Sucrose lethal gene-based halophilic bacterium plasmid and application thereof

Abstract

The invention relates to a halophilic bacterium plasmid based on a sucrose lethal gene. The invention also provides a method for obtaining engineering halophilic bacteria without plasmids by using the halophilic bacteria plasmids.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and more specifically, to a halophilic bacteria plasmid based on a sucrose lethal gene and application thereof. Background Art

[0002] Halophiles are extreme microorganisms that have the ability to grow normally in high-salt, high-pH environments. Using halophiles as the base cells, the next generation of industrial biotechnology based on halophiles can be constructed, which can achieve sterilization-free, energy-saving and water-saving, open continuous fermentation, and biomanufacturing production of polyhydroxyalkanoates (PHA), tetrahydropyrimidine, acetoin, γ-aminobutyric acid and other compounds. It is extremely important to establish an efficient gene editing method in halophiles. The reported gene editing tools based on the type II CRISPR-cas9 system are used for genome editing of halophiles to achieve knockout, insertion or replacement of target positions. In order to obtain stable, resistance-free recombinant strains, the edited strains need to be plasmid-discarded.

[0003] The current method of removing plasmids takes a long time and has a low positive rate, which results in a long time for gene editing strains. The typical method of removing plasmids in the prior art includes the following steps: picking a monoclonal colony that has been successfully genetically modified in a medium without antibiotics, culturing it to the late logarithmic phase, then transferring it to a fresh medium without antibiotics and re-culturing it to the late logarithmic phase. After multiple culturing, diluting the cells by 10 6 The strain is spread on a plate without antibiotics for culture, and then a single clone is picked and streaked on plates with and without antibiotics to identify the strain that has lost the CRISPR plasmid. The entire plasmid removal process takes at least 3 days, which is time-consuming and has low plasmid removal efficiency. Summary of the invention

[0004] In view of the above problems existing in the prior art, the inventors express the sacB gene by using a promoter (preferably an endogenous promoter of halophilic bacteria) to integrate it into a gene editing plasmid, and culture it on a plate containing a certain concentration of sucrose or in a liquid culture medium to assist in plasmid removal. This method does not require multiple subcultures, saves time, and has high plasmid removal efficiency.

[0005] Therefore, according to one aspect of the present invention, there is provided a plasmid for halophilic bacteria, comprising a sucrose lethal gene sacB operably linked to a promoter.

[0006] In one embodiment, the promoter is an inducible promoter or a constitutive promoter, preferably the inducible promoter is selected from lux promoter, lac promoter, trp promoter, tac promoter or a combination thereof, more preferably the promoter of the sacB gene itself, or an endogenous promoter of halophile, even more preferably the promoter is selected from the wild-type Pporin promoter or a mutant thereof, wherein the mutant is selected from Pporin42, Pporin51, Pporin58, Pporin68, Pporin140, Pporin141, Pporin 194, Pporin221, Pporin226 and Pporin278, most preferably Pporin140.

[0007] In another embodiment, the amino acid sequence encoded by the sucrose lethal gene sacB is shown as SEQ ID No.1, and preferably the nucleotide sequence of sacB is shown as SEQ ID No.2.

[0008] In a preferred embodiment, the sequence of the promoter of the sacB gene itself is shown as SEQ ID No.3.

[0009] In a preferred embodiment, the sequence of the wild-type Pporin promoter is shown as SEQ ID No.4.

[0010] In a preferred embodiment, the sequence of the Pporin42 promoter is shown as SEQ ID No.5.

[0011] In a preferred embodiment, the sequence of the Pporin51 promoter is shown as SEQ ID No.6.

[0012] In a preferred embodiment, the sequence of the Pporin58 promoter is shown as SEQ ID No.7.

[0013] In a preferred embodiment, the sequence of the Pporin68 promoter is shown as SEQ ID No.8.

[0014] In a preferred embodiment, the sequence of the Pporin140 promoter is shown as SEQ ID No.9.

[0015] In a preferred embodiment, the sequence of the Pporin141 promoter is shown as SEQ ID No.10.

[0016] In a preferred embodiment, the sequence of the Pporin 194 promoter is shown as SEQ ID No.11.

[0017] In a preferred embodiment, the sequence of the Pporin221 promoter is shown as SEQ ID No.12.

[0018] In a preferred embodiment, the sequence of the Pporin226 promoter is shown as SEQ ID No.13.

[0019] In a preferred embodiment, the sequence of the Pporin278 promoter is shown as SEQ ID No.14.

