CN113106050A - Method for reducing vancomycin resistance and application of method in cofactor production - Google Patents

Method for reducing vancomycin resistance and application of method in cofactor production Download PDF

Info

Publication number
CN113106050A
CN113106050A CN202110469210.5A CN202110469210A CN113106050A CN 113106050 A CN113106050 A CN 113106050A CN 202110469210 A CN202110469210 A CN 202110469210A CN 113106050 A CN113106050 A CN 113106050A
Authority
CN
China
Prior art keywords
gene cluster
wza
galf
gene
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202110469210.5A
Other languages
Chinese (zh)
Other versions
CN113106050B (en
Inventor
王小元
王建莉
马文渐
陈闪闪
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to CN202110469210.5A priority Critical patent/CN113106050B/en
Publication of CN113106050A publication Critical patent/CN113106050A/en
Application granted granted Critical
Publication of CN113106050B publication Critical patent/CN113106050B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • 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
    • C07K14/245—Escherichia (G)
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/36—Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
    • 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 discloses a method for reducing vancomycin resistance and application thereof in cofactor production, and belongs to the field of genetic engineering and medical engineering. According to the invention, a core sugar gene cluster gmhD-waaQ and an O-antigen gene cluster wbbL-rmlB of lipopolysaccharide on a genome of Escherichia coli W3110, an extracellular polysaccharide gene cluster galF-wza and a type 4 capsular polysaccharide synthetic gene cluster yccC-ymcD are knocked out, so that a recombinant strain WJW04 is obtained; obtaining a strain WJW09 by knocking out an extracellular polysaccharide gene cluster galF-wza of escherichia coli W3110; the MICs of WJW09 and WJW04 on the vancomycin are 600mg/L and 76.8mg/L respectively, and the MICs of WJW09 and WJW04 on the vancomycin are 1000mg/L lower than the MICs of the wild strain W3110 on the vancomycin, so that the technical scheme disclosed by the invention is widely applied to the pharmaceutical industry.

Description

Method for reducing vancomycin resistance and application of method in cofactor production
Technical Field
The invention relates to a method for reducing vancomycin resistance and application thereof in cofactor production, and belongs to the fields of genetic engineering and biological medicine.
Background
Bacterial resistance refers to the phenomenon that bacteria are not sensitive to antibacterial drugs, and is a special manifestation of the survival process of bacteria. The overuse of antibiotics in agriculture, animal husbandry and clinical settings leads to the production of a large number of resistant bacteria, which puts a great strain on the choice of drugs. In order to deal with the worldwide problem of bacterial resistance, a thorough understanding of the properties of the target enzyme for bacterial resistance is needed to guide the design and synthesis of enzyme inhibitors and to act synergistically with clinical antibiotics to combat resistant bacteria. Because of the presence of drug resistance, pathogenic strains are not susceptible to antibiotics, which require frequent replacement during the course of disease treatment.
Vancomycin (Vancomycin) has strong potency, is called as the last king brand of antibiotics, can be used for treating infection of super-drug-resistant bacteria, and is the last line of defense for human beings to deal with super-drug-resistant strains at present. However, food contamination of vancomycin is caused by abuse of vancomycin in a farm, and adverse reactions such as toxic reaction of ears, kidneys and nervous systems, skin allergy, organ dysfunction and the like can be caused by long-term or excessive consumption of animal-derived food containing vancomycin. Meanwhile, the number of resistant bacteria of vancomycin is increased due to abuse of the vancomycin, and more serious antibiotic resistance is generated, which is extremely adverse to prevention and treatment of human diseases and influences safety, public health, ecological balance and export of animal-derived foods. In addition, the synthesis of vancomycin is synthesized by microbial fermentation, the synthesis yield is low, the synthesis regulation and control are complex, and the extraction process is complex. Therefore, the discovery of a vancomycin resistance system or structure has great significance for preventing and controlling vancomycin resistance. Vancomycin kills bacteria by inhibiting synthesis of a peptidoglycan layer in a cell wall, the peptidoglycan layer in the cell wall of Escherichia coli is located between an outer membrane and an inner membrane of the cell, and vancomycin needs to penetrate through the outer membrane to enter a peptidoglycan layer action target spot, so that the outer membrane has a vital function of blocking vancomycin from entering the cell as a barrier. The method is of great significance for preventing and controlling the drug resistance of the vancomycin by exploring the drug-resistant outer membrane structure of the vancomycin.
