferably, the genus Chromohalobacterium 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, Halomonas desiderata, 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 Cobetia marina 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.

[0105] Preferably, the halophilic microorganisms include mild halophiles, moderate halophiles or extreme halophiles, and more preferably moderate halophiles.

[0106] Preferably, the halophilic microorganism is a halophilic microorganism that can grow under conditions containing a salt concentration of 0.5 mol / L to 2.5 mol / L, preferably a halophilic microorganism that grows stably or grows best.

[0107] Preferably, the expression method includes selective pressure such as adding antibiotics or not adding antibiotics.

[0108] 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.

[0109] Preferably, the endogenous plasmid is knocked out or knocked down in the engineered bacteria, or the endogenous plasmid is rendered non-functional.

[0110] Preferably, the knockout or knockdown method comprises overexpressing hbpB while knocking out or knocking down hbpC.

[0111] Preferably, the engineered bacteria include halophilic microorganisms;

[0112] Preferably, the halophilic microorganism is a bacterium.

[0113] Preferably, the halophilic microorganisms include but are not limited to Chromohalobacter, Halomonas, Halovibrio, Vibrio, Cobetia, Kushneria, Salicola, Halotela, or Carnimonas.

[0114] Preferably, the genus Chromohalobacterium 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, Halomonas desiderata, 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 Cobetia marina 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.

[0115] Preferably, the halophilic microorganisms include mild halophiles, moderate halophiles or extreme halophiles, and more preferably moderate halophiles.

[0116] Preferably, the halophilic microorganism is a halophilic microorganism that can grow under conditions containing a salt concentration of 0.5 mol / L to 2.5 mol / L, preferably a halophilic microorganism that grows stably or grows best.

[0117] In a ninth aspect, a method for preparing the above-mentioned engineered bacteria is provided, which comprises introducing a recombinant plasmid into the engineered bacteria.

[0118] Preferably, the introduction is chemical transformation, electroporation transformation or conjugation transformation.

[0119] In the tenth aspect, a method is provided for providing the use of 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.

[0120] The gene editing is halophilic microbial gene editing.

[0121] Preferably, the biosynthetic products include but are not limited to PHA, tetrahydropyrimidine, amino acids, inositol, 1,3-propanediol, 3-hydroxypropionic acid, 1,5-pentanediamine, 5-aminolevulinic acid, lipase, amylase, saccharifying 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.

[0122] 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, a 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.

[0123] In the eleventh aspect, a fermentation method is provided, which comprises fermenting and culturing the above-mentioned engineered bacteria.

[0124] In a twelfth aspect, a method for product biosynthesis is provided, wherein the method comprises culturing the above-mentioned engineered bacteria.

[0125] No antibiotics were added to the cultures.

[0126] Through the above technical solution, the present invention has the following advantages:

[0127] 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.

[0128] The terms "comprising" or "including" as used in the present invention are open-ended. 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.

[0129] The "homology" mentioned in the present invention refers to the fact that when using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the sequences used have (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%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 112%, %, 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.

[0130] The "halophilic microorganisms" of the present invention are microorganisms whose optimal growth salt concentration is greater than 0.2 mol / L. Based on their salt needs, microorganisms can be divided into non-halophilic microorganisms, weak halophilic microorganisms, moderate halophilic microorganisms, and extreme halophilic microorganisms.

[0131] The "mild halophile" of the present invention is a bacterium that can grow under conditions of 0.2 mol / L to 0.5 mol / L salt concentration, preferably with stable growth or optimal growth, provided that other culture conditions except the salt concentration of the bacterium are kept consistent.

[0132] "Moderately halophilic bacteria" are bacteria that can grow in a salt concentration of 0.5 mol / L to 2.5 mol / L, preferably with stable growth or the best growth, provided that all other culture conditions except the salt concentration are kept constant.

[0133] "Extreme halophiles" are bacteria that can grow in a salt concentration of 2.5 mol / L to 5.2 mol / L, preferably grow stably or grow best, when other culture conditions except salt concentration are kept consistent.