[0020] In another preferred embodiment, 1) the plasmid is an expression plasmid or a gene editing plasmid, preferably a CRISPR-Cas9 gene editing plasmid; and / or 2) the plasmid further comprises an antibiotic resistance gene, such as spectinomycin, kanamycin, or chloramphenicol.

[0021] In another preferred embodiment, in the CRISPR-Cas9 gene editing plasmid, the sucrose lethal gene sacB is located outside the CRISPR-Cas9 gene editing functional region, that is, it will not be integrated into the genome of the halophilic bacteria after CRISPR-Cas9 gene editing. In another preferred embodiment, in the expression plasmid, sacB and the expressed target gene are under the control of the same or different (independent of each other) promoters.

[0022] According to another aspect of the present invention, a method for obtaining an engineered halophilic bacterium is provided, the method comprising introducing the plasmid according to the present invention into a halophilic bacterium cell.

[0023] In one embodiment, the method further comprises culturing the engineered halophile in a medium containing sucrose after expressing the target gene or after completing gene editing to remove the plasmid.

[0024] In another embodiment, the culture medium is a plate medium or a liquid medium and does not contain antibiotics, wherein the concentration of sucrose in the culture medium is 5 to 100 g / L (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 g / L), preferably 10 to 50 g / L, more preferably less than 10 g / L.

[0025] In another preferred embodiment, the halophile is Halomonas, preferably Halomonas is Halomonas bluephagenesis, Halomonas aydingkolgenesis or Halomonas campaniensis, more preferably Halomonas bluephagenesis TD1.0 (Zhao, H. et al. (2017) Novel T7-like expression systems used for Halomonas. Metab Eng 39, 128-140), Halomonas bluephagenesis TD01 (strain collection number CGMCC No. 4353), Halomonas aydingkolgenesis M1 (strain collection number CGMCC No. 19880), Halomonas campaniensis LS21 (strain collection number CGMCC No. 6593) (see YE J, HU D, CHE X, JIANG X, LI T, CHEN J, ZHANG HM, CHEN GQ. Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose[J]. Metabolic Engineering, 2018,47:143-52).

[0026] In another preferred embodiment, the plasmid is introduced into the halophile cell by conjugative transformation.

[0027] The present invention utilizes the endogenous promoter of halophilic bacteria to over-express the sucrose lethal gene (sacB) to assist in plasmid removal of the recombinant strain, and can quickly and efficiently remove the plasmid to complete the construction of the recombinant halophilic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0029] Figure 1 is the pB01 plasmid map;

[0030] Figure 2 For colony PCR verification;

[0031] Figure 3 is the pB07 plasmid map;

[0032] Figure 4 Plasmid elimination comparison is shown. DETAILED DESCRIPTION

[0033] Unless otherwise indicated, the terms used herein have the general technical meanings understood by those skilled in the art. For definitions and terms in the art, it is particularly recommended that the technician refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0034] In the present invention, the term "halophiles" refers to a type of archaea living in a high salinity environment. In a preferred embodiment of the present invention, the halophiles include but are not limited to the genus Halomonas. Preferably, the genus Halomonas includes but is not limited to Halomonas bluephagenesis, Halomonasaydingkolgenesis or Halomonas campaniensis or its derivatives, more preferably Halomonasbluephagenesis TD1.0, Halomonas bluephagenesis TD01 (strain collection number CGMCC No.4353), Halomonas aydingkolgenesis M1 (strain collection number CGMCC No.19880), Halomonascampaniensis LS21 (strain collection number CGMCC No.6593).

[0035] In the present invention, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that enables a specific gene to be transcribed. The promoter can be recognized by RNA polymerase and begin to transcribe and synthesize RNA. In ribonucleic acid (RNA) synthesis, the promoter can interact with the transcription factor that regulates gene transcription to control the start time and degree of gene expression (transcription). The promoter includes a core promoter region and a regulatory region, which is located in the regulatory sequence that controls gene expression and upstream of the gene transcription start site (5' direction of the DNA antisense strand), and has no compilation function itself. According to its mode of action and function, it is divided into three categories: constitutive promoters (maintaining continuous activity in most or all tissues), specific promoters (tissue specificity or developmental period specificity) and inducible promoters (regulated by external chemical or physical signals).

[0036] In the present invention, the term "operably connected" generally refers to placing the regulatory sequence necessary for the expression of the coding sequence in a suitable position relative to the coding sequence so as to achieve the expression of the coding sequence. For example, when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence, the first nucleic acid sequence is operably connected to the second nucleic acid sequence. In some embodiments, the arrangement of the coding sequence and the transcription control element in the expression vector can be represented. The control element can include a promoter, an enhancer and a termination element. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is operably connected to the coding sequence. In some embodiments, "operably connected" can also refer to that the target gene is connected to the vector so that the transcription and translation control sequences in the vector play their intended functions of regulating the transcription and translation of the target gene.

