CN115976088A - Low endotoxin-containing fungus Roche and application thereof - Google Patents

Low endotoxin-containing fungus Roche and application thereof Download PDF

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CN115976088A
CN115976088A CN202210864606.4A CN202210864606A CN115976088A CN 115976088 A CN115976088 A CN 115976088A CN 202210864606 A CN202210864606 A CN 202210864606A CN 115976088 A CN115976088 A CN 115976088A
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丰婧
杨晓妍
尹进
李腾
张浩千
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Shenzhen Blue Crystal Biotechnology Co ltd
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Abstract

The invention relates to the technical field of microorganisms, in particular to a low-endotoxin-content Eubacterium rolfsii and application thereof. The invention provides application of expression and/or enzyme activity reduction of a H16_ A0228 protein and/or an H16_ B0917 protein of a Eumycota rolfsii in reducing endotoxin content of the Eumycota rolfsii. The invention discovers that the inactivation of proteins H16_ A0228 and H16_ B0917 of the Eumycota rolfsii can obviously reduce the endotoxin content of the Eumycota rolfsii. The engineered Eumycota rolfsii with the H16_ A0228 protein and the H16_ B0917 protein lacking functions not only has the obvious reduction of the endotoxin content, but also has the obvious improvement of the cell dry weight and the PHA yield, provides new genes and strain resources for the development of the engineered PHA strain, provides an effective method for reducing the endotoxin content of PHA and has important significance for the expansion of the application of PHA in medical materials.

Description

Low endotoxin-containing fungus Roche and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to a low-endotoxin-content Eubacterium rolfsii and application thereof.
Background
Endotoxin is a component of the outer cell membrane of most gram-negative bacteria, which is an asymmetric lipid bilayer consisting mainly of phospholipids as the inner layer and lipopolysaccharides as the outer layer. Lipopolysaccharides consist of hydrophobic Lipid A (Lipid A), hydrophilic non-specific Core polysaccharides (Core polysaccharides) and long-chain O-antigen polysaccharides (O-antipigen), wherein the Core polysaccharides comprise an exohexose region and an endo-heptose region which are respectively connected with specific polysaccharides and Lipid A, the long-chain O-antigen polysaccharides endow the strain with a specific surface antigen and consist of repeated oligosaccharide subunits, and the Lipid A is a key component for causing endotoxic toxicity. Lipid A defects have been reported in the literature to be lethal to most other gram-negative bacteria (Clementz, T.inhibition of lipolysis and cell growth fermentation activation of the kdtA gene in Escherichia coli [ J ]. Journal of Biological Chemistry,1995,270 (46): 27646.).
Polyhydroxyalkanoates (PHA) is a high molecular polymer synthesized by microorganisms, has multi-material chemical properties, is widely applied to the fields of medical treatment, agriculture, environmental protection, chemical industry and the like, and is considered to be a material with a wide application prospect due to excellent material properties in the field of medical materials. However, the biologically synthesized PHA is not in direct contact with the human body and pyrogen components (e.g., endotoxins) must be removed to obtain a medical grade PHA material.
The fungus Eutropha rolfsii (Ralstonia eutropha, also known as Cupriavidus dicator), which is one of gram-negative bacteria, is also one of the strains that can be used for PHA synthesis. However, the residual endotoxin (endoxin) in PHA material greatly limits the application of PHA in medical materials, and thus purified PHA needs to reduce the content of endotoxin to the maximum extent. At present, no report of genetic modification of the Eubacterium rolfsii to reduce the content of endotoxin is found.
Disclosure of Invention
It is an object of the present invention to provide a method for reducing endotoxin content in a fungus belonging to the genus Eubacterium. Another objective of the invention is to provide an engineered Eubacterium reuteri with reduced endotoxin content.