[0134] The "salt" may be a salt commonly used in the prior art for microbial culture, and the salt is an inorganic salt used to maintain the osmotic pressure required by the microorganism. Examples include potassium salts and / or sodium salts. The potassium salts include, but are 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 salts include, but are 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

[0135] Figure 1: Effects of orfF2 and orfF3 overexpression on the growth of Halomonas.

[0136] Figure 2: Knockout system of the toxin-encoding gene hbpC, wherein, Figure 2A: pSEVA321 plasmid expresses Cas9, Figure 2B: pSEVA341 plasmid expresses gRNA and homology arms, Figure 2C: gRNA is designed on the toxin-encoding gene hbpC gene, oriT: conjugative transfer initiation site; HR (Homologous arm): homology arm; CmR: chloramphenicol resistance protein; KanR: kanamycin resistance protein; SpeR: spectinomycin resistance protein.

[0137] Figure 3: Knockout results of the toxin-encoding gene hbpC. Using CRISPR / Cas9 editing technology, Figure 3A directly knocks out hbpC, and Figure 3B overexpresses hbpB while knocking out hbpC.

[0138] Figure 4: Knockout of the stable endogenous plasmid pHbCP does not affect the growth rate of Halomonas.

[0139] Figure 5: Expression of the toxin-antitoxin system in H. campaniensis.

[0140] Figure 6: The toxin-antitoxin system hbpB / hbpC is a key stabilizing element of pHbPBC.

[0141] Figure 7: Plasmid replication initiation site and replication initiation protein suitable for Halomonas and Escherichia coli. Figure 7A is the replication initiation site and replication initiation protein from the endogenous plasmid pRO1600 of Pseudomonas aeruginosa; Figure 7B is the replication initiation site and replication initiation protein from the endogenous plasmid pHbCP of Halomonas H.bluephagenesis; Figure 7C is the replication initiation site suitable for Escherichia coli S17-1 strain; Figure 7D is the conjugative transfer initiation site.

[0142] Figure 8: Stable recombinant plasmid vector pHbPBC with the exogenous target protein sfGFP encoding gene inserted, Promoter HbpBC-HbpB-HbpC: toxin-antitoxin stable element expression system; pMB1 ori: replication origin suitable for Escherichia coli S17-1 strain; pHbCP ori: replication origin suitable for Halomonas; pHbCP RepB: replication initiation protein; oriT: conjugative transfer origin; rrnB T1 terminator-J23119 promoter-RBS-sfGFP-lambda t0 terminator: sfGFP expression system; KanR: kanamycin resistance protein; SpeR: spectinomycin resistance protein.

[0143] Figure 9: The sfGFP encoding gene is integrated into the genome, Figure 9A: pSEVA321 plasmid expresses Cas9; Figure 9B: pSEVA341 plasmid expresses Grna, oriT: conjugative transfer initiation site; HR-L / R: upstream and downstream homology arms of the 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.

[0144] Figure 10: The sfGFP encoding gene was inserted into the vector plasmid pSEVA341, pMB1 ori: replication origin suitable for Escherichia coli S17-1 strain; pRO1600 oriⅤ: replication origin suitable for Halomonas; pRO1600 RepB: replication initiation protein; oriT: conjugative transfer origin; rrnB T1 terminator-J23119 promoter-RBS-sfGFP-lambda t0 terminator: sfGFP expression system; KanR: kanamycin resistance protein; SpeR: spectinomycin resistance protein.

[0145] Figure 11: The target gene is integrated into the genome and remains stably present 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.

[0146] Figure 12: The stability of pSEVA341 depends on the selective pressure of antibiotics. The diamond icon indicates the average percentage of fluorescent cells in the pSEVA341-sfGFP group under antibiotic-free culture conditions; the triangle icon indicates the average percentage of fluorescent cells in the pSEVA341-sfGFP group under antibiotic culture conditions.