[0037] In the present invention, the term "sucrose lethal gene sacB" refers to a gene encoding levansucrase, for example from Bacillus subtilis. Since there is a lack of genes for processing levan in Gram-negative bacteria, when the sacB gene is expressed in Gram-negative bacteria, the product levan of the sacB gene will accumulate in the Gram-negative bacteria, leading to bacterial death.

[0038] In the present invention, the term "gene editing" refers to a method of using a site-directed nuclease to cut DNA at a precise target position in the genome, thereby producing single-stranded or double-stranded DNA breaks at a specific position in the genome. Such breaks can be regularly repaired by endogenous cellular processes, such as HDR and non-homologous end joining (NHEJ). The third repair mechanism can be microhomology-mediated end joining (Microhomology-Mediated End Joining, MMEJ), also known as "alternative NHEJ (ANHEJ)", in which small deletions and insertions may occur at the cutting site, and its genetic results are similar to NHEJ.

[0039] In the present invention, the term "CRISPR / Cas system" or "CRISPR-Cas system" generally refers to a nuclease system composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (i.e., Cas proteins), which can cut almost all genomic sequences adjacent to the protospacer-adjacent motif (PAM) in the cell. "CRISPR / Cas system" can be used to collectively refer to transcripts involving CRISPR-associated ("Cas") genes, as well as other elements involved in their expression or guiding their activity, which may include sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or its active portion), tracr partner sequences (in the context of endogenous CRISPR / Cas systems, covering "direct repeats" and processed partial direct repeats), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR / Cas systems), or other sequences and transcripts from CRISPR loci. Five types of CRISPR systems have been identified (e.g., type I, type II, type III, type U, and type V).

[0040] In this application, the term "Cas9 protein" or "Cas9 nuclease", also known as Csn1 or Csx12, generally refers to a class of proteins in the type II CRISPR / Cas system that are involved in both crRNA biosynthesis and the destruction of invading DNA. The Cas9 protein generally includes a RuvC nuclease domain and a HNH nuclease domain, which cut two different strands of a double-stranded DNA molecule, respectively. Cas9 proteins have been described in different bacterial species such as S. thermophiles, Listeria innocua, and S. pyogenes. For example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein is shown in the SwissProt database accession number Q99ZW2; the amino acid sequence of the Neisseria meningitides Cas9 protein is shown in the UniProt database number A1IQ68; the amino acid sequence of the Streptococcus thermophilus Cas9 protein is shown in the UniProt database number Q03LF7; and the amino acid sequence of the Staphylococcus aureus Cas9 protein is shown in the UniProt database number J7RUA5.

[0041] CRISPR / Cas systems can include many short repetitive sequences, called "repeat". When expressed, repetitive sequences can form secondary structures (e.g., hairpins) and / or contain unstructured single-stranded sequences. Repetitive sequences usually appear in clusters and often diverge between species due to evolution. These repetitive sequences are regularly spaced with a unique intermediate sequence called a "spacer", thereby forming a repeat-spacer-repeat (repeat-spacer-repeat) locus structure. The spacer is identical to or has a high degree of homology with a known foreign invader sequence. The spacer-repeat unit encodes crispRNA (crRNA), which is processed into a mature form of the spacer repeat unit. The crRNA contains a "seed" or spacer sequence (a naturally occurring form in prokaryotes, where the spacer sequence targets foreign invader nucleic acids) targeting a target nucleic acid. The spacer sequence is located at the 5' or 3' end of the crRNA.

[0042] In the present application, the term "sequence encoding gRNA" generally refers to a DNA sequence from which the gRNA can be obtained by transcription. The gRNA described in the present application can bind to a sequence in a target nucleic acid. The nucleic acid (or part thereof) targeting the genome can interact with the target nucleic acid in a sequence-specific manner by hybridization (i.e., base pairing). The gRNA described in the present application can be a single-stranded guide RNA (sgRNA), and the nucleotide sequence of the sgRNA can vary according to the sequence of the target nucleic acid.

