CN115976088B - Low endotoxin content eutrophic rogowski bacteria and application thereof - Google Patents
Low endotoxin content eutrophic rogowski bacteria and application thereof Download PDFInfo
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- CN115976088B CN115976088B CN202210864606.4A CN202210864606A CN115976088B CN 115976088 B CN115976088 B CN 115976088B CN 202210864606 A CN202210864606 A CN 202210864606A CN 115976088 B CN115976088 B CN 115976088B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
- C12P7/625—Polyesters of hydroxy carboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of microorganisms, in particular to a low endotoxin content truffle and application thereof. The invention provides an application of the expression and/or enzyme activity reduction of H16_A0228 protein and/or H16_B0917 protein of Eutrophic bacteria in reducing endotoxin content of the Eutrophic bacteria. The invention discovers that the inactivation of H16_A0228 and H16_B0917 proteins of the eutrophic bacteria of the Ralstonia can obviously reduce the endotoxin content of the eutrophic bacteria of the Ralstonia. The engineering rogowski bacteria with the H16_A0228 protein and the H16_B0917 protein with the lost functions not only have the remarkably reduced endotoxin content, but also have the remarkably improved cell dry weight and PHA yield, provide new genes and strain resources for the development of engineering strains of PHA, provide an effective method for reducing the endotoxin content of PHA, and have important significance for expanding the application of PHA in medical materials.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a low endotoxin content truffle and application thereof.
Background
Endotoxin is a component of the outer membrane of most gram-negative bacteria, which is an asymmetric lipid bilayer consisting essentially of phospholipids as the inner layer and lipopolysaccharides as the outer layer. Lipopolysaccharide comprises hydrophobic Lipid A (Lipid A), hydrophilic non-specific core polysaccharide (Core polysaccharides) and long-chain O-antigen polysaccharide (O-anti), wherein the core polysaccharide comprises an outer hexose region and an inner heptose region, which are respectively connected with specific polysaccharide and Lipid A, the long-chain O-antigen polysaccharide endows the strain with a specific surface antigen, the long-chain O-antigen polysaccharide consists of repeated oligosaccharide subunits, and the Lipid A is a key component causing endotoxin toxicity. Lipid A defects have been reported to be fatal to most other gram-negative bacteria (Clementz, T.inhibition of lipopolysaccharide biosynthesis and cell growth following inactivation of the kdtA gene in Escherichia coli. [ J ]. Journal of Biological Chemistry,1995,270 (46): 27646.).
Polyhydroxyalkanoates (PHAs) are high molecular polymers synthesized by microorganisms, have multi-element material properties, are widely applied to the fields of medical treatment, agriculture, environmental protection, chemical industry and the like, and are considered to be a material with wide application prospect due to excellent material properties. However, the biosynthesized PHA cannot be brought into direct contact with the human body, and the pyrogenic components (e.g., endotoxin) must be removed to achieve medical grade PHA material.
The fungus eutrophic (Ralstonia eutropha, also known as Cupriavidus necator) is one of the gram-negative bacteria and one of the strains that can be used for the synthesis of PHA. However, residual endotoxin in PHA material greatly limits the use of PHA in medical materials, and therefore the purified PHA needs to be minimized in endotoxin content. There is no report of genetic modification of eutrophic rogowski bacteria to reduce endotoxin content thereof.
Disclosure of Invention
It is an object of the present invention to provide a method for reducing endotoxin content in eutrophic rogowski bacteria. It is another object of the present invention to provide an engineered eutrophic rogowski bacterium having reduced endotoxin content.