[0147] Figure 13: The stability of pHbPBC is independent of antibiotic selection pressure. The circular graph indicates the average percentage of fluorescent cells in the pHbPBC-sfGFP group under antibiotic-free culture conditions; the square graph indicates the average percentage of fluorescent cells in the pHbPBC-sfGFP group under antibiotic culture conditions.

[0148] Figure 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; and the triangular icon indicates the average proportion of fluorescent cells in the pSEVA341-sfGFP group under antibiotic culture conditions.

[0149] Figure 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-integrated 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; and the circle icon indicates the average fluorescence intensity of single cells in the pHbPBC-sfGFP group under antibiotic-free culture conditions.

[0150] Figure 16: The addition of antibiotics negatively affects the expression level of the target protein. The circular graph indicates the mean fluorescence intensity of single cells in the pHbPBC-sfGFP group under antibiotic-free culture conditions; the square graph indicates the mean fluorescence intensity of single cells in the pHbPBC-sfGFP group under antibiotic-containing culture conditions.

[0151] Figure 17: Expression of the toxin-antitoxin system in V. natriegens.

[0152] Figure 18: Expression of toxin-antitoxin systems from H. venusta and H. alkaliantarctica.

[0153] Figure 19: Expression of the toxin-antitoxin system in a moderately halophilic environment. DETAILED DESCRIPTION

[0154] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0155] Table 1 Sources of bacteria used in the examples

[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, while hbpB can inhibit the toxicity of hbpC and protect cells to maintain normal growth.

[0158] Bacteria were cultured in a 96-well plate with a 200 μL system in a microplate reader, and the absorbance of the bacterial solution at a wavelength of 600 nm (OD 600 ) to characterize bacterial growth. Taking Halomonas as an example, sfGFP, encoding the green fluorescent protein, the orfF2 (i.e., hbpB) sequence, the orfF3 (i.e., hbpC) sequence, and the full-length orfF2+3 (i.e., hbpB+C) sequence were overexpressed in Halomonas bluephagenesis TD01. 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] The results are shown in Figure 1. Compared to the strain overexpressing green fluorescent protein, the strain overexpressing the orfF2 sequence maintained a normal growth rate and did not show growth slowdown or death. This may be because the orfF2 sequence is short, only 231 bp in length, and the molecular weight of the encoded product is also small. Compared with the full-length 238aa sfGFP (protein shown in SEQ ID NO: 9, coding sequence shown in SEQ ID NO: 10), the metabolic pressure on the bacteria is less, resulting in a slightly faster growth rate than the control group. In contrast, the expression of the orfF3 sequence directly caused bacterial death, indicating that the orfF3 expression product was lethal to H. bluephagenesis TD01. It is noteworthy that overexpressing the orfF2 / orfF3 system does not cause lethality to bacteria. When orfF2 and orfF3 are located on the same operon, regulated by the same promoter, and overexpressed simultaneously in bacteria, orfF2 can inhibit the damage caused by orfF3 to cells, allowing the bacteria to grow and reproduce normally. This means that orfF2 / orfF3 conforms to the mode of action of a toxin-antitoxin system. In this toxin-antitoxin system, hbpC is lethal to cells, while hbpB inhibits the toxicity of hbpC, protecting cells from normal growth.

[0160] 2. The toxin-antitoxin system hbpB / hbpC is a key element in maintaining the stability of endogenous plasmids.

[0161] Halophilic microorganisms contain an endogenous plasmid, pHbCP, which replicates stably as the bacteria grow and multiply without the need for additional selective pressure. pHbCP also harbors a toxin-antitoxin system, hbpB / hbpC. To investigate the role of the toxin-antitoxin system, hbpB / hbpC, in pHbCP, a knockout experiment was conducted to eliminate the toxin-encoding gene, hbpC.