Escherichia coli (Escherichia coli), which is a typical industrial production strain of gram-negative bacteria, is commonly used for synthesis and production of intracellular polyhydroxyalkanoates, organic acids, amino acids, pigments and other products, and the products belong to the fields of food and medicine. Researches find that the drug resistance of the escherichia coli can cause microbial pollution and potential safety hazards of product eating. Coli is essential for repairing damage of cells and improving their viability in harsh environments. Coli has found some resistance to vancomycin. Therefore, how to improve the drug resistance of escherichia coli has great significance for medical research.
Disclosure of Invention
The technical problem is as follows:
the technical problem to be solved by the application is to provide a method capable of reducing the vancomycin resistance of escherichia coli, provide application of knocking out a composition gene cluster, provide a drug action target point for reducing the vancomycin resistance of escherichia coli, provide a drug for reducing the vancomycin resistance of escherichia coli, and provide application of the composition gene cluster serving as the drug action target point in developing the drug for reducing the vancomycin resistance of escherichia coli.
The technical scheme is as follows:
in order to solve the technical problems, the invention provides a drug for reducing the drug resistance of escherichia coli to vancomycin, wherein the drug can inhibit or down-regulate the expression of a composition gene cluster; the composition gene cluster is as follows: core saccharide gene cluster gmhD-waaQ and O-antigen gene cluster wbbL-rmlB of lipopolysaccharide, extracellular polysaccharide gene cluster galF-wza and type 4 capsular polysaccharide synthesis gene cluster yccC-ymcD;
or the composition gene cluster is: the extracellular polysaccharide gene cluster galF-wza.
In one embodiment of the present invention, the core carbohydrate gene cluster gmhD-waaQ genes are waaQ, waaG, waaP, waaS, waaB, waaO, waaR, waaY, waaZ, waaU u, waaL, waaC, waaF, gmdD; the WbbL-rmlB genes of the O-antigen gene cluster are respectively WbbL, IS5, WbbK, WbbJ, WbbI, wzy, glf, wzx, rmlC, rmlA, rmlD and rmlB; the extracellular polysaccharide gene cluster galF-wza genes are wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, wcaG, wcaH, wcaI, manC, manB, wcaJ, wzx, wcaK, wcaL, wcaM and galF respectively; the type 4 capsular polysaccharide synthetic gene cluster yccC-ymcD genes are yccC, etp, yccZ, ymcA, ymcB, ymcC and ymcD respectively.
The invention provides a drug action target for reducing the drug resistance of escherichia coli to vancomycin, wherein the action target is a composition gene cluster; the composition gene cluster is as follows: core saccharide gene cluster gmhD-waaQ of lipopolysaccharide, O-antigen gene cluster wbbL-rmlB, extracellular polysaccharide gene cluster galF-wza and type 4 capsular polysaccharide synthesis gene cluster yccC-ymcD;
or the composition gene cluster is: the extracellular polysaccharide gene cluster galF-wza.
The invention provides an application of a deletion composition gene cluster in reducing the drug resistance of escherichia coli to vancomycin, wherein the composition gene cluster is as follows: a core saccharide gene cluster gmhD-waaQ of lipopolysaccharide, an O-antigen gene cluster wbbL-rmlB, an extracellular polysaccharide gene cluster galF-wza and a type 4 capsular polysaccharide synthesis gene cluster yccC-ymcD.
In one embodiment of the present invention, the core carbohydrate gene cluster gmhD-waaQ is waaQ, waaG, waaP, waaS, waaB, waaO, waaR, waaY, waaZ, waaU u, waaL, waaC, waaF, gmdD, NCBI accession number of the gene sequence is BAE77660.1, BAE77661.1, BAE77662.1, BAE77663.1, BAE77664.1, BAE77665.1, BAE77666.1, BAE77667.1, BAE77668.1, BAE77669.1, BAE77670.1, BAE77671.1, BAE77672.1, BAE77673.1, respectively.