The invention takes the eutrophic bacteria of Roche as a research object, and develops a method for reducing the endotoxin content of the eutrophic bacteria by genetic engineering. In the research and development process, the invention discovers that the molecular weight, the chemical structure of fatty acid chain structure and the like of lipid A of the eutrophic rogowski bacteria have great differences from other gram-negative bacteria such as escherichia coli, for example: lipid A of the fungus is increased by one more 4-amino-4-deoxy-L-arabinose, the primary acyl chain of the fatty acid chain of the fungus is C14, and two secondary acyl chains are generated at the 2 and 2' positions respectively, and the two secondary acyl chains are also C14 (the primary acyl chain of the escherichia coli is C14, and one of the two secondary acyl chains is C12). Whereas currently discovered synthetases of the fatty acid chains of gram-negative bacterial lipid a generally have substrate specificity, the synthesis of fatty acid chains of different lengths is catalyzed by different enzymes (e.g., lauroyl transferase catalyzes the synthesis of C12 fatty acid chains, myristoyl transferase catalyzes the synthesis of C14 fatty acid chains). Thus, it is deduced that the enzymes involved in the synthesis of lipid A and its fatty acid chains in eutrophic bacteria and their synthesis and regulation mechanisms may be significantly different from those of other gram-positive bacteria such as E.coli, but no report has been made on the synthetic pathways and related enzymes of lipid A and its fatty acid chains in eutrophic bacteria.
In addition, the present invention has been developed in an attempt to reduce endotoxin toxicity or content of gram-negative bacteria such as E.coli and B.pertussis, for example, by using methods disclosed in the prior art, in the case of eubacteria of the genus Roche, for example: knocking out msbB and pagP genes for regulating fatty acid chain transfer to a carbon skeleton of lipid a while overexpressing Francisella tularensis-derived endomembrane phosphatase lpxEft to change the structure of lipid a, and expressing an acyltransferase LpxDpa, lpxApa from pseudomonas aeruginosa source to change the acyl chain length at different positions, however, none of the above methods can achieve the purpose of reducing endotoxin content in eutrophic bacteria, and further prove that the chemical structural characteristics of lipid a and its fatty acid chains in eutrophic bacteria may be significantly different from other gram-positive bacteria such as escherichia coli.
Through continuous attempts, the invention surprisingly discovers that attenuation or knockout of the H16_A0228 protein and the H16_B0917 protein of the eutrophic bacteria of Roche can obviously reduce the endotoxin content of the eutrophic bacteria, and is also beneficial to the improvement of PHA yield and biomass.
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides the use of the expression and/or reduced enzymatic activity of a H16 a0228 protein and/or a H16B 0917 protein of a eutrophic bacterium of the genus rochnia for reducing endotoxin content of the eutrophic bacterium of the genus rochnia.
In a second aspect, the invention provides the use of reduced expression and/or enzymatic activity of a H16 a0228 protein and/or a H16B 0917 protein of eutrophic bacteria to increase PHA production by eutrophic bacteria.
In a third aspect, the invention provides the use of the expression and/or reduced enzymatic activity of a H16 a0228 protein and/or a H16B 0917 protein of a eutrophic bacterium of rochanterium to increase biomass of the eutrophic bacterium of rochanterium.
In a fourth aspect, the present invention provides the use of reduced expression and/or enzyme activity of the H16_A0228 protein and/or the H16_B0917 protein of Eutrophic bacteria to reduce endotoxin content of the Eutrophic bacteria while increasing PHA yield and biomass of the Eutrophic bacteria.
In some embodiments of the invention, the invention provides the use of reduced expression and/or enzymatic activity of a H16 a0228 protein of eutrophic rochanterium in reducing endotoxin content of eutrophic rochanterium, increasing PHA yield of eutrophic rochanterium, and/or increasing biomass of eutrophic rochanterium.
In some embodiments of the invention, the invention provides the use of reduced expression and/or enzymatic activity of a H16B 0917 protein of eutrophic roller to reduce endotoxin content of eutrophic roller, increase PHA production by eutrophic roller, and/or increase biomass of eutrophic roller.
In some embodiments of the invention, the invention provides the use of reduced expression and/or enzyme activity of the H16 a0228 protein and the H16B 0917 protein of eutrophic bacteria to reduce endotoxin content of the eutrophic bacteria, increase PHA production of the eutrophic bacteria, and/or increase biomass of the eutrophic bacteria.
In the above applications, the increase in biomass may be expressed as an increase in dry cell weight.