[0043] In the CRISPR / Cas system of the present application, the gRNA sequence can be designed to hybridize with a target nucleic acid adjacent to a PAM sequence recognizable by the Cas protein used in the system. The gRNA may completely match or mismatch the target sequence. Cas proteins usually have a specific PAM sequence that can be recognized in the target DNA. The gRNA used in the CRISPR system described in the present application can be synthesized by chemical methods, for example, high performance liquid chromatography. For example, two or more RNA molecules are connected together. Longer RNA (e.g., RNA encoding Cas9) can be obtained by enzymatic reaction. In the art, various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic synthesis of RNA, for example, to enhance stability, reduce innate immune response and / or enhance modifications of other properties.

[0044] The CRISPR / Cas9-based gene editing system includes at least one gRNA scaffold. The gRNA scaffold promotes the binding of Cas9 to the gRNA and the endonuclease activity. The gRNA scaffold is a polynucleotide sequence that follows the gRNA targeting sequence. The gRNA targeting sequence and the gRNA scaffold together form a polynucleotide.

[0045] The present invention is further described in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The chemical substances used in the following reactions are all commercially available products unless otherwise indicated.

[0046] Escherichia coli was grown in LB medium, and the medium composition contained: 10 g / L sodium chloride, 10 g / L peptone (OXID Company, UK, product catalog number LP0042), and 5 g / L yeast extract (OXID Company, UK, product catalog number LP0021).

[0047] Halomonas bluephagenesis TD01 was cultured on LB60 medium, which had the same ingredients as LB except that the concentration of sodium chloride was changed to 60 g / L.

[0048] Halomonas bluephagenesis TD01 See Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01 (Tan et al, Bioresource Technology, 2011, 102(17), 8130-6).

[0049] For the CRISPR genome editing method, please refer to the literature CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018, 47, 219-229).

[0050] For the genome editing sites described in this patent, refer to the literature CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018, 47, 219-229).

[0051] The promoter and related gene sequences used in the examples are shown below:

[0052] SacB amino acid sequence: (SEQ ID No.1)

[0053] MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISHITRHDMLQIPEQQKNEKYQVPEFDSSTIK

[0054] NISSAKGLDVWDSWPLQNADGTVANYHGYHIVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDK

[0055] FDANDSILKDQTQEWSGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG

[0056] KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKAYYGKSTSFFRQESQK

[0057] LLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDEIERANVFKMNGKWYLFTDSRGSKMTIDGIT

[0058] SNDIYMLGYVSNSLTGPYKPLNKTGLVLKMDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSVVKDSILEQGQLTVNK*

[0059] SacB gene sequence: (SEQ ID No.2)

[0060] atgaacatcaaaaagtttgcaaaacaagcaacagtattaacctttactaccgcactgctggcaggaggcgcaac

[0061] tcaagcgtttgcgaaagaaacgaaccaaaagccatataaggaaacatacggcatttcccatattacacgccatg

[0062] atatgctgcaaatccctgaacagcaaaaaaatgaaaaatatcaagttcctgagttcgattcgtccacaattaaa

[0063] aatatctcttctgcaaaaggcctggacgtttgggacagctggccattacaaaacgctgacggcactgtcgcaaa

[0064] ctatcacggctaccacatcgtctttgcattagccggagatcctaaaaatgcggatgacacatcgatttacatgt

[0065] tctatcaaaaagtcggcgaaacttctattgacagctggaaaaacgctggccgcgtctttaaagacagcgacaaa

[0066] ttcgatgcaaatgattctatcctaaaagaccaaacacaagaatggtcaggttcagccacatttacatctgacgg

[0067] aaaaatccgtttattctacactgatttctccggtaaacattacggcaaacaaacactgacaactgcacaagtta

[0068] acgtatcagcatcagacagctctttgaacatcaacggtgtagaggattataaatcaatctttgacggtgacgga

[0069] aaaacgtatcaaaatgtacagcagttcatcgatgaaggcaactacagctcaggcgacaaccatacgctgagaga

[0070] tcctcactacgtagaagataaaggccacaaatacttagtatttgaagcaaacactggaactgaagatggctacc

[0071] aaggcgaagaatctttatttaacaaagcatactatggcaaaagcacatcattcttccgtcaagaaagtcaaaaa

[0072] cttctgcaaagcgataaaaaacgcacggctgagttagcaaacggcgctctcggtatgattgagctaaacgatga

[0073] ttaacacactgaaaaaagtgaatgaaaccgctgattgcatctaacacagtaacagatgaaattgaacgcgcgaacg

[0074] tctttaaaatgaacggcaaatggtatctgttcactgactcccgcggatcaaaatgacgattgacggcattacg

[0075] tctaacgatatttacatgcttggttatgtttctaattctttaactggcccatacaagccgctgaacaaaactgg

[0076] ccttgtgttaaaaaatggatcttgatcctaacgatgtaacctttacttactcacattcgctttacctcaagcga