The invention takes the Eumycota rolfsii as a research object and develops a method for reducing the content of endotoxin by genetic engineering modification. In the development process, the invention finds that the molecular weight, the fatty acid chain structure and other chemical structures of lipid A of the eumycete roche have great difference with other gram-negative bacteria such as escherichia coli, for example: lipid a of eubacterium reuteri has one more 4-amino-4-deoxy-L-arabinose, and the first acyl chain of the fatty acid chain of eubacterium reuteri is C14, and at the same time, one secondary acyl chain is generated at each of the 2 and 2' positions, and both the secondary acyl chains are C14 (the first acyl chain of e.coli is C14, and one of the secondary acyl chains is C12). Whereas the presently discovered synthases for fatty acid chains of gram-negative lipid a generally have substrate specificity, the synthesis of fatty acid chains of different lengths is catalyzed by different enzymes (e.g., lauroyltransferase catalyzes the synthesis of C12 fatty acid chains, myristoyltransferase catalyzes the synthesis of C14 fatty acid chains). It is inferred that the enzyme participating in the synthesis of lipid A and the fatty acid chain thereof in Eutropha rolfsii and the synthesis and regulation mechanism thereof may be obviously different from other gram-positive bacteria such as Escherichia coli, but no report about the synthesis pathway of lipid A and the fatty acid chain thereof in Eutropha rolfsii and related enzymes is found at present.
In addition, in the course of development, the present inventors have tried to reduce endotoxin toxicity or content of gram-negative bacteria such as escherichia coli and bordetella pertussis by using the methods disclosed in the prior art in the eumycetes roche, for example: knocking out msbB and pagP genes used for regulating and controlling fatty acid chains to be transferred to a carbon skeleton of lipid A, and simultaneously over-expressing Francisella tularensis-derived inner membrane phosphatase lpxEft to change the structure of the lipid A, and expressing acyltransferase LpxdP and Lpxafrom pseudomonas aeruginosa to change the lengths of acyl chains at different positions, however, the method can not realize the purpose of reducing the content of endotoxin in the eubacterium rosenbergii, and further proves that the chemical structure characteristics of the lipid A and the fatty acid chains in the eubacterium rosenbergii cause that the synthetase and the synthesis and regulation mechanisms of the synthetase are possibly obviously different from other gram-positive bacteria such as escherichia coli.
Through continuous attempts, the invention unexpectedly discovers that the weakening or knockout of the H16_ A0228 protein and the H16_ B0917 protein of the eubacterium rolfsii can obviously reduce the content of endotoxin, and is beneficial to the improvement of PHA yield and biomass.
Specifically, the invention provides the following technical scheme:
in a first aspect, the invention provides the use of reduced expression and/or enzymatic activity of a H16_ A0228 protein and/or H16_ B0917 protein of Eubacterium rolfsii for reducing the endotoxin content of Eubacterium rolfsii.
In a second aspect, the present invention provides the use of reduced expression and/or enzymatic activity of the H16_ A0228 protein and/or H16_ B0917 protein of Eubacterium rolfsii for increasing PHA production by Eubacterium rolfsii.
In a third aspect, the invention provides the use of reduced expression and/or enzyme activity of the H16_ A0228 and/or H16_ B0917 proteins of Eumycota rolfsii for increasing the biomass of Eumycota rolfsii.
In a fourth aspect, the invention provides the use of reduced expression and/or enzymatic activity of the H16_ A0228 and/or H16_ B0917 proteins of Eubacterium rolfsii in reducing the endotoxin content of Eubacterium rolfsii while increasing PHA production and biomass of Eubacterium rolfsii.
In some embodiments of the invention, the invention provides for the use of reduced expression and/or enzymatic activity of a eubacterium reuteri H16_ a0228 protein to reduce endotoxin content, increase PHA production and/or increase biomass of eubacterium reuteri.
In some embodiments of the invention, the invention provides for the use of reduced expression and/or enzymatic activity of the roche fungi H16_ B0917 protein to reduce endotoxin content, increase PHA production and/or increase biomass of the roche fungi.
In some embodiments of the invention, the invention provides for the use of reduced expression and/or enzymatic activity of the H16_ a0228 and H16_ B0917 proteins of eubacterium rolfsii in reducing endotoxin levels, increasing PHA production and/or increasing biomass of eubacterium rolfsii.
In the above applications, the increase in biomass may be expressed as an increase in dry weight of the cells.
