CN114480453B - Escherichia coli for synthesizing monophosphoryl lipid A containing only 3 fatty acid chains - Google Patents
Escherichia coli for synthesizing monophosphoryl lipid A containing only 3 fatty acid chains Download PDFInfo
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- CN114480453B CN114480453B CN202210111489.4A CN202210111489A CN114480453B CN 114480453 B CN114480453 B CN 114480453B CN 202210111489 A CN202210111489 A CN 202210111489A CN 114480453 B CN114480453 B CN 114480453B
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- 241000588724 Escherichia coli Species 0.000 title claims abstract description 30
- 125000005313 fatty acid group Chemical group 0.000 title abstract description 9
- 229940035032 monophosphoryl lipid a Drugs 0.000 title abstract description 5
- 230000002194 synthesizing effect Effects 0.000 title abstract description 4
- 125000002252 acyl group Chemical group 0.000 claims abstract description 46
- 108090000992 Transferases Proteins 0.000 claims abstract description 22
- 241000894006 Bacteria Species 0.000 claims abstract description 19
- 150000002632 lipids Chemical class 0.000 claims abstract description 19
- 101150052479 lpxP gene Proteins 0.000 claims abstract description 14
- 101150060640 lpxM gene Proteins 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 13
- 101150011311 lpxL gene Proteins 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- 102000004357 Transferases Human genes 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 12
- 108091033409 CRISPR Proteins 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 6
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- 239000013604 expression vector Substances 0.000 claims description 5
- 101150109249 lacI gene Proteins 0.000 claims description 5
- 101150083361 lpxE gene Proteins 0.000 claims description 5
- 101150050459 pagL gene Proteins 0.000 claims description 5
- 238000010354 CRISPR gene editing Methods 0.000 claims description 3
- 241001052560 Thallis Species 0.000 claims description 3
- 238000013518 transcription Methods 0.000 claims description 3
- 230000035897 transcription Effects 0.000 claims description 3
- 230000004568 DNA-binding Effects 0.000 claims description 2
- 241000607142 Salmonella Species 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 241000589601 Francisella Species 0.000 claims 1
- GZQKNULLWNGMCW-PWQABINMSA-N lipid A (E. coli) Chemical group O1[C@H](CO)[C@@H](OP(O)(O)=O)[C@H](OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)[C@@H](NC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCC)[C@@H]1OC[C@@H]1[C@@H](O)[C@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OP(O)(O)=O)O1 GZQKNULLWNGMCW-PWQABINMSA-N 0.000 abstract description 42
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 239000013612 plasmid Substances 0.000 description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 15
- 238000000132 electrospray ionisation Methods 0.000 description 15
- 239000002609 medium Substances 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
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- 238000010276 construction Methods 0.000 description 8
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- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 7
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- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
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- XAOLJGCZESYRFT-ICVWGWAISA-N (2r,5r)-2-[(2r,4r,5r)-2-carboxy-6-[(1r)-1,2-dihydroxyethyl]-5-hydroxy-2-[[(3s,5s,6r)-6-[[(3s,5s,6r)-3-hydroxy-5-[[(3r)-3-hydroxytetradecanoyl]amino]-4-[(3r)-3-hydroxytetradecanoyl]oxy-6-phosphonooxyoxan-2-yl]methoxy]-5-[[(3r)-3-hydroxytetradecanoyl]amino] Chemical compound O[C@H]1C(OC(=O)C[C@H](O)CCCCCCCCCCC)[C@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OP(O)(O)=O)OC1CO[C@H]1[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)C(OC(=O)C[C@H](O)CCCCCCCCCCC)[C@H](OP(O)(O)=O)C(CO[C@]2(OC([C@H](O)[C@H](O[C@]3(OC([C@H](O)C(O)C3)[C@H](O)CO)C(O)=O)C2)[C@H](O)CO)C(O)=O)O1 XAOLJGCZESYRFT-ICVWGWAISA-N 0.000 description 2
- 101100234243 Aquifex aeolicus (strain VF5) kdtA gene Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 2
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- -1 WaaC Proteins 0.000 description 2
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- 238000006555 catalytic reaction Methods 0.000 description 2
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- 239000000411 inducer Substances 0.000 description 2
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- HEHQDWUWJVPREQ-XQJZMFRCSA-N lipid X Chemical compound CCCCCCCCCCC[C@@H](O)CC(=O)N[C@H]1[C@@H](OP(O)(O)=O)O[C@H](CO)[C@@H](O)[C@@H]1OC(=O)C[C@H](O)CCCCCCCCCCC HEHQDWUWJVPREQ-XQJZMFRCSA-N 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 229960000268 spectinomycin Drugs 0.000 description 2
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
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- 238000012546 transfer Methods 0.000 description 2