[0162] First, using H. bluephagenesis TD01 as an example, CRISPR / Cas9 editing technology was used to design gRNA (SEQ ID NO: 25) using hbpC as the target sequence on the endogenous plasmid pHbCP for direct knockout (Figure 2A, B). The results showed that this strategy could not successfully knock out the toxin-encoding gene hbpC, and the experiment failed to obtain effective single clones (Figure 3A). Secondly, CRISPR / Cas9 editing technology was also used, and the same gRNA as for direct knockout was used, but hbpB was overexpressed in Halomonas at the same time during the knockout (Figure 2A, C). The experiment proved that this knockout strategy was effective, and a large number of normally growing strains were obtained. However, colony PCR and sequencing verification showed that it was not the toxin-encoding gene hbpC on the stable plasmid pHbCP that was knocked out in these strains, but the endogenous stable plasmid pHbCP had been lost (Figure 3B). It is speculated that this phenomenon is because CRISPR / Cas9 editing itself targets the toxin-encoding gene hbpC, destroying the stable elements of the endogenous stable plasmid pHbCP. Therefore, when Cas9 cuts pHbCP at the corresponding position, the homologous recombination step based on the homologous arms fails to be completed, and the linearized pHbCP is directly lost during the growth and reproduction of bacteria. Therefore, the knockout of the toxin-encoding gene hbpC in this experiment is actually the knockout of the stable plasmid pHbCP.

[0163] This result is consistent with the classic "postsegregational killing" (PSK) model. Under appropriate growth conditions, the toxin is relatively stable within the cell, while the antitoxin is relatively unstable. When the plasmid is lost, the antitoxin is first degraded, and the toxin loses its inhibitory effect, leading to cell death. In other words, cells that fail to inherit a copy of the plasmid are killed by the toxin-antitoxin module (Gerdes et al., 1986; Ogura and Hiraga, 1983; Unterholzner et al., 2013). In knockout experiments conducted in Halomonas, CRISPR / Cas9 editing creates double-strand breaks in the endogenous plasmid pHbCP. This prevents pHbCP from replicating and propagating normally during bacterial growth. The hbpB expression product on it is first degraded, and the concentration of the free hbpC expression product increases, exerting a toxic effect and causing bacterial death. Therefore, when pHbCP is directly knocked out using CRISPR / Cas9 editing technology, all edited bacteria die, and no effective single clones can be obtained. When hbpB was continuously overexpressed while pHbCP was knocked out, the endogenous hbpB expression products that were first degraded were effectively replenished. 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.

[0164] 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.

[0165] Example 2: Construction of a recombinant stable plasmid vector based on the toxin-antitoxin system hbpB / hbpC

[0166] 1. Construction of the LCP of the plasmid-free Halomonas bluephagenesis

[0167] 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 construction and improve its stability, the endogenous plasmid pHbCP using 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 Halomonas strain without plasmids (including the stable endogenous plasmid pHbCP, the exogenous plasmids pSEVA321 (Figure 2A) and pSEVA341 (Figure 2C) used for editing in Example 1) was obtained. It will be named Halomonas Halomonas bluephagenesis LCP (H.bluephagenesis LCP) below.

[0168] Table 2 gRNA sequences used for knockout

[0169] The growth rate of Halomonas bluephagenesis LCP was measured using a medium containing 1.03 mol / L NaCl. The growth curves indicate that knocking out the stable endogenous plasmid pHbCP had no significant effect on the growth rate of Halomonas bluephagenesis (Figure 4). Under optimal culture conditions, H. bluephagenesis LCP maintained normal growth.

[0170] 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.

[0171] 2. Construction of a recombinant stable plasmid vector based on the toxin-antitoxin system hbpB / hbpC

[0172] 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.

[0173] 3. The toxin-antitoxin system hbpB / hbpC is a key stabilizing element

[0174] This study discovered a novel toxin-antitoxin system, hbpB / hbpC, on an endogenous plasmid from a halophilic microorganism. This genetic module maintains the stability of the plasmid through passage without the addition of antibiotics or other selective pressures. Therefore, the toxin-antitoxin system hbpB / hbpC was used as a key stabilizing element in the construction of the recombinant plasmid vector pHbPBC. Because hbpB and hbpC are located in the same operon, and the toxin-antitoxin complex often interacts directly with a 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 multiple publications (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 multiple models, this study selected the 250 bp upstream of hbpB / hbpC as the promoter region ( Figure 6 ). The sequences of the promoter and RBS regions are shown in SEQ ID NO: 5. In addition, other promoter and RBS regulatory sequences that can increase the expression of hbpB / hbpC can also be selected, such as the sequence shown in SEQ ID NO: 11, wherein the partial promoter sequence is SEQ ID NO: 22, and the RBS regulatory sequence is SEQ ID NO: 23.