[0077] aaagcaacaatgtcgtgattacaagctatatgacaaacagaggattctacgcagacacaacaatcaacgtttgcg

[0078] cccagcttcctgctgaacatcaaaggcaagaaaacatctgttgtcaaagacagcatccttgaacaaggacaatt

[0079] aacagtaacaaata

[0080] P sacB :(SEQ ID No.3)

[0081] tgaatggcgtttgaattgtgattaaaaaggcaactttatgcccatgcaacaagaaactataaaaaatagaagagaa

[0082] tgaaaagaaacagatagattttttagttctttaggcccgtagtctgcaaatccttttatgattttctatcaaac

[0083] aaaaaaggaaaatagaccagttgcaatccaaacgagagtctaatagaatgaggtcgaaaagtaatcgcgcggg

[0084] tttgttactgataaagcaggcaagacctaaaatgtgtaaagggcaaagtgtatactttggcgtcaccccttaca

[0085] tattttaggtctttttttattgtgcgtaactaacttgccatcttcaaacaggagggctggaagaagcagaccgc

[0086] taacacagtacataaaaaaggagacatgaacg

[0087] Pporin:(SEQ ID No.4)

[0088] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatagagtTTGACCTGCGAGCAaGC

[0089] TGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAga

[0090] gttactagagaaagaggagaaatactag

[0091] Pporin42:(SEQ ID No.5)

[0092] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCACTAtagagtTTGACCTGCGAGCAaGC

[0093] TGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAga

[0094] gttactagagaaagaggagaaatactag

[0095] Pporin51:(SEQ ID No.6)

[0096] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCATTTtagagtTTGACCTGCGAGCAaGC

[0097] TGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAga

[0098] gttactagagaaagaggagaaatactag

[0099] Pporin58:(SEQ ID No.7)

[0100] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAATAtagagtTTGACCTGCGAGCAaGC

[0101] TGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAga

[0102] gttactagagaaagaggagaaatactag

[0103] Pporin68:(SEQ ID No.8)

[0104] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAGACtagagtTTGACCTGCGAGCAaGC

[0105] TGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAga

[0106] gttactagagaaagaggagaaatactag

[0107] Pporin140:(SEQ ID No.9)

[0108] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatAAGAtTTGACCTGCGAGCAaGC

[0109] TGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAga

[0110] gttactagagaaagaggagaaatactag

[0111] Pporin141:(SEQ ID No.10)

[0112] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatATACtTTGACCTGCGAGCAaGC

[0113] TGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAga

[0114] gttactagagaaagaggagaaatactag

[0115] Pporin194:(SEQ ID No.11)

[0116] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatCTAAtTTGACCTGCGAGCAaGC

[0117] TGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAga

[0118] gttactagagaaagaggagaaatactag

[0119] Pporin221:(SEQ ID No.12)

[0120] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatGCTAtTTGACCTGCGAGCAaGC

[0121] TGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAga

[0122] gttactagagaaagaggagaaatactag

[0123] Pporin226: (SEQ ID No.13)

[0124] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatAAAGtTTGACCTGCGAGCAaGC

[0125] TGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAga

[0126] gttactagagaaagaggagaaatactag

[0127] Pporin278: (SEQ ID No.14)

[0128] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatTAAAtTTGACCTGCGAGCAaGC

[0129] TGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAga

[0130] gttactagagaaagaggagaaatactag

[0131] SgRNA sequence: (SEQ ID No.15)

[0132] TTGACAGCTAGCTCAGTCCTAGGTATAATACTAGTattgtgcttgccccacacttGTTTTAGAGCTAGAAATAG

[0133] CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAA

[0134] The primers used in the examples are shown in Table 1.

[0135] Table 1. PCR primer sequences

[0136]

[0137]

[0138] Example 1

[0139] Construction of pB01 plasmid carrying sacB gene

[0140] The backbone of the pB01 plasmid is pSEVA341 (see the literature for plasmid: Silva-Rocha, Rafael, et al. "The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes." Nucleic Acids Research 41. D1 (2012): D666-D675). The sacB element is inserted after the lambda t0 terminator position on the pSEVA341 plasmid. The sequence of the sacB element is SEQ ID No. 2. The plasmid map is shown in Figure 1 The specific steps are as follows:

[0141] Using the pSEVA341 plasmid as a template, primers pSB-F / R were designed to amplify the vector backbone fragment; using the plasmid containing the sacB gene as a template, primers sacB-F / R were designed to amplify the sacB fragment; the above fragments were connected by Gibson, and the connection products were transferred to S17-1 competent cells (Beijing Zhuangmeng International Biogene Technology Co., Ltd.), and spread on LB plates containing kanamycin antibiotics (50μg / ml). After a single colony grows on the plate, a single colony of appropriate size is selected for PCR verification ( Figure 2 ) to obtain the recombinant plasmid pB01 (with sacB gene's own promoter SEQ ID No. 3) carrying the sacB gene.