- 239000012646 vaccine adjuvant Substances 0.000 description 2
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- 239000013598 vector Substances 0.000 description 2
- 101150040194 waaA gene Proteins 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- WIIZWVCIJKGZOK-IUCAKERBSA-N 2,2-dichloro-n-[(1s,2s)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide Chemical compound ClC(Cl)C(=O)N[C@@H](CO)[C@@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-IUCAKERBSA-N 0.000 description 1
- 102000057234 Acyl transferases Human genes 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
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- 108010078791 Carrier Proteins Proteins 0.000 description 1
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
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- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- 150000000963 Kdo2-lipid A derivatives Chemical class 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
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- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- CYKLRRKFBPBYEI-NQQHDEILSA-N UDP-alpha-D-glucosamine Chemical compound O1[C@H](CO)[C@@H](O)[C@H](O)[C@@H](N)[C@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 CYKLRRKFBPBYEI-NQQHDEILSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 102000005922 amidase Human genes 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000002019 anti-mutation Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- 108700014210 glycosyltransferase activity proteins Proteins 0.000 description 1
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- 210000004020 intracellular membrane Anatomy 0.000 description 1
- 101150033242 lpxC gene Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000004712 monophosphates Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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Abstract
The invention discloses escherichia coli for synthesizing monophosphoryl lipid A with only 3 fatty acid chains, belonging to the fields of genetic engineering and biological engineering. The invention knocks out secondary acyl chain transferase genes lpxL, lpxP and lpxM on the genome of the escherichia coli HW 003. Recombinant HWJ003, which is a lipid A-type strain lacking the 1-position phosphoric acid, the 3-position primary acyl chain, the 2 '-position secondary acyl chain and the 3' -position secondary acyl chain, is provided. The lipid A structure provides a method for identifying the function of a lipid A secondary acyl chain transferase gene of gram-negative bacteria in Escherichia coli.
Description
Technical Field
The invention relates to escherichia coli for synthesizing monophosphoryl lipid A with only 3 fatty acid chains, belonging to the fields of genetic engineering and bioengineering.
Background
Coli is a gram-negative model in Lipid a biosynthesis studies, wherein Lipid a synthesis starts with UDP-glucosamine acetic anhydride (UDP-GlcNAc), and a 3-OH fatty acid chain is added at the C2 and C3 positions by successive catalysis of three soluble enzymes LpxA, lpxC and LpxD, respectively, to form UDP-diamido-glucose (UDP-diacetyl-GlcN), and then Lipid X is formed by hydrolysis of LpxH. One molecule of Lipid X and one molecule of UDP-Diacyl-GlcN are condensed into 1-Disaccharide phosphate (dispersoid-1-P) by LpxB, transferred to the inner side of an intracellular membrane, and subjected to a C4' phosphorylation reaction by LpxK catalysis to form Lipid IV A . Next, kdtA adds two molecules of Kdo groups to the C6' position, forming Kdo2-Lipid IVA. Kdo2-Lipid IVA is then acylated with LpxL and LpxM to form Kdo2-Lipid A (Kdo 2-LipidA). LpxK, kdtA, lpxL and LpxM are endomembrane proteins, the catalytic site being inside the endomembrane. LpxL adds a 12C secondary acyl chain at the C2 'position and LpxM adds a 14C secondary fatty acid chain at the C3' position. Subsequently, the core saccharide is linked to the Kdo group by glycosyltransferases such as WaaC, waaF, etc., forming the core polysaccharide-lipid a, which is turned outside the inner membrane by the action of the membrane transporter MsbA. O-antigen is synthesized in cytoplasm and transported to periplasmic space, and is connected to core polysaccharide-lipoid A under the action of WaaL to form complete LPS and transported to outer side of outer membrane under the action of various transport proteins. However, the intact E.coli lipid A has a higher molecular weight, and ESI/MS may produce more structural components when identifying its structure, and wild-type E.coli cannot be used to identify lipid A acylases of other gram-negative bacteria. It has been reported that lipid A can be obtained by knocking out the gene lacI encoding DNA binding transcription repressor on the genome of E.coli, the lipid A synthesis gene lpxM, and overexpressing the heptyltransferase encoding genes waaC and waaF.