[0175] 4. Replication origin and replication initiation protein of recombinant stable plasmid vector

[0176] 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 in the stable recombinant plasmid vector pHbPBC to ensure replication and passage in H. bluephagenesis LCP.

[0177] The 861-bp repB gene (SEQ ID NO: 7) is the only known functional sequence on the endogenous plasmid pHbCP, besides the newly discovered toxin-antitoxin system hbpB / hbpC. It encodes a replication initiation protein, RepB (SEQ ID NO: 8), which is also found in other halophilic microorganisms.

[0178] However, due to the incomplete research on the genetic background of Halomonas H. bluephagenesis TD01, the replication start site sequence of its endogenous plasmid is still unknown, and attempts to compare the current replication start site public database and use the prediction methods reported in the literature have failed to determine the replication start site region on pHbCP. Therefore, pHbCP was ligated into the pSEVA341 backbone, but the pSEVA341 vector's original replication origin sequence suitable for Halomonas was deleted. The pSEVA341 vector's broad-host replication origin pRO1600 oriV (SEQ ID NO: 12) and the corresponding replication initiator protein pRO1600 Rep coding region (SEQ ID NO: 13) were deleted. The sequence of the expressed replication initiator protein pRO1600 Rep is shown in SEQ ID NO: 24 ( FIG7A ). The pHbCP sequence was then segmented, initially in 500-bp segments and then gradually reduced in size. It was discovered that a 360-bp sequence upstream of the gene encoding the replication initiator protein RepB is essential for plasmid persistence and replication in Halomonas. Therefore, this segment was used as the replication origin pHbCP ori (SEQ ID NO: 6) and retained to construct the stable recombinant plasmid vector pHbPBC ( FIG7B ).

[0179] In addition, to transform plasmids into Halomonas, chemical transformation, electroporation transformation, and conjugation transformation are currently the three most commonly used methods for transferring exogenous DNA into bacteria. This example uses conjugation as an example to transform plasmids into Halomonas. Conjugation transformation also requires the addition of additional replication origins to pHbPBC.

[0180] To construct a multi-host shuttle plasmid capable of replication and passage in both Halomonas and the E. coli S17-1 strain used for conjugative transformation, we selected the optimized replication origin pMB1 ori from the pUC series of plasmids as the replication origin for the stable recombinant plasmid vector pHbPBC in E. coli S17-1. pMB1 ori relies on the replication initiator protein encoded by the host bacterial genome, eliminating the need for additional addition (Figure 7C). oriT is the initiation site for plasmid transfer during conjugation and is also an essential sequence on pHbPBC (Figure 7D).

[0181] In addition, other high copy number regulatory element sequences may be selected as needed, such as the broad-host replication origin site pRO1600 oriV (SEQ ID NO: 12), and the corresponding replication initiator protein pRO1600Rep coding region (SEQ ID NO: 13).

[0182] 5. Other sequences

[0183] 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 , used for positive clone screening on Escherichia coli S17-1 and Halomonas bluephagenesis LCP plates, respectively. This resistance selection marker is only used during plasmid construction and conjugation transformation; no antibiotics are required during the subsequent production phase of exogenous target gene expression. pHbPBC inserted with the target gene fragment relies on its inherent toxin-antitoxin system, hbpB / hbpC, to maintain stable replication and propagation in host cells.

[0184] 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.

[0185] Thus, this study successfully constructed a stable recombinant plasmid vector pHbPBC suitable for halophilic microorganisms (the stable recombinant plasmid vector with the target gene and resistance gene inserted is shown in Figure 8).