In the invention, H16_A0228 and H16_B0917 are the locus_tag of the encoding genes of the proteins in GenBank, and the sequences of the H16_A0228 and H16_B0917 proteins and the encoding genes thereof can be obtained in GenBank.
Specifically, the coding gene sequence of the H16_B0917 protein is shown in SEQ ID NO.3, and the amino acid sequence of the H16_B0917 protein is shown in SEQ ID NO. 4. The coding gene sequence of the H16_A0228 protein is shown as SEQ ID NO.5, and the amino acid sequence of the H16_A0228 protein is shown as SEQ ID NO. 6.
The amino acid sequence of the H16_B0917 protein (SEQ ID NO. 4) is specifically as follows:
MKHRLQAALTIAVFKLVAALPYGVTARLGDAIGKLLYRIPSRRRRIVHTNLSLCFPDMDADTRDKLARNHFGHVLRSYLERGVQWFGSAERLGKLVELDSRIDLASCAEHPTIFMGFHFVGIEAGCMFYSMRHPVASLYTRMSSQMLEDISRTQRGRFGAEMIPRSGSGKQVVRTLRAGCPVMLASDMDFGINDSVFVPFFGVPACTLTSASRLASMTGARVVPFTTEVLPDYRGYRLRIFDPLEGFPSGSVEEDSRRMNAFLEAQIATMPEQYYWIHRRFKNRPAGMPSVY。
the amino acid sequence of the H16_A0228 protein (SEQ ID NO. 6) is specifically as follows:
MSRVFTWLGIGLLTVLGKLPYPFVARFGEALGSLLYLVPSERRRVVQANLRLCFPDRTEAEIDELSRQSFRILFRSFAERGIFWTGSEAQMRRWVQIDDQAGLVALDGTPHILVTLHLSGVEAGAIRLTIDLREHLGRSGASLYTRQKNDLFDHFLKHARGRFGANMISRNDSARDILRCLKKGEALQLIADMDFGERDSEFVPFFGVQALTLTSVSRLARLTGAKVVPIYTEMLPDYQGYVLRILPPWEDYPGASVTDDTRRMNAFFEDCIRPRVPEYYWVHKRFKHRLPGEPEIY。
in such applications, the reduction in expression and/or enzymatic activity includes attenuating the expression and/or enzymatic activity of the protein, or alternatively, causing the protein to not be expressed or inactivated.
The means for achieving the reduction of the expression and/or the enzyme activity is not particularly limited, and for example, a target protein, a gene encoding the same, a regulatory element thereof and/or a regulatory gene or protein thereof may be modified by genetic engineering means so that the expression amount and/or the enzyme activity of the target protein is reduced.
In some embodiments of the invention, the expression and/or reduced enzymatic activity of h16_a0228 protein, h16_b0917 protein is achieved by a combination of any one or more of the following:
(1) Mutating the amino acid sequence of the protein such that expression and/or enzymatic activity of the protein is reduced;
(2) Mutating the nucleotide sequence of the encoding gene of the protein so that the expression and/or enzymatic activity of the protein is reduced;
(3) The transcriptional and/or translational regulatory elements of the gene encoding the protein are replaced with less active elements such that the expression level of the protein is reduced.
Mutations in the amino acid sequences described above include deletions, insertions or substitutions of one or more amino acids.
Mutations in the nucleotide sequences described above include deletions, insertions or substitutions of one or more nucleotides.
The transcriptional and translational regulatory elements described above include promoters, ribosome binding sites, and the like.
In some embodiments of the invention, the reduced expression and/or enzymatic activity of the h16_a0228 protein, h16_b0917 protein is achieved by inactivating the protein.
In some embodiments of the invention, the reduced expression and/or enzymatic activity of the h16_a0228 protein, h16_b0917 protein is achieved by deleting the gene encoding the protein (h16_b0917 gene, h16_a0228 gene).
In a fifth aspect, the present invention provides an engineered eutrophic bacterium modified such that expression and/or enzymatic activity of the h16_a0228 protein and/or the h16_b0917 protein therein is reduced.