In the invention, H16_ A0228 and H16_ B0917 are locus _ tag of protein coding genes in GenBank, and the H16_ A0228 and H16_ B0917 proteins and sequences of the coding genes thereof can be obtained in GenBank.
Specifically, the coding gene sequence of the H16_ B0917 protein is shown as SEQ ID NO.3, and the amino acid sequence of the H16_ B0917 protein is shown as 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 (SEQ ID NO. 4) of the H16_ B0917 protein is specifically as follows:
MKHRLQAALTIAVFKLVAALPYGVTARLGDAIGKLLYRIPSRRRRIVHTNLSLCFPDMDADTRDKLARNHFGHVLRSYLERGVQWFGSAERLGKLVELDSRIDLASCAEHPTIFMGFHFVGIEAGCMFYSMRHPVASLYTRMSSQMLEDISRTQRGRFGAEMIPRSGSGKQVVRTLRAGCPVMLASDMDFGINDSVFVPFFGVPACTLTSASRLASMTGARVVPFTTEVLPDYRGYRLRIFDPLEGFPSGSVEEDSRRMNAFLEAQIATMPEQYYWIHRRFKNRPAGMPSVY。
the amino acid sequence (SEQ ID NO. 6) of the H16_ A0228 protein is specifically as follows:
MSRVFTWLGIGLLTVLGKLPYPFVARFGEALGSLLYLVPSERRRVVQANLRLCFPDRTEAEIDELSRQSFRILFRSFAERGIFWTGSEAQMRRWVQIDDQAGLVALDGTPHILVTLHLSGVEAGAIRLTIDLREHLGRSGASLYTRQKNDLFDHFLKHARGRFGANMISRNDSARDILRCLKKGEALQLIADMDFGERDSEFVPFFGVQALTLTSVSRLARLTGAKVVPIYTEMLPDYQGYVLRILPPWEDYPGASVTDDTRRMNAFFEDCIRPRVPEYYWVHKRFKHRLPGEPEIY。
in the above applications, the reduction of expression and/or enzymatic activity comprises reducing the expression and/or enzymatic activity of the protein, or rendering the protein non-expressed or inactive.
The mode for achieving the reduction of the expression and/or the enzyme activity is not particularly limited in the present invention, and for example, the 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 reduction in expression and/or enzymatic activity of the H16_ a0228 protein, H16_ B0917 protein is achieved by a combination of any one or more of the following (1) - (3):
(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 a gene encoding a protein such that the expression and/or enzymatic activity of the protein is reduced;
(3) The transcription and/or translation regulatory elements of the gene encoding the protein are replaced with less active elements so that the expression amount of the protein is reduced.
The mutation of the above-mentioned amino acid sequence includes deletion, insertion or substitution of one or more amino acids.
The mutation of the nucleotide sequence described above includes deletion, insertion or substitution of one or more nucleotides.
The above-mentioned transcription and translation regulatory elements include promoters, ribosome binding sites and the like.
In some embodiments of the invention, the reduction in 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 reduction in expression and/or enzymatic activity of the H16_ a0228 protein, H16_ B0917 protein is achieved by deleting the genes encoding the protein (H16 _ B0917 gene, H16_ a0228 gene).
In a fifth aspect, the present invention provides an engineered eubacterium reuteri modified such that expression and/or enzyme activity of H16_ a0228 protein and/or H16_ B0917 protein therein is reduced.
In some embodiments of the invention, the engineered eubacterium rolfsii 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 eubacterium rolfsii is modified such that the expression and/or enzymatic activity of the H16_ B0917 protein therein is reduced.
In some embodiments of the invention, the engineered rogowski fungi are modified such that the expression and/or enzymatic activity of the H16_ a0228 protein and the H16_ B0917 protein therein is reduced.
Such reduction in expression and/or enzyme activity includes attenuating expression and/or enzyme activity of the protein, or alternatively, rendering the protein unexpressed or inactive.