Disclosure of Invention
In order to solve the technical problems, the invention knocks out genes encoding three lipid A secondary acyl chain transferases on the genome in escherichia coli W3110, knocks in genes encoding lipid A3-O-deacylase from salmonella and genes encoding lipid A1-phosphatase from Francisco, provides escherichia coli HWJ003 of monophosphate lipid A which lacks 1-position phosphoric acid, 3-position primary acyl chain, 2 '-position secondary acyl chain and 3' -position secondary acyl chain and contains only 3 fatty acid chains, wherein the 3 fatty acid chains are 2-position primary acyl chain, 2 '-position primary acyl chain and 3' -position primary acyl chain.
The invention provides a genetic engineering bacterium with a simplified lipoid A structure, which takes escherichia coli as an initial strain, knocks out a gene lacI of a coding DNA combined transcription repressor on a genome, three genes lpxL, lpxP and lpxM of lipoid A secondary acyl chain transferase, and overexpresses a gene pagL of lipoid A3-O-deacylase and a gene lpxE of lipoid A1-phosphatase.
In one embodiment, the sequence of the lacI has an accession number "NP-414879.3" on NCBI.
In one embodiment, the sequences of lpxL, lpxP, and lpxM have accession numbers "np_415572.1", "np_416879.4", and "np_416369.1" in order on NCBI.
In one embodiment, the NCBI accession number of the sequence of the gene pagL encoding lipid A3-O-deacylase is NP-461188.1.
In one embodiment, the sequence encoding the lipid A1-phosphatase gene lpxE has the NCBI accession number WP_003041686.1.
In one embodiment, the E.coli comprises E.coli W3110.
The invention provides a method for preparing lipoid A with a simplified structure, which comprises the following specific steps: (1) Inoculating the genetically engineered bacteria into a culture medium to obtain bacterial liquid; (2) Collecting thallus and extracting lipoid A in the Bligh-Dyer mixed system.
In one embodiment, the lipid a is structured as a monophosphate lipid a lacking the 1 st, 3 rd, 2 'and 3' second acyl chains based on the wild type e.
The invention also provides application of the genetically engineered bacterium in identifying genes for encoding lipid A secondary acyl chain transferase, wherein the application is that the expression vector is utilized to express the genes for encoding the lipid A secondary acyl chain transferase in the genetically engineered bacterium to obtain recombinant bacterium, the lipid A is induced to be expressed and extracted, and then the genes for encoding the lipid A secondary acyl chain transferase are identified through ESI/MS.
In one embodiment, the gene encoding a lipid a secondary acyl chain transferase is selected from gram negative bacteria.
In one embodiment, the expression vector comprises pBAD33.
In one embodiment, the conditions for inducing expression are inoculating the recombinant bacterium into a medium and inducing expression using an inducer.
In one embodiment, the inducer comprises arabinose.
In one embodiment, the extraction is extraction of lipid A using a Bligh-Dyer mixing system.
The invention also provides application of the genetically engineered bacterium in preparation of vaccine adjuvants.
The beneficial effects are that:
the lipid A structure-simplified genetically engineered bacterium can rapidly and effectively identify genes of other strains for encoding lipid A secondary acyl chain transferase, and can also be used as chassis strains for further modifying the lipid A structure to prepare vaccine adjuvants. The genetically engineered bacterium of the invention can be used for biosynthesis of the monophosphoryl lipid A with the simplest structure and containing only three fatty acid chains.
Drawings
Fig. 1: HWJ003 gene knockout flow chart; x is the target genes lpxL, lpxP and lpxM.
Fig. 2: HWJ003 knockdown results verify.
Fig. 3: ESI/MS analysis of HWJ003 lipidA samples.
Fig. 4: ESI/MS analysis of HWJ003-pBAD-RS01045 lipdA samples.