[0142] Example 2

[0143] Testing the sensitivity of sacB gene expression to sucrose in halophilic bacteria

[0144] Plasmid pB01 was transferred into Halomonas bluephagenesis TD01 by conjugation transformation, and then spread on a 60LB plate containing spectinomycin antibiotic (100μg / ml). After a single clone grew on the plate, the halophilic strain TDS1 with the sacB gene was obtained. TD01 and TDS1 strains were inoculated in 60LB and 60LB+spectinomycin (100μg / ml) liquid culture medium, respectively, and the bacterial liquid was spread on a 60LB plate containing sucrose. On plates with a sucrose concentration of less than 500g / L, both strains without and with the sacB gene grew single clones; on plates with a sucrose concentration of more than 500g / L, strains without the sacB gene grew normally on the plate, while strains with the sacB gene could not grow on the plate. The results showed that halophiles are insensitive to sucrose; halophiles with the sacB gene are insensitive to low concentrations of sucrose, but sensitive to high concentrations of sucrose.

[0145] Example 3

[0146] Optimizing sacB gene expression to improve the sensitivity of recombinant halophilic bacteria to sucrose

[0147] The promoter of the sacB gene itself was used to express in halophiles, but it was not sensitive to low concentrations of sucrose. The reason may be that the gene expression level was low. Therefore, the gene expression level was improved by replacing the original promoter with the endogenous promoter of halophiles. The selected promoter was the wild type P in halophiles. porin (SEQ ID No.4), mutant P porin221 (SEQ ID No. 12), P porin68 (SEQ ID No.8) and P porin140 (SEQ ID No.9).

[0148] (1) Construction of sacB gene plasmids expressed by different promoters

[0149] Using pB01 plasmid as template, primers pSB-F1 / R1 were designed to amplify the vector backbone fragment; porin , P porin221 , P porin68 , P porin140 The plasmid of the gene was used as a template and primers P porin -F / R amplify the corresponding promoter fragment; connect the above fragments by Gibson, transfer the connection product to S17-1 competent cells, and spread it on LB plates containing kanamycin antibiotics. After a single colony grows on the plate, pick a single colony of appropriate size for sequencing verification, and obtain P porin , P porin221 , P porin68 , Pporin140 Plasmids pB02, pB03, pB04, and pB05 expressing the sacB gene.

[0150] (2) Construction of strains containing the sacB gene expressed by different promoters

[0151] Plasmids pB02, pB03, pB04, and pB05 were transformed into Halomonas blue phagenesis TD01 by conjugation, and then spread on 60LB plates containing spectinomycin antibiotics. After single colonies grew on the plates, P porin , P porin221 , P porin68 , P porin140 The strains TDS02, TDS03, TDS04, and TDS05 expressing the sacB gene were inoculated in 60LB+spectimycin liquid medium and the bacterial liquid was spread on 60LB plates containing different sucrose concentrations. The results showed that P porin , P porin221 The strain expressing the sacB gene had no single colony growing on the plates containing 100 g / L and 200 g / L sucrose, but had more single colonies growing on the 50 g / L plate; P porin68 The strain expressing the sacB gene had no single colony growing on the plates containing 100 g / L and 200 g / L sucrose concentrations, and a few single colonies grew on the plates containing 50 g / L sucrose concentrations; P porin140 The strain expressing the sacB gene did not grow a single colony on the plates containing 50 g / L, 100 g / L, and 200 g / L sucrose concentrations, indicating that the strain expressing the sacB gene using the endogenous promoter of halophilic bacteria was sensitive to sucrose. This result shows that P porin140 The expressed sacB gene is particularly suitable for the subsequent auxiliary removal of halophilic bacteria resistance plasmids.