In some embodiments of the invention, the engineered eutrophic bacterium is modified such that expression and/or enzymatic activity of the h16_a0228 protein therein is reduced.
In some embodiments of the invention, the engineered eutrophic bacterium is modified such that expression and/or enzymatic activity of the h16_b0917 protein therein is reduced.
In some embodiments of the invention, the engineered eutrophic bacterium is modified such that expression and/or enzymatic activity of the h16_a0228 protein and the h16_b0917 protein therein is reduced.
Such reduced expression and/or enzymatic activity includes attenuating expression and/or enzymatic activity of the protein or alternatively, rendering the protein non-expressed or inactive.
In some embodiments of the invention, the expression and/or reduced enzymatic activity of h16_a0228 protein, h16_b0917 protein is achieved by a combination of any one or more of the following:
(1) Mutating the amino acid sequence of the protein such that expression and/or enzymatic activity of the protein is reduced;
(2) Mutating the nucleotide sequence of the encoding gene of the protein so that the expression and/or enzymatic activity of the protein is reduced;
(3) The transcriptional and/or translational regulatory elements of the gene encoding the protein are replaced with less active elements such that the expression of the protein is reduced.
Mutations in the amino acid sequences described above include deletions, insertions or substitutions of one or more amino acids.
Mutations in the nucleotide sequences described above include deletions, insertions or substitutions of one or more nucleotides.
The transcriptional and translational regulatory elements described above include promoters, ribosome binding sites, and the like.
In some embodiments of the invention, the h16_a0228 protein and/or the h16_b0917 protein are inactivated in the engineered eutrophic bacterium, or the engineered eutrophic bacterium does not express the h16_a0228 protein and/or the h16_b0917 protein.
In some embodiments of the invention, the engineered eutrophic rogowski is deficient in genes encoding the h16_a0228 protein and/or the h16_b0917 protein.
In a sixth aspect, the invention provides the use of an engineered eutrophic bacterium as described above in the fermentative production of a biochemical.
The above-mentioned biochemicals include, but are not limited to, polyesters, alcohols, amino acids, polypeptides, proteins, nucleic acids, saccharides, lipids, etc.
In some embodiments of the invention, there is provided the use of the engineered eutrophic bacterium described above in the fermentative production of PHA or a derivative thereof.
In some embodiments of the invention, the fermentative production of PHA material using the engineered truffle described above is performed with a vegetable oil (including, but not limited to, one or more of palm oil, palm kernel oil, peanut oil, soybean oil, linseed oil, rapeseed oil, cottonseed oil, castor oil, corn oil) as a carbon source.
The medium used for fermentation production may also contain nitrogen sources (including but not limited to ammonium salts), inorganic salts (including but not limited to disodium hydrogen phosphate, potassium dihydrogen phosphate), trace elements (including but not limited to magnesium, calcium, zinc, manganese, cobalt, boron, copper, nickel, molybdenum).
In a seventh aspect, the present invention provides a method for constructing the engineered eutrophic rogowski bacterium described above, the method comprising: the eutrophic organism is modified such that the expression and/or enzymatic activity of the h16_a0228 protein and/or the h16_b0917 protein therein is reduced.
In an eighth aspect, the present invention provides a method of reducing endotoxin content of eutrophic bacteria of the genus rochanterium, the method comprising: the eutrophic organism is modified such that the expression and/or enzymatic activity of the h16_a0228 protein and/or the h16_b0917 protein therein is reduced.
In some embodiments of the invention, the reduction in expression and/or enzyme activity is inactivation of the h16_a0228 protein and/or the h16_b0917 protein, or is such that the eutrophic rochanterium does not express the h16_a0228 protein and/or the h16_b0917 protein.