In one embodiment of the invention, the O-antigen gene cluster wbBL-rmlB IS wbBL:IS 5, wbbK, wbbJ, wbbI, wzy, glf, wzx, rmlC, rmlA, rmlD, rmlB, NCBI accession numbers of the gene sequences are BAA15873.1, BAA15874.1, BAA15875.1, BAA15876.1, BAA15877.1, BAA15878.1, BAA15879.1, BAA15880.1, BAA15881.1, BAA15882.1, BAA15883.1, BAA15884.1 in this order.
In one embodiment of the invention, the extracellular polysaccharide gene cluster galF-wza is wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, wcaG, wcaH, wcaI, manC, manB, wcaJ, wzx, wcaK, wcaL, wcaM, galF, NCBI accession numbers of the gene sequences are BAE76576.1, BAE76575.1, BAA15913.1, BAA15912.1, BAA15911.1, BAE76574.1, BAE76573.1, BAE76572.1, BAA15910.1, BAA15909.1, BAA15908.1, BAA15907.1, BAA15906.1, BAA15905.1, BAA15901.1, BAA15900.1, BAA15899.1, BAE76571.1, BAA15898.1, BAA15897.1, BAA15896.1, respectively.
In one embodiment of the invention, the type 4 capsular polysaccharide synthesis gene cluster yccC-ymcD is yccC, etp, yccZ, ymcA, ymcB, ymcC, ymcD, NCBI accession numbers of the gene sequences are BAA35746.1, BAA35747.2, BAA35748.1, BAA35749.1, BAA35750.2, BAA35751.1, and BAA35752.2 in sequence.
In one embodiment of the invention, the Escherichia coli is E.coli K-12W 3110.
In one embodiment of the invention, the deletion is a knock-out or no expression.
The invention also provides application of the composition gene cluster as a drug action target in preparing a drug for reducing the drug resistance of escherichia coli to antibiotics, and is characterized in that the composition gene cluster is as follows: core saccharide gene cluster gmhD-waaQ of lipopolysaccharide, O-antigen gene cluster wbbL-rmlB, extracellular polysaccharide gene cluster galF-wza and type 4 capsular polysaccharide synthesis gene cluster yccC-ymcD;
or the composition gene cluster is: the extracellular polysaccharide gene cluster galF-wza.
In one embodiment of the invention, the agent is capable of inhibiting or down-regulating the expression level of a gene cluster of the composition.
The invention also provides a method for improving the preparation of acetyl coenzyme A and cofactor NADH/NAD by using escherichia coli+And NADPH/NADP+A method for the production of a combination gene cluster or exopolysaccharide gene cluster galF-wza in the genome of escherichia coli by knocking out, said combination gene cluster being: the composition gene cluster is as follows: fat and oilThe core saccharide gene cluster gmhD-waaQ of the polysaccharide, the O-antigen gene cluster wbbL-rmlB, the extracellular polysaccharide gene cluster galF-wza and the type 4 capsular polysaccharide synthesis gene cluster yccC-ymcD.
Advantageous effects
(1) The extracellular polysaccharide gene cluster on the W3110 genome of wild type escherichia coli is knocked out to knock out galF-wza to obtain a strain WJW 09; knocking out a core sugar gene cluster gmhD-waaQ, an O-antigen gene cluster wbbL-rmlB, an extracellular polysaccharide gene cluster galF-wza and a type 4 capsular polysaccharide synthetic gene cluster yccC-ymcD of lipopolysaccharide on a genome of wild type Escherichia coli W3110 to finally obtain a strain WJW 04. Under normal LB culture conditions, the Minimum Inhibitory Concentrations (MIC) of the recombinant bacteria WJW09 and WJW04 on vancomycin are 600mg/L and 76.8mg/L respectively, while the Minimum Inhibitory Concentration (MIC) of W3110 on vancomycin is 1000mg/L, wherein the recombinant bacteria WJW04 is reduced by 13 times compared with the wild control bacteria W3110(MIC is 1000 mg/L).