[0152] Example 4

[0153] Verification of sacB gene loss on helper plasmid

[0154] (1) Construction of knockout plasmid pB06

[0155] Using pSEVA341 as a template, primers pF / pR were used to amplify the vector backbone; using TD01 strain as a template, primers HL-F / R and HR-F / R were used to amplify the upstream and downstream homology arms of the knockout lpxM gene (lipid Abiosynthesis acyltransferase (EGP20428.1)) respectively; a plasmid containing sgRNA (SEQ ID No.15) (plasmid pW06, see Wang Z, Zheng Y, Ji M, Zhang X, Wang H, Chen Y, Wu Q, Chen GQ. Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defective Halomonas blue phagenesis grown in bioreactors.MicrobBiotechnol.2022May;15(5):1586-1597) was used as a template, and the fragment was amplified using primers P01-F / R; the above fragments were connected by Gibson, and the connection product was transferred to S17-1 competent cells and coated on LB plates containing kanamycin antibiotics. After a single colony grew on the plate, a single colony of appropriate size was selected for PCR verification to obtain the knockout plasmid pB06.

[0156] (2) Construction of knockout plasmid pB07 with sacB gene

[0157] Using pB05 as a template, primers pF / pR were used to amplify the vector backbone; using TD01 strain as a template, primers HL-F1 / R1 and HR-F1 / R1 were used to amplify the upstream and downstream homology arms of the knockout lpxM gene respectively; using the plasmid containing sgRNA as a template, primers P01-F / R were used to amplify the fragment; the above fragments were connected by Gibson, and the connection product was transferred to S17-1 competent cells and coated on LB plates containing kanamycin antibiotics. After a single colony grew on the plate, a single colony of appropriate size was selected for PCR verification to obtain the knockout plasmid pB07 (plasmid map as shown in Figure 3 shown).

[0158] (3) Construction of lpxM gene knockout strain

[0159] The embodiment uses the pQ08 plasmid (see Qin Q, Ling C, Zhao Y, et al. CRISPR / Cas9 editing genome of extremophile Halomonas spp [J]. Metabolic engineering, 2018, 47: 219-229). The pQ08 plasmid itself is very easy to lose after the strain is propagated. The plasmid pB06 and the pQ08 plasmid were respectively transferred into Halomonas bluephagenesis TD01 by conjugation transformation. PCR and sequencing verification proved that the lpxM gene had been successfully knocked out to obtain TDM01. The plasmid pB07 and the pQ08 plasmid were respectively transferred into Halomonas bluephagenesis TD01 by conjugation transformation. PCR and sequencing verification proved that the lpxM gene had been successfully knocked out to obtain TDM02.

[0160] The successfully edited TDM01 strain was cultured in 60LB liquid medium and transferred every 12 hours. After three consecutive subcultures, the bacterial solution was diluted 10 with 60LB medium. 6 Multiple clones were picked from the 60LB non-antibiotic plate and streaked onto 60LB+spectimycin (100 μg / ml), 60LB+chloramphenicol (25 μg / ml), and 60LB non-antibiotic plate, and cultured at 37°C. The colonies that could grow well on the 60LB non-antibiotic plate but could not grow on the spectinomycin and chloramphenicol antibiotic plates successfully eliminated the plasmid.

[0161] The successfully edited TDM02 strain was cultured in 60LB liquid medium, and then the bacterial solution was diluted 10 6 The clones were plated on 60LB+sucrose plates and cultured. After that, several single clones on 60LB+sucrose plates were picked and streaked on 60LB+spectimycin, 60LB+chloramphenicol, and 60LB antibiotic-free plates in turn and cultured at 37°C. The colonies that could grow well on 60LB antibiotic-free plates but could not grow on spectinomycin and chloramphenicol antibiotic plates successfully eliminated the plasmid.

[0162] The successfully edited TDM02 strain was cultured in 60LB+sucrose liquid medium, and then the bacterial solution was diluted 10 with 60LB medium. 6The clones were then plated on 60LB plates and cultured. After that, several single clones on the 60LB plates were picked and streaked on 60LB+spectimycin, 60LB+chloramphenicol, and 60LB antibiotic-free plates in turn and cultured at 37°C. The colonies that could grow well on the 60LB antibiotic-free plates but could not grow on the spectinomycin and chloramphenicol antibiotic plates successfully eliminated the plasmid.

[0163] Plasmid elimination efficiency Figure 4 After three passages, the plasmid elimination efficiency of TDM01 was 3.33%; after two sucrose-assisted plasmid elimination of TDM02, the plasmid elimination efficiency was 100%.