The invention has the beneficial effects that: the inactivation of the H16_A0228 protein and the H16_B0917 protein of the eutrophic bacteria provided by the invention can obviously reduce the endotoxin content of the eutrophic bacteria. The engineering rogowski bacteria with the H16_A0228 protein and the H16_B0917 protein with the lost functions not only have the remarkably reduced endotoxin content, but also have the remarkably improved cell dry weight and PHA yield, provide new genes and strain resources for the development of engineering strains of PHA, provide an effective method for reducing the endotoxin content of PHA, and have important significance for expanding the application of PHA in medical materials.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The experimental methods used in the following examples are conventional, unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Wherein, the enzyme reagent is purchased from New England Biolabs (NEB), the kit for extracting plasmids is purchased from Tiangen Biochemical technology (Beijing) Co., ltd, the kit for recovering DNA fragments is purchased from American omega company, the corresponding operation steps are strictly carried out according to the product specification, and all culture media are prepared by deionized water unless specified.
The medium formulation used in the following examples was as follows:
seed culture medium: 10g/L peptone,5g/L Yeast Extract,3g/L glucose.
Production medium: 1.0% palm oil, 9.85g/L Na 2 HPO 4 〃12H 2 O,1.5g/L KH 2 PO 4 ，3.0g/L NH 4 Cl,10mL/L trace element solution I and 1mL/L trace element solution II. Wherein the trace element solution I comprises the following components: 20g/LMgSO 4 ，2g/L CaCl 2 . The trace element solution II comprises the following components: 100mg/L ZnSO 4 〃7H 2 O，30mg/LMnCl 2 〃4H 2 O，300mg/L H 3 BO 3 ，200mg/L CoCl 2 〃6H 2 O，10mg/L CuSO 4 〃5H 2 O，20mg/LNiCl 2 〃6H 2 O，30mg/L NaMoO 4 〃2H 2 O. The above reagents were purchased from national drug group chemical reagent company.
EXAMPLE 1H16_B0917 construction and identification of deletion mutant
In the embodiment, the eutrophic bacteria H16 of Roche is taken as a starting bacteria, and the H16_B0917 gene is knocked out, which comprises the following steps:
step one: construction of the basic plasmid
PCR amplification was performed using the genome of Eutrophic bacteria H16 as a template, using 917H1-F and 917H1-R to obtain an upstream homology arm 917-H1 of H16_B0917, and using 917H2-F and 917H2-R to obtain a downstream homology arm 917-H2 of H16_B0917; the modified plasmid pK18mob is used as a template, pK-F, pK-R is used as a primer for PCR amplification to obtain a vector fragment, 917-H1 and 917-H2 are connected with the vector fragment by a Gibson Assembly method to obtain a recombinant plasmid pKO-H16_B0917 (the sequence is shown as SEQ ID NO. 1). The primers used above are shown in Table 1.
TABLE 1
Step two: construction of H16_B0917 deletion mutant target Strain
The recombinant plasmid pKO-H16_B0917 obtained in the first step is transformed into escherichia coli S17-1, and then transferred into the eutrophic rochanterium H16 by a joint transformation method, and positive clones are screened out by using an LB plate simultaneously containing 500 mug/mL of spectinomycin and 100 mug/mL of apramycin by utilizing the characteristic that the suicide plasmid cannot replicate in host bacteria. The recombinant plasmid with homologous fragments in the positive clone is integrated into the genome at the specific position of H1 and H2, and is the first homologous recombinant bacterium. The first homologous recombinant was streaked on LB plates containing 100mg/mL sucrose, clones without spectinomycin resistance were selected from these monoclonal strains, and primer 917-H1FP (SEQ ID NO. 19) was used: ATGTCGCTGACCGACGACCATGTC and 917-H1RP (SEQ ID NO. 20) TTGGCACCACCAGCCTGACCAATG PCR-identifying the recombinant strain knocked out by H16_B0917, and the obtained recombinant strain is the Ralstonia rhodesiense Re01 knocked out by H16_B0917.