In some embodiments of the invention, the reduction in expression and/or enzymatic activity of the H16_ a0228 protein, H16_ B0917 protein is achieved by a combination of any one or more of the following (1) - (3):
(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 a gene encoding the protein such that expression of the protein and/or enzymatic activity 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.
The mutation of the above-mentioned amino acid sequence includes deletion, insertion or substitution of one or more amino acids.
The mutation of the nucleotide sequence described above includes deletion, insertion or substitution of one or more nucleotides.
The above-mentioned transcriptional and translational regulatory elements include promoters, ribosome binding sites and the like.
In some embodiments of the invention, the engineered eubacterium rolfsii is inactivated for H16_ a0228 protein and/or H16_ B0917 protein, or does not express H16_ a0228 protein and/or H16_ B0917 protein.
In some embodiments of the invention, the engineered r.reuteri lacks genes encoding the H16_ a0228 protein and/or the H16_ B0917 protein.
In a sixth aspect, the present invention provides the use of the engineered eubacterium rolfsii as described above in the fermentative production of a biochemical.
The above-mentioned biochemical substances include, but are not limited to, polyesters, alcohols, amino acids, polypeptides, proteins, nucleic acids, saccharides, lipids, and the like.
In some embodiments of the invention, there is provided the use of an engineered Eumycota rolfsii as described above in the fermentative production of PHA or derivatives thereof.
In some embodiments of the invention, the fermentation production of PHA using the above-described engineered Eubacterium reuteri is conducted with a vegetable oil (including, but not limited to, a mixture of 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 the fermentative 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 the above method for constructing engineered eubacterium rolfsii, the method comprising: the eubacterium rolfsii is modified such that expression and/or enzyme activity of H16_ a0228 protein and/or H16_ B0917 protein therein is reduced.
In an eighth aspect, the present invention provides a method of reducing endotoxin content of a fungus selected from the group consisting of: the eubacterium rolfsii is modified such that expression and/or enzyme activity of H16_ a0228 protein and/or H16_ B0917 protein therein is reduced.
In some embodiments of the invention, the expression and/or enzymatic activity is reduced to inactivate the H16_ a0228 protein and/or the H16_ B0917 protein, or such that the H16_ a0228 protein and/or the H16_ B0917 protein is not expressed by the eumycete rolfsii.
The invention has the beneficial effects that: the inactivation of the protein H16_ A0228 and the protein H16_ B0917 of the eubacterium rolfsii provided by the invention can obviously reduce the endotoxin content of the eubacterium rolfsii. The engineered Eutyphus roseus with the H16_ A0228 protein and the H16_ B0917 protein lacking functions not only has the endotoxin content remarkably reduced, but also has the cell dry weight and the PHA yield remarkably improved, provides new genes and strain resources for the development of PHA engineered strains, provides an effective method for reducing the endotoxin content of PHA, and has important significance for expanding the application of PHA in medical materials.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The experimental procedures 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 plasmid is purchased from Tiangen Biotechnology (Beijing) Co., ltd, the kit for recovering DNA fragment is purchased from American omega, the corresponding operation steps are strictly carried out according to the product instruction, and all culture media are prepared by deionized water if no special instruction exists.
The media formulations used in the following examples were as follows:
seed culture medium: 10g/L peptone,5g/L Yeast Extract, and 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 of the trace element solution I and 1mL/L of the trace element solution II. Wherein the microelement solution I comprises the following components: 20g/LMgSO 4 ，2g/L CaCl 2 . The composition of the trace element solution II is as follows: 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 And O. The reagents are all purchased from chemical reagents of national drug group.