[0164] Example 5

[0165] Optimization of sucrose concentration in solid culture medium

[0166] Prepare 60LB plates with sucrose concentrations of 5 g / L, 10 g / L, 15 g / L, and 20 g / L in sequence, inoculate a single clone of the TDM02 strain carrying the sacB gene into the 60LB medium for culture, and then dilute the bacterial solution with 60LB medium by 10 6 times, and spread them on 60LB plates with 5g / L, 10g / L, 15g / L, and 20g / L sucrose concentrations. Then, multiple single clones on 60LB plates with 5g / L, 10g / L, 15g / L, and 20g / L sucrose concentrations were picked, and streaked on 60LB+spectinomycin, 60LB+chloramphenicol, and 60LB non-antibiotic plates in turn, and cultured at 37°C. The plasmid elimination efficiency is shown in the table below. When adding sucrose concentrations above 10g / L, the plasmid elimination efficiency can reach 100%, so the 60LB plate with a sucrose concentration of 10g / L was selected for screening.

[0167] Table 2. Comparison of plasmid removal in recombinant halophilic bacteria at different sucrose concentrations

[0168]

[0169] Example 6

[0170] Optimization of sucrose concentration in liquid culture medium

[0171] 60LB liquid culture medium with sucrose concentrations of 5 g / L, 10 g / L, 15 g / L, and 20 g / L was prepared in sequence, and a single clone of the TDM02 strain carrying the sacB gene was inoculated into the 60LB culture medium for culture, and then the bacterial solution was diluted 10 6times, and spread them on 60LB plates respectively. Then pick multiple single clones on the 60LB plate, streak them on 60LB+spectinomycin, 60LB+chloramphenicol, and 60LB non-antibiotic plates in turn, and culture at 37°C. The plasmid elimination efficiency is shown in the table below. When adding sucrose concentrations above 10g / L, the plasmid elimination efficiency can reach 100%, so 60LB+10g / L sucrose liquid medium is selected for screening.

[0172] Table 3. Comparison of plasmid removal in recombinant halophilic bacteria at different sucrose concentrations

[0173]

[0174] Those skilled in the art should understand that, although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught by the present invention, without departing from the spirit of the present invention, those skilled in the art can make appropriate modifications or improvements, and the equivalent embodiments obtained therefrom are all within the scope of the present invention.

Claims

1. A plasmid for halophilic bacteria, comprising a sucrose lethal gene sacB operably linked to a promoter, wherein the promoter is a promoter of the sacB gene itself or an endogenous promoter of the halophilic bacteria.

2. The plasmid according to claim 1, wherein the promoter is an endogenous promoter of halophilic bacteria selected from the wild-type Pporin promoter or its mutants, wherein the mutants are selected from Pporin42, Pporin51, Pporin58, Pporin68, Pporin140, Pporin141, Pporin194, Pporin221, Pporin226 and Pporin278, preferably Pporin140.

3. The plasmid according to claim 1 or 2, wherein the amino acid sequence encoded by the sucrose lethal gene sacB is as shown in SEQ ID No.1, and preferably the nucleotide sequence of sacB is as shown in SEQ ID No.

2.

4. The plasmid according to any one of claims 1 to 3, wherein: 1) The plasmid is an expression plasmid or a gene editing plasmid, preferably a CRISPR-Cas9 gene editing plasmid; and / or 2) The plasmid also contains an antibiotic resistance gene, such as spectinomycin, kanamycin, and chloramphenicol.

5. The plasmid according to claim 4, wherein in the CRISPR-Cas9 gene editing plasmid, the sucrose lethal gene sacB is located outside the CRISPR-Cas9 gene editing functional region, that is, it will not be integrated into the genome of the halophilic bacteria after CRISPR-Cas9 gene editing.

6. A method for obtaining an engineered halophilic bacterium, the method comprising introducing the plasmid according to any one of claims 1 to 5 into a halophilic bacterium cell.

7. The method according to claim 6, further comprising culturing the engineered halophilic bacteria in a medium containing sucrose after expressing the target gene or after completing gene editing to remove the plasmid.

8. The method according to claim 7, wherein the culture medium is a plate culture medium or a liquid culture medium and does not contain antibiotics, wherein the concentration of sucrose in the culture medium is 5 to 100 g / L, preferably 10 to 50 g / L, more preferably below 10 g / L.

9. according to the method described in any one in claim 6 to 8, wherein the halophile is Halomonas, preferably the Halomonas is Halomonas bluephagenesis, Halomonas aydingkolgenesis or Halomonascampaniensis, more preferably Halomonas bluephagenesis TD1.0, Halomonas bluephagenesisTD01 (strain collection number CGMCC No.4353), Halomonas aydingkolgenesis M1 (strain collection number CGMCC No.19880) or Halomonas campaniensis LS21 (strain collection number CGMCC No.6593).

10. The method according to any one of claims 6 to 9, wherein the plasmid is introduced into the halophile cell by conjugative transformation.

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