(2) By adopting the technical scheme of the invention, the Escherichia coli wild type W3110 can synthesize 60.20 mug/L of acetyl coenzyme A, while WJW09 can synthesize the acetyl coenzyme A with the concentration of 75.65 mug/L, which is improved by 25.7 percent compared with a wild type strain; recombinant strain WJW09 synthesized coenzyme NADH/NAD+The ratio was 0.0911, compared to the wild-type control strain W3110 (NADH/NAD)+0.0507) by 79.8%; recombinant strain WJW09 synthesized coenzyme NADPH/NADP+The ratio was 0.0840, compared to the wild-type control strain W3110 (NADPH/NADP)+Ratio 0.0347) by a factor of 1.42.
By adopting the method, the recombinant strain WJW04 can synthesize acetyl coenzyme A with the concentration of 460.2 mug/L, which is 6.6 times higher than that of the wild W3110 strain; the recombinant strain WJW04 can synthesize NADH/NAD+The ratio was 0.453, and NADPH/NADP was synthesized+The ratio of the strain is 0.0580, which is respectively improved by 7.94 times and 67.0 percent compared with the wild strain; shows that the deletion of the extracellular polysaccharide structure can improve the cofactor NADH/NAD in the escherichia coli cells+And NADPH/NADP+The ratio of (a) to (b).
Acetyl coenzyme A, coenzyme NADH/NAD+And coenzyme NADPH/NADP+The improvement of the proportion is beneficial to the improvement of the production efficiency of the strains.
Drawings
FIG. 1: MIC determination of each strain for vancomycin.
FIG. 2: determination of intracellular acetyl-CoA Synthesis by wild type Strain W3110 and recombinant Strain WJW 09.
FIG. 3: synthesis of intracellular coenzyme NADH/NAD by wild type strain W3110 and recombinant strain WJW09+And (4) measuring the proportion.
FIG. 4: synthesis of intracellular coenzyme NADPH/NADP by wild type strain W3110 and recombinant strain WJW09+And (4) measuring the proportion.
FIG. 5: determination of intracellular acetyl-CoA Synthesis by recombinant Strain WJW 04.
FIG. 6: recombination strain WJW04 synthesizes intracellular coenzyme NADH/NAD+Ratio and NADPH/NADP+And (4) measuring the proportion.
Detailed Description
The wild-type E.coli W3110 referred to in the following examples was purchased from ATCC with accession number ATCC 39936.
The strains involved in the following examples are shown in table 1:
TABLE 1 strains
Figure BDA0003044696230000041
Figure BDA0003044696230000051
(1) Gene knockout method
And (3) knocking out the large fragment gene cluster by adopting a combined site-specific recombination method and a CRISPR/Cas9 knocking-out system. Introducing a lox L-Kan fragment at one end of a target gene cluster through Red recombination, then introducing a cat-lox R fragment at the other end through Red recombination, then expressing through Cre enzyme, identifying sites lox L and lox R, carrying out site recombination, adopting AMP resistance screening, obtaining transformants without Kan and Cm resistance through resistance verification, and further adopting PCR to verify the genotype, thereby determining the correctness of the transformants. The method for simplifying FRT sites is similar, FRT sites (two sites can be used) are introduced at two ends of a target gene cluster, then the FRT sites are identified through Flp enzyme expression, and correct knockout mutant strains can be obtained through resistance screening. The Flp enzyme can continuously recognize the FRT site until the most distal FRT site that does not contain the essential gene is recombined.