[0186] Example 3: Stability Characterization of Recombinant Stable Plasmid Vector pHbPBC

[0187] 1. Exogenous target gene expression strategy

[0188] 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.

[0189] Strategy 1 (Figure 9): Integrate the exogenous target gene into the genome of the H. bluephagenesis LCP strain, culture it in the absence of antibiotics, and measure the target cell ratio and target protein expression. This is the most commonly used strategy for engineering bacteria. It provides good transformation stability and does not require additional selection pressure. However, the transformation cycle is long, the operation is cumbersome, and multiple manipulations can easily interfere with genome stability.

[0190] Strategy 2 (Figure 10): Insert the exogenous target gene into a general plasmid expression vector and introduce it into the Halomonas bluephagenesis LCP strain via conjugation. Cultivate the cells with and without antibiotics, and measure the plasmid retention rate 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 damage the integrity of the genome. However, since additional selection pressure such as antibiotics is required to maintain the stable existence of the expression vector in the bacteria, it increases production costs and is harmful to the ecological environment.

[0191] 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 in the presence and absence of antibiotics, and the plasmid retention ratio and target protein expression level were measured.

[0192] 2. Stable replication of the recombinant plasmid vector pHbPBC in H. bluephagenesis LCP

[0193] This study used flow cytometry to detect fluorescence in individual cells and measured the average fluorescence intensity of each cell. Of the three exogenous target gene expression strategies described above, Strategy 1 involves integrating the gene encoding the green fluorescent protein (sfGFP) into the bacterial genome. In Strategies 2 and 3, the gene encoding the green fluorescent protein (sfGFP) is located on a plasmid. Therefore, cells carrying the plasmid will emit fluorescence, indicating that the plasmid is stably present in the cell.

[0194] Using a culture medium containing 1.03 mol / L NaCl, H. bluephagenesis LCP strains modified using three different strategies were subcultured for 24 h × 7 days. That is, the bacterial liquid was subcultured 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. Three biological replicates were set up for each group, and 30,000 cell data were recorded for each sample each time.

[0195] First, for the sample group using strategy 1, using the unmodified H. bluephagenesis LCP that does not contain the green fluorescent protein sfGFP encoding gene as a control, basically no fluorescent cells were detected (Figure 11); however, in the H. bluephagenesis sfGFP group with the green fluorescent protein sfGFP encoding gene integrated into the genome, after 7 consecutive days of transfer culture, the average proportion of fluorescent cells remained above 95%. This result was in line with expectations, that is, the target gene can be integrated into the genome and remain stable without loss (Figure 11, Table 3).

[0196] Table 3 The target gene can be integrated into the genome and remain stable without loss

[0197] For Strategy 2, pSEVA341-sfGFP was introduced into H. bluephagenesis LCP by conjugation and subcultured for 7 days with and without antibiotics. Flow cytometric analysis showed that in the antibiotic group, the average percentage of fluorescent cells decreased slightly over the 7 days, from 99.4% on Day 1 to 91.9% on Day 7. Overall, the percentage remained high, above 90%, but slightly lower than the 95.8% on Day 7 of the H. bluephagenesis sfGFP group in Strategy 1. In the antibiotic-free group, the average percentage of fluorescent cells dropped significantly from 99.5% on Day 1 to 25.6% on Day 7, with nearly three-quarters of the cells no longer expressing detectable fluorescence, indicating that the recombinant plasmid pSEVA341-sfGFP had been lost (Figure 12, Table 4).

[0198] Table 4 The stability of pSEVA341 depends on the selective pressure of antibiotics

[0199] This proves that for general expression plasmid vectors without special stabilizing elements, such as pSEVA341, their stability in host cells mainly depends on additional selective pressure, such as the corresponding antibiotics that 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 selective pressure, general expression plasmid vectors cannot maintain stability in cells and will gradually be lost as the bacteria grow and reproduce.