Example 1H16 u B0917 construction and identification of deletion mutants
In this embodiment, the eumycete rochei H16 is used as an initiating bacterium to knock out the gene H16_ B0917, which specifically includes the following steps:
the method comprises the following steps: construction of the base plasmid
Taking a genome of the Eutropha rolfsii H16 as a template, performing PCR amplification by using 917H1-F and 917H1-R to obtain an upstream homologous arm 917-H1 of H16_ B0917, and performing PCR amplification by using 917H2-F and 917H2-R to obtain a downstream homologous arm 917-H2 of H16_ B0917; PCR 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, and 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
Figure BDA0003758079260000071
Figure BDA0003758079260000081
Step two: construction of target strains for H16_ B0917 deletion mutation
Transforming the recombinant plasmid pKO-H16_ B0917 obtained in the step one into Escherichia coli S17-1, transferring the recombinant plasmid into the Eubacteria H16 by a conjugative transformation method, and screening positive clones by using an LB plate simultaneously containing 500 mu g/mL spectinomycin and 100 mu g/mL 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 a specific position of H1 and H2 on a genome, and is a first homologous recombinant bacterium. The first homologous recombinant strain was subjected to single colony culture on LB plate containing 100mg/mL of sucrose, and clones having no spectinomycin resistance were selected from these single colonies, and the strain was cultured using primer 917-H1FP (SEQ ID NO. 19): ATGTCGCTGACCGACGACCATGTC and 917-H1RP (SEQ ID NO. 20): TTGGCACCACCAGCCTGACCAATG was used to perform PCR to identify recombinant strains with H16_ B0917 gene knockout, and the recombinant strain finally obtained was H16_ B0917 gene knockout Eutropha rolfsii Re01.
Example 2 construction and identification of H16 _A0228deletion mutant
In this embodiment, the eumycete roche H16 is used as an initiating bacterium to knock out the gene H16_ a0228, and the method specifically includes the following steps:
the method comprises the following steps: construction of the base plasmid
Performing PCR amplification by using a genome of the eubacterium rolfsii H16 as a template and using 228H1-F and 228H1-R to obtain an upstream homology arm 228-H1 of H16_ A0228, and performing PCR amplification by using 228H2-F and 228H2-R to obtain a downstream homology arm 228-H2 of H16_ A0228; carrying out PCR amplification 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 are connected with the vector fragment by a Gibson Assembly method to obtain a recombinant plasmid pKO-H16_ A0228 (the sequence is shown as SEQ ID NO. 2). The primers used are shown in Table 2.
TABLE 2
Figure BDA0003758079260000082
Figure BDA0003758079260000091
Step two: construction of H16_ A0228 deletion mutant target Strain
And (3) transforming the recombinant plasmid pKO-H16_ A0228 obtained in the step one into escherichia coli S17-1, transferring the escherichia coli S17-1 into a eumycete Roche by a conjugative transformation method, and screening positive clones by using an LB plate simultaneously containing 500 mu g/mL spectinomycin and 100 mu g/mL apramycin by utilizing the characteristic that the suicide plasmid cannot be replicated in a host bacterium. The recombinant plasmid with homologous fragments in the positive clone is integrated into a specific position of H1 and H2 on a genome, and is a first homologous recombinant bacterium. The first homologous recombinant strain is subjected to single clone culture on an LB plate containing 100mg/mL sucrose, clones without spectinomycin resistance are screened out from the single clones, PCR identification is carried out on a recombinant strain with H16_ A0228 gene knockout by using primers 228-H1FP (SEQ ID NO. 21): ATCGATACCACCGAGATCCATTCG and 228-H1RP (SEQ ID NO. 22): AGCTGCATGGCTTTGACGACTACC, and finally the obtained recombinant strain is H16_ A0228 knockout Roche eumycete Re02.
Example 3 construction and identification of double deletion mutants of H16 \ u B0917 and H16_ A0228
In this example, the H16_ B0917 gene is knocked out by using the eubacterium roche Re02 constructed in example 2 as a starting bacterium, and the specific steps are as follows:
the recombinant plasmid pKO-H16_ B0917 obtained in example 1 was transformed into E.coli S17-1, and then transferred into the fungal strain Re02 of example 2 by the conjugative transformation method, and positive clones were selected by using LB plates containing both 500. Mu.g/mL spectinomycin and 100. Mu.g/mL apramycin, taking advantage of the property that the suicide plasmid cannot replicate in the host bacteria. The recombinant plasmid with homologous fragments in the positive clone is integrated into a specific position of H1 and H2 on a genome, and is a first homologous recombinant bacterium. The first homologous recombinant strain was subjected to single colony culture on an LB plate containing 100mg/mL sucrose, clones free from spectinomycin resistance were selected from these single clones, and the DNA fragment was isolated using primers 917-H1FP: ATGTCGCTGACCGACGACCATGTC and 917-H1RP: TTGGCACCACCAGCCTGACCAATG PCR was performed to identify recombinant strains with H16_ B0917 gene knockout, and the final recombinant strain obtained was a Roche eutropha Re03 with H16_ B0917 and H16_ A0228 knockout.