Fig. 5: A. ESI/MS analysis of Vibrio parahaemolyticus (Vibriopahaemeolyticus) ATCC 33846. B. ESI/MS analysis of vibrio parahaemolyticus VP-RS 01045 gene knockout strain.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Unless otherwise indicated, the reagents and materials used in the following examples are commercially available or may be prepared by known methods.
The following examples relate to the following media:
ddH was used for the medium 2 And (3) preparing O, and sterilizing for 15-20 min at 121 ℃ after the preparation is finished.
LB medium (g/L): yeast powder 5, peptone 10 and NaCl 10.
LBC medium (g/L): yeast powder 5, peptone 10,NaCl 10,Cm 0.005.
TABLE 1 primer sequences according to the following examples
TABLE 2 strains and plasmids involved in the examples below
EXAMPLE 1 construction of knockout plasmid
Three genes related to lipid a secondary acyl chain transferase in escherichia coli were knocked out using CRISPR/Cas9 knockout system, requiring construction of 3 knockdown plasmids: pTargetF-lpxL, pTargetF-lpxP and pTargetF-lpxM, the construction process of these plasmids is specifically as follows:
(1) Selecting 20nt N complementary with target sequence of target gene 20 Sequence, in particular N 20 The sequences are respectively underlined sequences of the primers pTargetF-lpxL, pTargetF-lpxP and pTargetF-lpxM, and the sequences are modified to the 5' end of the forward primer and the reverse primer of the plasmid pTargetF to obtain the forward primer pTargetF-lpxL-F, pTargetF-lpxP-F, pTargetF-lpxM-F and the reverse primer pTargetF-lpxL-R, pTargetF-lpxP-R, pTargetF-lpxM-R.
The plasmid pTargetF is used as a template, forward and reverse primers of pTargetF-lpxL, pTargetF-lpxP and pTargetF-lpxM are used for PCR amplification, and N is introduced into the PCR amplification 20 A ring-opened plasmid of the sequence. And (5) carrying out electrophoresis verification and purification recovery on the PCR amplification product.
(2) Since the recovered product may contain the template plasmid pTarget-F, which affects the subsequent experiments, dpnI was added to the recovered product and reacted at 37℃for 2 hours, and the template plasmid was digested.
(3) The recovered product after digestion of the template plasmid was phosphorylated using T4 polynucleotide kinase (T4 PNK), reacted at 37℃for 30min, and then heat-inactivated at 65℃for 10min.
(4) 1 mu LT4 DNA ligase is added into the phosphorylated reaction system, and the reaction is carried out for 4 hours at 22 ℃ to obtain a connecting solution. After the reaction is finished, the competent cells of the escherichia coli JM109 are taken out and melted on ice, and the connecting solution is added into the competent cells and is gently blown and sucked for uniform mixing, and the mixture is subjected to ice bath for 30min. Competent cells were then heat shocked in a 42℃water bath for 90s, ice-bath for 2min, and 1mL LB medium was added rapidly. The transformants were selected by resuscitating at 37℃for 1 hour at 100rpm, then plating on LB plates supplemented with 50mg/L of Qcomycin (Spe), and culturing at 37℃in an inverted state. Colony PCR was performed on transformants using pTargetF as negative control to verify whether the knocked-out plasmid was successfully constructed. The correct transformants were inoculated into LB liquid tubes to which spectinomycin (Spe) was added, and plasmids were extracted to obtain knockout plasmids pTargetF-gene (pTargetF-lpxL, pTargetF-lpxP and pTargetF-lpxM).
EXAMPLE 2 construction of E.coli lipid A reduced construction strain HWJ003
The CRISPR/Cas9 knockout system is adopted to knock out the related gene of the E.coli HW003 lipoid A secondary acyl chain transferase. The specific knockout procedure is as follows (fig. 1):
(1) Preparation of E.coli electrotransformation knockout competent cells HW003/pCas
Transforming plasmid pCas into escherichia coli HW003 to obtain recombinant escherichia coli HW003/pCas containing pCas plasmid, activating the escherichia coli HW003/pCas on an LB solid plate added with 30mg/L kanamycin (Kan), inoculating to an LB (Kan+) test tube, and culturing overnight to obtain seed liquid; the seed solution was transferred to 25mL of LB (Kan+) medium at 1% (v/v), and cultured at 30℃and 200rpm to OD 600 =0.2, induced by adding 500 μ L L-arabinose solution, and continued culture to OD 600 =0.5, ice bath for 30min; centrifuging at 4000rpm at 4 ℃ for 10min to collect thalli, and washing the thalli three times by using a precooled 10% glycerol solution; mu.L of 10% glycerol solution was added to resuspend the cells and the cells were aliquoted into sterile 1.5mL EP tubes, 80. Mu.L/tube.