[0200] For Strategy 3, the newly constructed pHbPBC-sfGFP construct was introduced into Halomonas bluephagenes LCP via conjugation and subcultured for 7 days with and without antibiotics. Flow cytometric analysis revealed that the average percentage of fluorescent cells in the antibiotic group decreased slightly over the 7 days, from 99.4% on Day 1 to 93.3% on Day 7. In the antibiotic-free group, the average percentage of fluorescent cells also decreased slightly over the 7 days, from 99.5% on Day 1 to 95.5% on Day 7. However, both groups maintained high levels of integration overall, and compared to the standard plasmid vectors used in Strategy 2, their stability was closer to that of genomic integration in Strategy 1 (Figure 13, Table 5).

[0201] Table 5 The stability of pHbPBC is not dependent on the selective pressure of antibiotics

[0202] Two commonly used strategies for engineering bacterial transformation—integrating the target gene into the genome and culturing under antibiotic-free conditions (i.e., strategy 1, no antibiotics added group), and inserting the target gene into a conventional expression vector to form a recombinant plasmid and culturing under antibiotic-added conditions (i.e., strategy 2, antibiotics added group)—were compared in stability (Figure 14) with the newly developed expression strategy: inserting the target gene into the stable recombinant plasmid pHbPBC, a stable expression vector based on the toxin-antitoxin system hbpB / hbpC, and culturing under antibiotic-free conditions (i.e., strategy 3, no antibiotics added group). The toxin-antitoxin system hbpB / hbpC, discovered in this study on the endogenous plasmid pHbCP of H. bluephagenesis TD01, is effective in maintaining the plasmid's stability and preventing loss. The recombinant plasmid expression vector pHbPBC, with this key stabilizing element, replicates and propagates stably with host cell growth and reproduction without the need for antibiotics, demonstrating similar stability to genomic integration.

[0203] 3. High-level expression of the recombinant plasmid vector pHbPBC in H. bluephagenesis LCP

[0204] Using the same medium containing 1.03 mol / L NaCl, flow cytometry was used to record the average fluorescence intensity of individual cells in each sample group during the 7-day transfer and passage experiment to characterize the expression level of the target protein, green fluorescent protein (sfGFP). The results showed that the stable recombinant plasmid pHbPBC-sfGFP expressed relatively high levels of the target protein, with expression levels 3.5-5 times higher than those obtained using the genomic integration strategy under the same conditions, and 1.5-2 times higher than those obtained using the pSEVA341-sfGFP group (Figure 15).

[0205] Because the expression level of genes on plasmids within cells is directly related to their intracellular copy number, this study measured the copy number of the stable recombinant plasmid vector pHbPBC within H. bluephagenesis LCP. Real-time quantitative PCR revealed that pHbPBC had a copy number of only 3, indicating a low-copy-number plasmid. However, pSEVA341, a high-copy plasmid, was measured to have an intracellular copy number exceeding 300. However, compared to a genome with a copy number of 1, the expression level of the inserted target gene was only 2-2.5 times that of the genomic integration plasmid, and was lower than that of the recombinant plasmid vector pHbPBC with a lower copy number (Figure 15). This may be due to the negative interference of antibiotics added as selective pressure during culture on cell growth and metabolism. Therefore, using the newly constructed pHbPBC-sfGFP strategy as an example, the expression level of the green fluorescent protein (sfGFP) was measured with and without antibiotics.

[0206] 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 added, which means that the addition of antibiotics during the culture process will indeed lead to a decrease in the expression level of the target protein (Figure 16).

[0207] 4. The recombinant plasmid vectors pHvPBC and pHaPBC composed of hbpB / hbpC from Halomonas venusta and Halomonas alkaliantarctica stably replicate in H. bluephagenesis LCP