Tools for CRISPR/Cas9 knockout systems plasmids pCas and pTargetF were purchased from vast panacea. The knockout procedure is referenced to the method of Jiang et al (Jiang, Y., Chen, B., Duan, C., et al., multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system [ J]Appl Environ Microbiol 2015,81(7): 2506-1410.1128/AEM 04023-14). The specific knockout process is as follows: firstly, transferring pCas plasmid into Escherichia coli outbreak W3110, and then preparing electrotransformation competence after L-arabinose induction for knockout; secondly, connecting upstream and downstream homology arms through overlapping PCR to construct a knockout fragment; then, 20nt of N which is the same as the target sequence of the target gene is introduced by reconstruction of pTargetF plasmid20Constructing a knockout plasmid pTargetF-gene; and simultaneously transferring the knockout fragment and the knockout plasmid into a corresponding strain containing pCas, screening to obtain a knockout transformant, and finally removing the knockout plasmid and the pCas to obtain a correct non-resistant traceless knockout mutant strain.
(2) MIC determination method
In 96-well plates, different concentrations of novobiocin were added to each well by a two-fold dilution method. For each concentration, 180. mu.L of LB broth plus 20. mu.L of antibiotic stock was added to the first well, mixed well, 100. mu.L transferred to the second well with 100. mu.L of LB, 100. mu.L transferred from the second well to the third well with 100. mu.L of LB, and so on.
The strains were cultured in LB liquid medium overnight to late logarithmic phase (OD)6003.00), the sample was diluted with fresh LB medium to OD600Diluting to 1 × 10-3 Add 100. mu.L to a 96-well plate. The first well is a blank LB control well with only LB medium and no antibiotics and bacteria solution, and the last well is a blank control well with only bacteria solution and no antibiotics. After determining the MIC value range, the MIC refinement experiment is carried out. The detailed method of the thinning experiment still adopts a 96-pore plate for determination, but the design gradient is not more than 2 times of the dilution method,MIC values are typically determined by isocratic methods, i.e., within the range of MIC values, concentration gradients with small concentration differences are designed to minimize MIC errors, typically with gradient differences less than or equal to 10% of their MIC values, to determine their MIC values accurately with as little error as possible.
(3) Determination of intracellular acetyl-CoA
The bacterial strain is cultured by adopting an LB culture medium to synthesize acetyl coenzyme A.
Extraction of intracellular acetyl coenzyme A: the main coa monomer assayed in this study is acetyl-coa. First, LB liquid culture cells were grown to mid-log phase (OD)6001.5), 20mL of the cells were collected by refrigerated centrifugation, quickly transferred to a pre-cooled 1.5mL EP tube, washed twice with ice-cold 0.9% NaCl solution, and resuspended in methanol pre-cooled to-20 ℃: acetonitrile: in an extract of water (45:45:10, v: v: v), 0.1M formic acid was contained in the extract, and the purpose of adding formic acid was to quench the coenzyme A monomer. And (3) under an ice bath condition, carrying out ultrasonic crusher crushing on the bacterial suspension for 10min, wherein the crushing procedure is to operate for 2s, pause for 3s, then carry out 12000rpm, centrifuge for 15min, discard impurities such as thallus fragments and the like, and absorb 800 mu L of supernatant into a sample bottle for immediately carrying out sample determination by a triple quadrupole liquid chromatography mass spectrometer (LC-MS).
Determination of acetyl-CoA: the quantification of intracellular coenzyme A is carried out in cooperation with a large instrument sharing test platform of the university of south China. The cell temperature was set at 4 ℃. The mobile phase A is 5mM chromatographic pure ammonium acetate, the mobile phase B is chromatographic pure methanol, and the elution speed is 0.2 mL/min. Mass spectrometry uses triple quadrupole mass spectrometry dedicated to quantitation, using Multiple Reaction Monitoring (MRM) mode, identifying target coa fragments by parent and daughter ions, acetyl coa parent m/z 810, daughter ion m/z 303, propionyl coa parent m/z 824, daughter ion m/z 317, propionyl coa as the internal standard, external standard acetyl coa. Mass spectral data were analyzed using a Waters C18 column (ACQUITY UPLC, BEH C18,1.7 μm, 2.1X 100mm, Ireland) using the Xcalibur software.
(4) Cellular synthesis of coenzyme NADH/NAD+Ratio and coenzymes NADPH/NADP+Determination of the ratio
Strain seed liquid adopts LB culture mediumCulturing and synthesizing coenzyme NADH/NAD+Ratio and coenzymes NADPH/NADP+The fermentation medium of the proportion is cultured by using M9G medium.