(2) Construction of homology arm knockout fragments
Extracting genome of escherichia coli W3110, taking the genome as a template, amplifying homologous arm primers lpxL-U-F/lpxL-U-R and lpxL-D-F/lpxL-D-R of a target gene lpxL to obtain an upstream homologous arm and a downstream homologous arm respectively, recycling glue, and carrying out overlap PCR on the lpxL-U-F/lpxL-D-R by using the primers to obtain homologous arm knockout fragments; amplifying the homologous arm primer lpxP-U-F/lpxP-U-R of the target gene lpxP to obtain an upstream homologous arm and a downstream homologous arm respectively, recovering glue, and carrying out overlap PCR on the lpxP-U-F/lpxP-D-R by using the primer pair to obtain homologous arm knockout fragments; the homologous arm primer lpxM-U-F/lpxM-U-R of the target gene lpxM is knocked out, the lpxM-D-F/lpxM-D-R is respectively amplified to obtain an upstream homologous arm and a downstream homologous arm, the glue is recovered, and overlapping PCR is carried out on the lpxM-U-F/lpxM-D-R by using the primer pair, so that a homologous arm knocked-out fragment is obtained.
(3) Electrotransfection and knockout of genes
Washing the electric shock cup with absolute ethyl alcohol for three times, drying, and pre-cooling for 20min. Melting the competent cells of E.coli HW003/pCas in step (1) on ice, adding 100ng of the knockout plasmid in example 1 and 500ng of the corresponding homologous arm knockout fragment in step (2), gently sucking and mixing, and sucking into a groove of a electric shock cup. Ice-bathing the electric shock cup for 10min, and electric shock is performed after wiping. 1mL of LB medium was then rapidly added to the cuvette, the whole broth was aspirated into a 1.5mL EP tube, resuscitated at 30℃and 100rpm for 1.5h, plated on LB plates containing 30mg/L Kan and 50mg/L Spe, and cultured upside down in a 30℃incubator. Colony PCR was performed on transformants using HW003 as a negative control with forward primers for the upstream homology arm and reverse primers for the downstream homology arm.
After the construction of HW003 DeltalpxL is completed, HW003 DeltalpxL/pCas competent cells are prepared according to the step (1) based on the strain, lpxP is knocked out according to the steps (2) and (3), and HW003 DeltalpxL DeltalpxP is constructed. Finally, HW 003. DELTA.lpxLDELTA.lpxP. DELTA.lpxM was constructed based on HW 003. DELTA.lpxLDELTA.lpxP in the same manner.
The results show that: as shown in FIG. 2, lanes 2, 4 and 6 are the wild type W3110 lpxL, lpxP and lpxM control bands, respectively, lane 1 is the band size after the lpxL knockout, and comparison lane 2 shows that lpxL knockout was successful; lane 3 is the band size after the lpxP knockout, and comparison of lane 4 indicates that lpxP knockout was successful; lane 5 is the band size after the lpxM knockout, and comparison of lane 6 indicates that the lpxM knockout was successful.
(4) Removal of knockout plasmid pTargetF-gene and temperature sensitive plasmid pCas
The correct knockdown transformant in step (3) was inoculated into LB test tubes to which 30mg/L Kan and 1mM IPTG were added, enzyme expression of the knockdown plasmid pTargetF-gene was removed by IPTG induction, and the mixture was cultured with shaking at 30℃for 12 hours, and single colonies were streaked on LB (Kan+) plates. Screening out single colony sensitive to spectinomycin to obtain mutant strain with pTargetF-gene knocked out plasmid, inoculating to LB (Kan+) test tube for seed preservation, and directly preparing competence for continuous knocking out. The mutant strain from which the knocked-out plasmid was removed was inoculated into an LB tube, cultured with shaking at 42℃and single colonies were streaked on an LB plate. Screening out single colony sensitive to kanamycin to obtain strain without anti-mutation for removing pCas, and inoculating to LB test tube for seed preservation.