EXAMPLE 2 construction and identification of H16_A0228 deletion mutant
In the embodiment, the eutrophic bacteria H16 of Roche is taken as a starting bacteria, and the H16_A0228 gene is knocked out, which comprises the following steps:
step one: construction of the basic plasmid
PCR amplification is carried out by using a genome of the eutrophic bacteria H16 of Roche as a template and 228H1-F and 228H1-R to obtain an upstream homology arm 228-H1 of H16_A0228, and PCR amplification is carried out by using 228H2-F and 228H2-R to obtain a downstream homology arm 228-H2 of H16_A0228; PCR (polymerase chain reaction) amplification is carried out by taking the modified plasmid pK18mob as a template and pK-F, pK-R as a primer to obtain a vector fragment; 228-H1 and 228-H2 were ligated to the vector fragment by Gibson Assembly method to obtain recombinant plasmid pKO-H16_A0228 (sequence shown in SEQ ID NO. 2). The primers used are shown in Table 2.
TABLE 2
Step two: construction of H16_A0228 deletion mutant target Strain
The recombinant plasmid pKO-H16_A0228 obtained in the step one is transformed into escherichia coli S17-1, and then transferred into the eutrophic rochanterium H16 by a joint transformation method, and positive clones are screened out by using an LB plate simultaneously containing 500 mug/mL of spectinomycin and 100 mug/mL of apramycin by utilizing the characteristic that suicide plasmids cannot replicate in host bacteria. The recombinant plasmid with homologous fragments in the positive clone is integrated into the genome at the specific position of H1 and H2, and is the first homologous recombinant bacterium. The first homologous recombinant was streaked on LB plates containing 100mg/mL sucrose, clones without spectinomycin resistance were selected from these monoclonal, and PCR was performed using primers 228-H1FP (SEQ ID NO. 21): ATCGATACCACCGAGATCCATTCG and 228-H1RP (SEQ ID NO. 22): AGCTGCATGGCTTTGACGACTACC to identify H16-A0228 knockout recombinant strains, and the resulting recombinant strain was H16-A0228 knockout Eutrophic bacteria Re02.
Example 3H16_B0917 and H16_A0228 double deletion mutant construction and identification
In this example, the H16B 0917 gene was knocked out using the truffle Re02 constructed in example 2 as a starting strain, and the specific steps were as follows:
the recombinant plasmid pKO-H16_B0917 obtained in example 1 was transformed into E.coli S17-1, and transferred into the eutrophic roller-type strain Re02 constructed in example 2 by the conjugation transformation method, and positive clones were selected from LB plates containing 500. Mu.g/mL spectinomycin and 100. Mu.g/mL apramycin, by utilizing the characteristic that the suicide plasmid could not replicate in the host strain. The recombinant plasmid with homologous fragments in the positive clone is integrated into the genome at the specific position of H1 and H2, and is the first homologous recombinant bacterium. The first homologous recombination was streaked on LB plates containing 100mg/mL sucrose, clones without spectinomycin resistance were selected from these monoclonal, and primers 917-H1FP were used: ATGTCGCTGACCGACGACCATGTC and 917-H1RP: TTGGCACCACCAGCCTGACCAATG, and identifying the recombinant strain with the H16-B0917 gene knocked out by PCR, and finally obtaining the recombinant strain which is the Ralstonia luoshiensis Re03 with H16-B0917 and H16-A0228 knocked out simultaneously.
Example 4 Performance verification of strains Re01, re02, re03
In the embodiment, the test of endotoxin content, biomass and PHB content after fermentation is carried out on Re01, re02 and Re03 by taking the eutrophic bacteria H16 of Roche as a control bacteria.
Step one: fermentation culture of strains Re01, re02 and Re03
Re01, re02, re03 and H16 were streaked on LB plates to obtain a monoclonal, which was inoculated into a seed medium (4 mL) and cultured for 12 hours. The bacterial liquid cultured overnight is transferred to a 100mL glass conical flask filled with 10mL of seed culture medium, and the final OD of inoculation is about 0.1, 30 ℃,220rpm, and the culture is carried out for 8 hours, thus the transfer culture can be carried out. The culture of PHA fermentation production is that the pre-culture seed solution with the OD value between 6 and 7 is inoculated into a 250mL shaking flask filled with 30mL of production culture medium according to the final inoculum size with the final OD of 0.1, and fermentation culture is carried out at 30 ℃ for 48h at 220 rpm. Each strain was set in 3 replicates.