The extraction and determination process comprises the following steps: determined by the kit method, i.e. NADP+/NADPH Assay Kit with WST-8(S0179,Beyotime),NAD+the/NADH Assay Kit with WST-8(S0175, Beyotime), following Kit instructions, is briefly summarized as follows: fermenting Escherichia coli cells in M9G culture medium, and culturing to middle logarithmic phase (OD)6002.5), 1mL of the culture broth was collected by centrifugation (4 ℃, 10000g), the supernatant was immediately and thoroughly removed to ensure the accuracy of the assay, and then immediately frozen with liquid nitrogen to quench the metabolism of the cells. At the same time, ATP standards were also prepared at various concentrations (0 to 10. mu.M). All samples and standards are prepared by using a tandem pipettor to operate simultaneously, so that the consistency and timeliness of the operation of all samples or standards are maintained as much as possible. The reaction was carried out using a 96-well opaque plate, and for the measurement of NADP and NAD, the reaction was carried out using a transparent 96-well plate, and after development for 30 minutes with a Microplate reader, the absorbance value was read at. lambda.450 nm. At the same time, the cellular protein concentration of the extraction mixture was also determined.
The media involved in the following examples are as follows:
LB liquid medium: 5g/L of yeast powder, 10g/L of tryptone and 10g/L of NaCl.
LB solid medium: 1.5 percent of agar powder is added on the basis of LB liquid culture medium.
M9G medium: 20g/L Glucose (Glucose), 17.1g/L disodium phosphate dodecahydrate (Na)2HPO4·12H2O), 3g/L potassium dihydrogen phosphate (KH)2PO4) 0.5g/L sodium chloride (NaCl), 1mM magnesium sulfate (MgSO)4) 0.1mM calcium chloride (CaCl)2) 10mg/mL Vitamin B1 (VB)1)。
Example 1: construction of engineering bacteria WJW00(E.coli W3110. DELTA. gmhD), WJW01, WJW02, WJW03, WJW04 and WJW09
The construction process of engineering bacteria WJW00(E.coli W3110. delta. gmhD), WJW01, WJW02, WJW03 and WJW04 is described in the Chinese invention patent document with the publication number CN 110387347B; the construction method of WJW09 strain is described in Chinese patent publication No. CN 110387346B; wherein:
WJW01 strain: lipopolysaccharide (LPS) comprises three parts, lipid A, core sugar and O-antigen, wherein lipid A is a highly conserved domain, and core sugar and O-antigen are composed of different sugar molecules. Wherein the core sugar-synthesizing gene cluster comprises 14 genes, which are waaQ, waaG, waaP, waaS, waaB, waaO, waaR, waaY, waaZ, waaU, waaL, waaC, waaF, and gmdD, respectively. Continuously knocking out the remaining 13 genes except the gmhD gene in the core carbohydrate cluster gmdD (gmhD) -waaFC-waaQGPSBORYZUL on the basis of the strain WJW 00; a single colony is picked to verify the sensitivity of the single colony to ampicillin and kanamycin, and a strain sensitive to both antibiotics is selected and named as WJW01 and is preserved.
WJW02 strain: the O-antigen synthesis gene cluster rmlBrmlBDACX-glf-wbHIJKL comprises 11 genes, namely wbbL, IS5, wbbK, wbbJ, wbbI, wzy, glf, wzx, rmlC, rmlA, rmlD and rmlB. Further knocking out O-antigen synthetic gene cluster on the basis of WJW01 to obtain WJW02, picking single colony to verify the sensitivity of the single colony to ampicillin, kanamycin and chloramphenicol, selecting a strain sensitive to three antibiotics, and naming the strain as WJW02 and preserving the strain.