Example 4 Performance verification of strains Re01, re02, re03
In this example, the content of endotoxin, biomass and PHB in Re01, re02 and Re03 after fermentation were tested by using Eubacterium reuteri H16 as a control.
The method comprises the following steps: fermentation culture of strains Re01, re02 and Re03
Re01, re02, re03 and H16 were streaked on LB plates to obtain single clones, which were inoculated into seed medium (4 mL) and cultured for 12 hours. The overnight cultured bacterial liquid was transferred to a 100mL glass Erlenmeyer flask containing 10mL seed medium, and then transferred culture was carried out by inoculating the final OD of about 0.1, 30 ℃,220rpm, and culturing for 8 hours. The PHA is cultured by inoculating preculture seed liquid with OD value of 6-7 into a 250mL shake flask containing 30mL production medium according to the final OD of 0.1, and performing fermentation culture at 30 ℃ and 220rpm for 48h. Each strain was set up in 3 replicates.
Step two: endotoxin content determination of strains Re01, re02 and Re03
Taking 1mL of fresh bacterial liquid fermented in the step one, centrifuging at 13400g for 1min to collect the bacteria, discarding the supernatant, then re-suspending the bacteria by using 30% ethanol solution, centrifuging, discarding the supernatant, repeating the steps twice, and washing the bacteria. Finally, the thalli is diluted to OD by using deionized water 600 0.5, the cells were lysed at 100 ℃ for 30min and then left at room temperature for 24 hours to release endotoxin sufficiently. Detecting the content of the endotoxin by using a ToxinSensor chromogenic LAL endotoxin detection kit. The results are shown in Table 3. Compared with a control strain Eutropha rolfsii H16, the endotoxin unit contents of Re01, re02 and Re03 are respectively reduced by 46 times, 52 times and 95 times.
The kit for detecting LAL endotoxin by the ToxinSensor color development method is purchased from Jinslei Biotechnology GmbH.
TABLE 3
Figure BDA0003758079260000101
Figure BDA0003758079260000111
Step three: biomass determination of strains Re01, re02 and Re03
Taking the fermentation liquid volume in the first step, measuring the volume of a bacterium liquid (marked as V, unit: mL) by using a 50mL measuring cylinder, placing the measured fermentation liquid volume in a weighed centrifuge tube (marked as m1, unit: g) at 8000rpm, centrifuging the solution for 10min at room temperature, discarding supernatant, and collecting thalli; then, using 15mL of 30% ethanol solution, resuspending the thalli, 8000rpm, room temperature, centrifuging for 10min, repeating twice, washing off grease and culture medium in the strain, finally placing the centrifugal tube for collecting the thalli in a 60 ℃ oven, drying to constant weight, and accurately weighing the weight (recorded as m2, unit: g) by using an analytical balance. And the dry weight thereof was calculated. The results are shown in Table 4. Compared with the control strain Eutropha rolfsii H16, the biomass of Re01, re02 and Re03 is respectively improved by 27.8 percent, 17.9 percent and 28.2 percent.
The above formula for dry weight calculation is M = (M2-M1)/V1000, unit is g/L.
TABLE 4
Bacterial strains Dry weight (g/L)
Eubacterium reuteri 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 the sample dried in the third step, accurately weighing 30-40 mg, placing the sample into a digestion tube, adding 2mL of esterification liquid and 2mL of chloroform, covering and sealing the esterification tube, reacting at 100 ℃ for 4 hours, standing and cooling to room temperature after the reaction is finished, adding 1mL of deionized water, performing vortex oscillation until the mixture is completely mixed, standing and layering, and taking a lower organic phase for gas chromatography analysis.