So far, the lipid A structure reduced strain HWJ003 is obtained by successfully knocking out lpxL, lpxM and lpxP genes from the genome of the escherichia coli HW003 by adopting a CRISP/Cas9 knocking-out method.
Example 3 extraction and validation of HWJ003 lipid A
(1) Lipid a extraction and purification method: lipidA is extracted by chloroform/methanol/water mixed phase extraction. Inoculating strain HWJ003 into LB liquid medium, culturing overnight at 37deg.C to obtain bacterial liquid, and regulating bacterial liquid to initial OD 600 Transfer to 1L LB liquid medium=0.02, culture at 37deg.C until late phase of thallus logarithm, centrifuging at 4000rpm for 20min to collect thallus, and culturing with Bligh-Dyer one-phase system chloroform/H 2 The cell pellet was suspended from 76mL of the mixture of O/methanol (1:0.8:2 v/v/v), magnetically stirred for 1h, centrifuged at 2000rpm for 30min to collect cell debris, 27mL of sodium acetate (pH 4.5) was added to the pellet, sonicated for 10min, and heated in a boiling water bath for 30min. The suspension was mixed with 60mL of methanol/chloroform (1:1 v/v) to form a Bligh-Dyer two-phase system suspension/methanol/chloroform (1.8:2:2 v/v/v), centrifuged at 2000rpm for 30min to separate phases, and finally the lower phase was collected and the organic reagent was evaporated with a rotary evaporator at 40℃for 3 min. Lipid A was dissolved in 1mL of chloroform/methanol (4:1 v/v) mixture. The organic solvent was evaporated with a nitrogen sparge to separate oxygen and prevent oxidation and the samples were stored at-20 ℃.
(2) ESI/MS analysis of lipid A: lipid A samples were dissolved in chloroform/methanol (4:1 v/v) and mass spectrometric detection was performed on a Waters SYNAPT mass spectrometer. The detection range is less than m/z 2500 using an electrospray ionization (ESI) source in negative ion mode. Ion collisional activation was performed at-150V. Mass spectral data were analyzed using masslynxv4.2 software. Coli HWJ003 can produce two types of lipid a. The first mass spectrum peak is at m/z 1323.9 and the second class of lipids A comprises m/z 1097.7 and m/z 1079.7. The former is that after the wild type m/z 1796.2 is changed to m/z 1716.2 by deleting the 1-position phosphoric acid, the secondary acyl chain is changed to m/z 1323.9 by deleting the 2 'and 3' positions. The latter was based on m/z 1323.9, which was changed to m/z 1097.7 by the deletion of the primary acyl chain at position 3 in addition to PagL expression. Thus, lipid A at m/z 1097.7 is the most simplified E.coli lipid A structure in this patent, and is a structure in which the 1-position phosphate, the 3-position primary acyl chain, the 2 '-position secondary acyl chain, and the 3' -position secondary acyl chain are deleted on the basis of m/z 1796.2 (FIG. 3).
Example 4 identification of a lipid A secondary acyl chain transferase Gene of gram-negative bacteria
The expression method and identification method of the lipid A secondary acyltransferase gene are described in this example using the lipid A secondary acyltransferase gene VP-RS 01045 gene of Vibrio parahaemolyticus ATCC 33846. The BLASTP result shows that VP_RS01045 of the vibrio parahaemolyticus is homologous gene of E.coli K-12lpxM, and the homology is 42%.
(1) Acquisition of VP-RS 01045 Gene: the VP_RS01045 gene with XbaI and SacI restriction enzyme sites introduced is amplified by using the V.ParahaameolyticusATCC33846 genome as a template and utilizing VP_RS01045-F and VP_RS01045-R primers for PCR amplification, and then electrophoresis verification and purification recovery are carried out.
(2) And (3) enzyme cutting of a carrier: the pBAD33 plasmid was digested with XbaI and SacI restriction enzymes, and purified.