Step two: endotoxin content determination of strains Re01, re02 and Re03
Taking 1mL of fresh bacterial liquid obtained after fermentation in the step one, centrifuging 13400g for 1min to collect bacterial cells, discarding the supernatant, and then re-suspending the bacterial cells by using 30% ethanol solutionThe cells were washed by centrifugation, discarding the supernatant, and repeating the above steps twice. Finally, deionized water is used for diluting the thalli to OD 600 0.5, cleaved at 100℃for 30min, and then allowed to stand at room temperature for 24 hours to allow for adequate endotoxin release. Endotoxin content was measured using the ToxinSensor chromogenic LAL endotoxin detection kit. The test results are shown in Table 3. Compared with the control strain eutrophic bacteria H16, the endotoxin unit contents of Re01, re02 and Re03 are respectively reduced by 46 times, 52 times and 95 times.
The above-described ToxinSensor chromogenic LAL endotoxin test kit was purchased from Kirsrui Biotech Co., ltd.
TABLE 3 Table 3
Step three: biomass determination of strains Re01, re02, re03
Taking the fermentation liquor volume (marked as V, unit: mL) of the fermentation liquor in the first step, putting the fermentation liquor volume into a weighed centrifuge tube (marked as m1, unit: g), centrifuging at 8000rpm at room temperature for 10min, discarding the supernatant, and collecting thalli; then 15mL of 30% ethanol solution is used to resuspend the thalli, 8000rpm, room temperature and centrifugation are carried out for 10min, the process is repeated twice, the grease and the culture medium in the strain are washed away, finally, a centrifuge tube for collecting the thalli is placed in a baking oven at 60 ℃, the thalli is baked to constant weight, and the weight (recorded as m2, unit: g) is accurately weighed by an analytical balance. And its dry weight was calculated. The results are shown in Table 4. Compared with the control strain eutrophic bacteria H16, the biomass of Re01, re02 and Re03 is respectively improved by 27.8%,17.9% and 28.2%.
The dry weight calculation formula is M= (M2-M1)/V.times.1000, and the unit is g/L.
TABLE 4 Table 4
| Strain | Dry weight (g/L) |
| Eutrophic bacteria H16 | 8.37±0.89 |
| Re01 | 10.70±1.80 |
| Re02 | 9.87±1.10 |
| Re03 | 10.73±2.35 |
Step four: PHB content determination of strains Re01, re02 and Re03
1. Sample treatment: weighing 30-40 mg of the dried sample in the third step, placing the sample in a digestion tube, adding 2mL of esterified liquid and 2mL of chloroform, capping and sealing the esterification tube for reaction for 4 hours at 100 ℃, standing and cooling to room temperature after the reaction is finished, adding 1mL of deionized water, vortex and shake until complete mixing, standing and layering, and taking a lower organic phase for gas chromatography analysis.
The preparation method of the esterified liquid comprises the steps of taking 485mL of absolute methanol, adding 1g/L of benzoic acid, and slowly adding 15mL of concentrated sulfuric acid to prepare 500mL of esterified liquid.
2. Standard substance treatment: poly [ (R) -3-hydroxybutyric acid ] is used for calibrating a 3HB unit, white powder, and the gradient values are 15mg, 25mg and 35mg, and the weighing mode and the processing mode are the same as the sample processing mode.
3. GC analysis of PHA composition and content: a GC-2014 type gas chromatograph from Shimadzu corporation was used. The chromatograph is configured to: HP-5 capillary chromatographic column, hydrogen flame ionization detector FID, SPL split-flow sample inlet; high-purity nitrogen is used as carrier gas, hydrogen is fuel gas, and air is fuel gas; an AOC-20S autosampler was used, acetone as the wash solution. The GC analysis procedure was set as follows: the temperature of the sample inlet is 240 ℃, the temperature of the detector is 250 ℃, the initial temperature of the column temperature is 80 ℃, and the sample inlet is maintained for 1.5 minutes; raising to 140 ℃ at a rate of 30 ℃/min and maintaining for 0 min; raising to 240 ℃ at a rate of 40 ℃/min and maintaining for 2 minutes; the total time was 8 minutes. The GC result adopts an internal standard normalization method to quantitatively calculate the PHB content according to the peak area. The results are shown in Table 5, and compared with the control strain, the PHB content of RE01, RE02 and RE03 is respectively improved by 7.02%,5.42% and 9.61%.