WJW03 strain: an extracellular polysaccharide synthesis and transport gene cluster (comprising 21 genes including wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, wcaG, wcaH, wcaI, manC, manB, wcaJ, wzx, wcaK, wcaL, wcaM and galF) is further knocked out on the basis of WJW02, and WJW03 is obtained. Because the exopolysaccharide synthesis and transport gene cluster and the O-antigen synthesis and transport gene cluster are adjacent on the W3110 genome, namely the adjacent gene of galF is the O-antigen synthesis gene rmlB, wza is continuously knocked out and lox RE is introduced in a strain which is singly knocked out of wbbL and is introduced with lox LE sites, site-specific recombination is carried out to delete all genes and resistance marker genes between the wbbL and wza, and finally, pKD-Cre plasmid is removed to obtain the nonreactive mutant strain WJW 03.
WJW04 strain: knocking out a type 4 capsular polysaccharide synthetic gene cluster yccC-ymcD (comprising 7 genes including yccC, etp, yccZ, ymcA, ymcB, ymcC and ymcD) from a WJW03 strain, obtaining a strain W3110 delta gmhD-waaQ delta wbbL-rmlB delta galF-wza delta yccC-ymcD after verification is correct, screening out a single colony sensitive to kanamycin, obtaining a mutation-free strain without pCas, and naming the strain as WJW 04.
WJW09 strain: 21 extracellular polysaccharide-encoding synthesis and transport genes (extracellular polysaccharide gene cluster galF-wza) are knocked out on the basis of Escherichia coli W3110, wherein the 21 extracellular polysaccharide-encoding synthesis and transport genes are wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, wcaG, wcaH, wcaI, manC, manB, wcaJ, wzx, wcaK, wcaL, wcaM and galF respectively, and are named as WJW 09.
Example 2: MIC analysis of recombinant strains for different antibiotics
1. Resistance analysis of E.coli W3110, WJW09, WJW02 and WJW04 to vancomycin
(1) In a 96-well plate, vancomycin at different concentrations was added to each well by a two-fold dilution method; for each concentration, 180. mu.L of LB broth plus 20. mu.L of antibiotic stock was added to the first well, mixed well, 100. mu.L transferred to the second well with 100. mu.L of LB, 100. mu.L transferred from the second well to the third well with 100. mu.L of LB, and so on.
The strains were cultured in LB liquid medium overnight to late logarithmic phase (OD)6003.00), the sample was diluted with fresh LB medium to OD600Diluting to 1 × 10-3 Add 100. mu.L to a 96-well plate.
The first well is a blank LB control well with only LB medium and no antibiotics and bacteria solution, and the last well is a blank control well with only bacteria solution and no antibiotics. Thus, the first initial antibiotic concentration is 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625mg/L in that order; sealing the 96-well plate by using a sealing film to prevent bacteria contamination and ensure the volatilization of water in the sample; finally, the concentrations of vancomycin in the 96-well plate were: 2000mg/L, 1000mg/L, 500mg/L, 250mg/L, 125mg/L, 62.5mg/L, 31.3mg/L, 15.6mg/L, 7.8mg/L, 3.9 mg/L.
(2) The 96-well plate was placed in a 37 ℃ incubator, and after 18 hours of incubation, the absorbance of the sample at a wavelength of 600nm was measured with a microplate reader, and the results are shown in Table 2.
Table 2: OD of each strain in presence of different vancomycin600Value of
Figure BDA0003044696230000081
(3) The minimum antibiotic concentration in the well that completely inhibited bacterial growth was taken as the MIC value of the strain for this antibiotic. Bacteria should grow significantly in all blank wells, and the liquid in the wells appears cloudy. The phenomenon of hole jumping should be avoided, and if multiple hole jumping occurs, repeated tests are required. Therefore, the concentration ranges of the vancomycin antibiotics of the W3110 strain and the WJW09 strain are determined to be 1000-5000 mg/L, and the concentration ranges of the vancomycin antibiotics of the recombinant strains WJW02 strain and WJW04 strain are respectively 250-125 mg/L and 125-62.5 mg/L.
After determining the range of the MIC, concentration gradients with smaller concentration differences were used to minimize the error in the MIC.