(3) Construction of the expression strain: the recovered VP-RS 01045 gene and pBAD33 vector were added into a reaction system of T4 DNA ligase, and reacted at 22℃for 4 hours to obtain a ligation mixture. After the reaction, the competent cells of the escherichia coli HWJ003 are taken out and melted on ice, and the connecting liquid is added into the competent cells and is gently blown and sucked for uniform mixing, and the ice bath is carried out for 30min. Competent cells were then heat shocked in a 42℃water bath for 90s, ice-bath for 2min, and 1mL LB medium was added rapidly. Resuscitates at 37℃for 1 hour at 100rpm, and then spreads on LB plates supplemented with 10mg/L chloramphenicol (Cm), and cultures were inverted at 37℃to select expression strains. Taking HWJ003 genome as negative control, picking colony for PCR to verify whether the expression vector is constructed successfully. The colony with correct PCR result was inoculated into LB liquid tube to which chloramphenicol (Cm) was added, and the colony was designated as HWJ-pBAD-RS 01045, and after overnight culture, it was stored with 30% glycerol.
(4) Expression of lipid a secondary acyl chain transferase gene: inoculating HWJ-pBAD-RS 01045 into LB liquid medium containing chloramphenicol (Cm), culturing overnight at 37deg.C to obtain seed solution, and adjusting the initial OD of the seed solution 600 =0.02 transfer to 1L LB broth, waiting for OD 600 When=0.1, 4.5g of arabinose was added to induce expression of recombinant vector, 3Culturing at 7℃until the bacterial cells are late logarithmic, and extracting lipid A according to the method of example 3.
(5) ESI/MS identification of the gram-negative lipid A secondary acyl chain transferase Gene: lipid A samples were dissolved in chloroform/methanol (4:1 v/v) and mass spectrometric detection was performed on a Waters SYNAPT mass spectrometer. The detection range is less than m/z 2500 using an electrospray ionization (ESI) source in negative ion mode. Ion collisional activation was performed at-150V. Mass spectral data were analyzed using MassLynxV4.2 software. HWJ003-pBAD-RS01045 can produce two lipid A. The first lipid A mass spectrum peak is at m/z 1307.9 and the second lipid A mass spectrum peak is at m/z 1534.1. The former is based on HWJ003 m/z 1097.7 structure, and a secondary acyl chain of C14:0 is added to become tetra-acylated lipid A. The latter is based on the m/z 1323.9 structure, with the addition of a C14:0 secondary acyl chain to become pentaacylated lipid A. Thus, it was identified from ESI/MS results that the VP-RS 01045 gene of Vibrio parahaemolyticus was C14:0 secondary acyl chain transferase (FIG. 4).
Comparative example: identification of VP_RS01045 in Vibrio parahaemolyticus.
(1) Knock-out of vp_rs01045 gene: the upstream and downstream fragments of VP-RS 01045, which are introduced with XbaI and SacI restriction sites, are amplified by PCR using the genome of V.Parahaameolyticus ATSC 33846 as a template, and the upstream and downstream fragments of VP-RS 01045 are ligated into a single fragment by fusion PCR, subjected to electrophoresis verification and purification recovery, and ligated with the pDS132 plasmid digested with XbaI and SacI restriction enzymes by T4 DNA ligase, and reacted at 22℃for 4 hours to obtain a ligation solution. After the reaction was completed, competent cells of E.coli CC118 (λpir) were transformed, and then LB plates containing 50ug/mL chloramphenicol were plated and cultured upside down at 37℃to select strains harboring the cloning vector. The colony with correct PCR result was inoculated into LB liquid tube added with chloramphenicol (Cm), and after overnight culture, recombinant pDS132-VP_RS01045 plasmid was extracted for use. The pDS132-VP_RS01045 plasmid was electrotransferred into E.coli S17-1 (λpir) by the method of example 2, a 50ug/mL chloramphenicol plate was applied, cultured at 37℃and the correct transformant was picked up to give strain S17-1-pDS132-VP_RS01045. S17-pDS132-VP_RS01045 was transferred to a wild-type strain of Vibrio parahaemolyticus ATCC33846 by conjugation transduction, plated with 10ug/mL chloramphenicol and 50u g/mL ampicillin double resistance plates, cultured at 37℃and subjected to allele exchange, and reverse screening on LB medium containing 10% sucrose to obtain chloramphenicol-sensitive clonal colonies. Finally, the deletion strain ATCC 33846. DELTA. VP-RS 01045 was obtained by PCR screening.