TABLE 5
| Strain | PHB％ |
| Eutrophic bacteria H16 | 70.59％ |
| Re01 | 75.55％ |
| Re02 | 74.42％ |
| Re03 | 77.38％ |
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. The application of the expression reduction of the H16_A0228 protein and/or the H16_B0917 protein of the Eutrophic bacterium of the Rogowski in reducing the endotoxin content of the Eutrophic bacterium of the Rogowski; the amino acid sequence of the H16_A0228 protein is shown as SEQ ID NO. 6; the amino acid sequence of the H16_B0917 protein is shown in SEQ ID NO. 4.
2. The application of the reduced expression of the H16_A0228 protein and/or the H16_B0917 protein of the Eutrophic bacteria in improving the PHA yield of the Eutrophic bacteria; the amino acid sequence of the H16_A0228 protein is shown as SEQ ID NO. 6; the amino acid sequence of the H16_B0917 protein is shown in SEQ ID NO. 4.
3. The application of the reduced expression of the H16_A0228 protein and/or the H16_B0917 protein of the Eutrophic bacteria in improving the biomass of the Eutrophic bacteria; the amino acid sequence of the H16_A0228 protein is shown as SEQ ID NO. 6; the amino acid sequence of the H16_B0917 protein is shown in SEQ ID NO. 4.
4. The application of the reduction of the expression of the H16_A0228 protein and/or the H16_B0917 protein of the eutrophic rochanteri in reducing the endotoxin content of the eutrophic rochanteri and simultaneously improving the PHA yield and biomass of the eutrophic rochanteri; the amino acid sequence of the H16_A0228 protein is shown as SEQ ID NO. 6; the amino acid sequence of the H16_B0917 protein is shown in SEQ ID NO. 4.
5. An engineered truffle, wherein the engineered truffle is modified such that expression of h16_a0228 protein and/or h16_b0917 protein therein is reduced;
the amino acid sequence of the H16_A0228 protein is shown as SEQ ID NO. 6; the amino acid sequence of the H16_B0917 protein is shown in SEQ ID NO. 4.
6. The engineered truffle of claim 5, wherein the h16_a0228 protein and/or the h16_b0917 protein in the engineered truffle is inactivated or the engineered truffle does not express the h16_a0228 protein and/or the h16_b0917 protein.
7. Use of the engineered eutrophic bacterium of claim 5 or 6 in the fermentative production of biochemicals.
8. The method for constructing the engineered eutrophic bacterium of claim 5 or 6, comprising the steps of: the eutrophic organism is modified such that the expression of the h16_a0228 protein and/or the h16_b0917 protein therein is reduced.
9. A method of reducing endotoxin content of eutrophic rogowski bacteria, the method comprising: modifying eutrophic bacteria to reduce the expression of the H16_A0228 protein and/or the H16_B0917 protein;
the amino acid sequence of the H16_A0228 protein is shown as SEQ ID NO. 6; the amino acid sequence of the H16_B0917 protein is shown in SEQ ID NO. 4.
10. The method of reducing endotoxin content of eutrophic bacteria of claim 9, wherein the reduction in expression is inactivation of h16_a0228 protein and/or h16_b0917 protein, or such that eutrophic bacteria of the genus h16_a0228 protein and/or h16_b0917 protein are not expressed.
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| CN115976088B (en) * | 2022-07-21 | 2023-09-19 | 深圳蓝晶生物科技有限公司 | Low endotoxin content eutrophic rogowski bacteria and application thereof |
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| WO2024017318A1 (en) | 2024-01-25 |
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