[0208] The plasmids pHvPBC-sfGFP and pHaPBC-sfGFP were generated by replacing the corresponding elements in the plasmid pHbPBC-sfGFP with the hbpB / hbpC genes and their upstream promoter regions from H. venusta and H. alkaliantarctica, respectively. Flow cytometry was used to detect fluorescence in individual cells. Halomonas bluephagenes LCP carrying the plasmids pHvPBC-sfGFP and pHaPBC-sfGFP were subcultured in a 1.03 mol / L NaCl-containing medium for 7 days, both in the absence and addition of antibiotics. The cultures were subcultured at a 1:1000 ratio every 24 hours for 7 days. The proportion of cells emitting fluorescence was recorded by flow cytometry every 24 hours. The results are shown in Figure 18. The proportion of fluorescent cells remained high in all four groups. This shows that in the Halomonas bacterium H. bluephagenesis LCP, the toxin-antitoxin system hbpB / hbpC derived from H. venusta and H. alkaliantarctica also has the effect of maintaining the stable existence of the plasmid.

[0209] 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 250 bp upstream of hbpB / hbpC were selected as the promoter region, and the sequence of the promoter and RBS region is shown in SEQ ID NO: 30.

[0210] 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 250 bp upstream of hbpB / hbpC were selected as the promoter region, and the sequence of the promoter and RBS region is shown in SEQ ID NO: 35.

[0211] 5. Stable replication of the recombinant plasmid vector pHbPBC in H. bluephagenesis LCP under moderately halophilic conditions

[0212] Flow cytometry was used to detect fluorescence in individual cells. Halomonas bacterium H. bluephagenesis LCP harboring the pHbPBC-sfGFP plasmid was subcultured for 7 days in medium containing either 0.51 mol / L or 2.50 mol / L NaCl, both without and with antibiotics. The culture was subcultured at a 1:1000 dilution ratio every 24 hours for 7 days. The percentage of cells emitting fluorescence was recorded by flow cytometry every 24 hours. The results are shown in Figure 19. The percentage of cells emitting fluorescence remained high in all four groups.

[0213] In addition, H. bluephagenesis has difficulty growing in culture media beyond a moderately halophilic environment. When the salt concentration is too high or too low, the bacteria grows slowly or cannot grow.

[0214] 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 bluephagenesis LCP, under a moderately halophilic environment, the toxin-antitoxin system hbpB / hbpC has the effect of maintaining the stable existence of the plasmid.

[0215] 6. Stable replication of the recombinant plasmid vector pHbPBC in Halomonas campaniensis

[0216] This study used flow cytometry to detect fluorescence in individual cells. Halomonas H. campaniensis carrying the plasmid pHbPBC-sfGFP was subcultured for 7 days in a culture medium containing 0.68 mol / L NaCl, both in the absence and addition of antibiotics. The bacterial solution was subcultured at a ratio of 1:1000 every 24 hours for a total of 7 days; the proportion of cells emitting fluorescence was recorded by flow cytometry every 24 hours. The results are shown in Figure 5. The proportion of cells emitting fluorescence in both groups remained at a high level. This shows that in Halomonas H. campaniensis, the toxin-antitoxin system hbpB / hbpC also has the effect of maintaining the stable presence of the plasmid.

[0217] 7. Recombinant plasmid vectors carrying toxin-antitoxin systems have increased replication stability in Vibrio natriegens

[0218] The plasmid pUC-HbPBC-sfGFP was generated by replacing the replicon of the pHbPBC-sfGFP plasmid with the pUC replicon. The plasmid pUC-sfGFP was also generated by deleting the toxin-antitoxin hbpB / hbpC genes in pUC-HbPBC-sfGFP. This work used flow cytometry to detect fluorescence in individual cells. V. natriegens carrying the plasmids pUC-HbPBC-sfGFP and pUC-sfGFP were subcultured for 7 days in a medium containing 0.51 mol / L NaCl in the absence of antibiotics. The cultures were subcultured at a 1:1000 ratio every 24 hours for 7 days. The percentage of cells emitting fluorescence was recorded by flow cytometry every 24 hours. The results are shown in Figure 17. The group carrying the toxin-antitoxin system maintained a higher percentage of fluorescent cells. This shows that in V. natriegens, the toxin-antitoxin system hbpB / hbpC also has the effect of maintaining the stable existence of the plasmid.

[0219] 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 modification 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.

[0220] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection 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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