(2) ESI/MS analysis ATCC33846 DeltaVP-RS 01045 lipid A: after lipid A was extracted and blow dried as in example 3, the sample was dissolved in chloroform/methanol (4:1 v/v) and mass spectrometric detection was performed on a Waters SYNAPT mass spectrometer. The detection range is less than m/z 2500 using an electrospray ionization (ESI) source in negative ion mode. Ion collisional activation was performed at-150V. Mass spectral data were analyzed using MassLynxV4.2 software. Compared with mass spectrum results of escherichia coli HWJ003 and HWJ003-pBAD-RS01045, the mass spectrum of the wild-type ATCC33846 and the mutant strain ATCC33846 delta VP_RS01045 has more main peaks, and the lipid A main structure of the escherichia coli has diversity. The major lipid A mass spectrum peaks of ATCC33846 are divided into 4 groups, and the major peaks of each group are m/z 2052.4, 2175.3, 2288.5 and 2411.4, respectively. The major lipid A mass spectrum peaks for ATCC 33846. DELTA. VP-RS 01045 are similarly found in 4 groups, with the major peaks for each group being m/z 2080.4, 2203.3, 2316.5 and 2439.4, respectively. As is clear from this, the knock-out of the secondary acyltransferase VP-RS 01045 in Vibrio parahaemolyticus resulted in a lipid A molecular weight of 28Da more, i.e.a molecular weight of 2 methylene groups (FIG. 5). While VP-RS 01045 is responsible for encoding the C14:0 secondary acyl chain transferase, predicted from BLASTP results, the theoretical lipid A molecular weight of ATCC 33846. DELTA. VP-RS 01045 should be reduced by 210Da over the wild-type. In view of the fact that the mass spectrum result of the vibrio parahaemolyticus lipoid A is not simple and clear, and the function of the gene can not be determined by knocking out the gene encoding the secondary acyl chain transferase, the invention provides a solution for identifying the secondary acyl chain transferase gene.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. The genetically engineered bacterium with a simplified lipoid A structure is characterized in that the genetically engineered bacterium takes escherichia coli as a starting strain, a gene lacI encoding a DNA binding transcription repressor on a genome is knocked out, three genes lpxL, lpxP and lpxM encoding lipoid A secondary acyl chain transferase are knocked out by a CRISPR/Cas9 knocking-out system, and a gene pagL encoding lipoid A3-O-deacylase and a gene lpxE encoding lipoid A1-phosphatase are overexpressed; the structure of the lipoid A is a monophosphoryl lipoid A only containing a 2-position primary acyl chain, a 2 '-position primary acyl chain and a 3' -position primary acyl chain;
accession numbers on NCBI of sequences of lpxL, lpxP and lpxM are "NP_415572.1", "NP_416879.4" and "NP_416369.1" in sequence;
the NCBI accession number of the sequence of lacI is "NP-414879.3";
the pagL is derived from salmonella and lpxE is derived from Francisella; the sequences of pagL have NCBI accession number "NP-461188.1" and lpxE have NCBI accession number "WP-003041686.1";
the E.coli includes E.coli W3110.
2. A method for preparing lipid a of reduced structure, comprising the specific steps of: (1) Inoculating the genetically engineered bacteria into a culture medium to obtain bacterial liquid; (2) Collecting thalli and extracting lipoid A by using a Bligh-Dyer mixed system; the structure of the lipoid A is monophosphoryl lipoid A only containing a 2-position primary acyl chain, a 2 '-position primary acyl chain and a 3' -position primary acyl chain.
3. The use of the genetically engineered bacterium of claim 1 in identifying genes encoding lipid a secondary acyl chain transferases, wherein expression vectors are used to express genes encoding lipid a secondary acyl chain transferases in the genetically engineered bacterium of claim 1 to obtain recombinant bacterium, and lipid a is induced to be expressed and extracted, and then the genes encoding lipid a secondary acyl chain transferases are identified by ESI/MS.
4. The use according to claim 3, wherein the expression vector comprises pBAD33.
5. The use according to claim 3, wherein the extraction is of lipid a using a Bligh-dyr mixed system.
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