The preparation method of the esterification solution comprises the steps of taking 485mL of anhydrous methanol, adding 1g/L of benzoic acid, and slowly adding 15mL of concentrated sulfuric acid to prepare 500mL of esterification solution.
2. Treating a standard substance: poly [ (R) -3-hydroxybutyric acid ], used to calibrate the 3HB unit, was a white powder, and the gradient values were 15mg, 25mg, and 35mg, which were weighed in the same manner as the sample treatment described above.
3. Analysis by GC of PHA composition and content: a gas chromatograph model GC-2014 from shimadzu was used. The chromatograph is configured to: an HP-5 type capillary chromatographic column, a hydrogen flame ionization detector FID and an SPL shunt sample inlet; high-purity nitrogen is used as carrier gas, hydrogen is fuel gas, and air is combustion-supporting gas; an AOC-20S autosampler was used, acetone being the wash liquid. The settings of the GC analysis program were: the sample inlet temperature is 240 ℃, the detector temperature is 250 ℃, the initial column temperature is 80 ℃, and the temperature is maintained for 1.5 minutes; raising the temperature to 140 ℃ at a rate of 30 ℃/min and maintaining the temperature for 0 min; ramping up to 240 ℃ at a rate of 40 ℃/min and maintaining for 2 min; the total time was 8 minutes. And quantitatively calculating the PHB content according to peak areas by adopting an internal standard normalization method in the GC result. As shown in Table 5, the PHB contents of RE01, RE02 and RE03 were increased by 7.02%,5.42% and 9.61%, respectively, as compared with the control strain.
TABLE 5
Bacterial strains PHB％
Eubacterium reuteri H16 70.59％
Re01 75.55％
Re02 74.42％
Re03 77.38％
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. Use of reduced expression and/or enzymatic activity of a H16_ A0228 protein and/or H16_ B0917 protein of Eubacterium rolfsii for reducing the endotoxin content of Eubacterium rolfsii.
2. Use of reduced expression and/or enzymatic activity of a H16_ A0228 protein and/or H16_ B0917 protein of a Eubacterium rolfsii in increasing PHA production by a Eubacterium rolfsii.
3. Use of reduced expression and/or enzymatic activity of a H16_ A0228 protein and/or H16_ B0917 protein of Eubacterium rolfsii for increasing biomass of Eubacterium rolfsii.
4. Use of reduced expression and/or enzymatic activity of a protein H16_ A0228 and/or H16_ B0917 of a Eubacterium rolfsii strain in reducing endotoxin content of the Eubacterium rolfsii strain while increasing PHA yield and biomass of the Eubacterium rolfsii strain.
5. Engineered eubacterium rolfsii, wherein said engineered eubacterium rolfsii is modified such that expression and/or enzymatic activity of H16_ a0228 protein and/or H16_ B0917 protein therein is reduced.
6. The engineered eubacterium rolfsii according to claim 5, wherein the H16_ a0228 protein and/or H16_ B0917 protein in the engineered eubacterium rolfsii is inactivated or the engineered eubacterium rolfsii does not express H16_ a0228 protein and/or H16_ B0917 protein.
7. Use of the engineered eubacterium rolfsii according to claim 5 or 6 in the fermentative production of a biochemical.
8. The method of constructing an engineered eubacterium reuteri according to claim 5 or 6, wherein said method comprises: the eubacterium rolfsii is modified such that expression and/or enzyme activity of H16_ a0228 protein and/or H16_ B0917 protein therein is reduced.
9. A method of reducing endotoxin content of a fungus selected from the group consisting of eubacterium rolfsii, said method comprising: the eubacterium rolfsii is modified such that expression and/or enzyme activity of H16_ a0228 protein and/or H16_ B0917 protein therein is reduced.
10. The method of reducing endotoxin content of eubacterium rolfsii according to claim 9, wherein said reduction in expression and/or enzyme activity is to inactivate H16_ a0228 protein and/or H16_ B0917 protein or to render eubacterium rolfsii not express H16_ a0228 protein and/or H16_ B0917 protein.
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