In MIC refinement experiments, the concentration range of vancomycin antibiotics of W3110 is 1000, 950, 900, 850, 800, 750, 700, 650, 600 and 550mg/L, the concentration range of vancomycin antibiotics of WJW02 is 250, 225, 200, 175, 150 and 125mg/L, and the concentration range of vancomycin antibiotics of WJW04 is 125, 112.5, 106.25, 100, 93.75, 87.5, 81.25, 75.0, 68.75 and 62.5 mg/L.
The MIC experiment showed that, as shown in fig. 1, the starting strain e.coli W3110 cells were highly resistant to vancomycin, with an MIC of 1000mg/L for vancomycin.
The MIC of the extracellular polysaccharide gene cluster galF-wza deletion recombinant strain WJW09 to vancomycin is 600mg/L, which is reduced by 40% compared with the wild control W3110; the MIC of WJW02 to vancomycin is 250mg/L, which is reduced by 3 times compared with the wild W3110; the MIC of WJW04 to vancomycin is 75.0mg/L, which is reduced by 2.33 times compared with the control bacteria WJW02 and 12.33 times compared with the wild control bacteria. The deletion of extracellular polysaccharide gene cluster and LPS gene cluster can improve the sensitivity of the strain to vancomycin. This indicates that the lipopolysaccharide structure and extracellular polysaccharide structural integrity of Escherichia coli play an important role in vancomycin resistance.
Example 3: strain W3110, extracellular polysaccharide knockout recombinant strain WJW09, and strain WJW04 for synthesizing acetyl coenzyme A
The method comprises the following specific steps:
(1) extraction of intracellular acetyl-CoA
The wild type strain W3110, strain WJW04, and strain WJW09 prepared in example 1 were inoculated into LB liquid medium and cultured at 37 ℃ and 200rpm to OD600＝1.5；
20mL of the cells were collected by refrigerated centrifugation, quickly transferred to a pre-chilled 1.5mL EP tube, washed twice with ice-cold 0.9% NaCl solution, and resuspended in methanol pre-chilled to-20 ℃: acetonitrile: in an extract of water (45:45:10, v: v: v), 0.1M formic acid was contained in the extract, and the purpose of adding formic acid was to quench the coenzyme A monomer. And (3) under an ice bath condition, carrying out ultrasonic crusher crushing on the bacterial suspension for 10min, wherein the crushing procedure is to operate for 2s, pause for 3s, then carry out 12000rpm, centrifuge for 15min, discard impurities such as thallus fragments and the like, and absorb 800 mu L of supernatant into a sample bottle for immediately carrying out sample determination by a triple quadrupole liquid chromatography mass spectrometer (LC-MS).
(2) Determination of acetyl-CoA
And extracting a characteristic peak of the acetyl coenzyme according to the measurement result, and calculating the concentration of the acetyl coenzyme A according to the peak area of the standard sample. The measurement results are shown in fig. 2 and 5.
The result shows that the Escherichia coli wild type W3110 can synthesize 60.20 mu g/L of acetyl coenzyme A, and the extracellular polysaccharide gene cluster galF-wza and the type 4 capsular polysaccharide gene cluster yccC-ymcD knockout strain WJW09 can synthesize the acetyl coenzyme A with the concentration of 75.65 mu g/L, which is improved by 25.7 percent compared with the wild type strain;
the recombinant strain WJW04 with LPS synthetic gene clusters of gmhD-waaQ and wbBL-rmlB knocked out in the W3110 genome and with galF-wza and ycC-ymcD knocked out further can synthesize acetyl coenzyme A with the concentration of 460.2 mug/L, which is 6.6 times higher than that of the wild W3110 strain.
The result shows that the deletion of the extracellular polysaccharide gene cluster and the LPS gene cluster can improve the yield of the bacterial strain to the acetyl coenzyme A. This indicates that the lipopolysaccharide structure and extracellular polysaccharide structural integrity of Escherichia coli play an important role in increasing the production of acetyl-CoA in the strain.
Example 4: production of cofactors by strain W3110, exopolysaccharide knock-out recombinant strain WJW09, strain WJW04
The wild type strain W3110, strain WJW04 and strain WJW09 prepared in example 1 were inoculated into LB liquid medium, and cultured at 37 ℃ and 200rpm for 4 hours to prepare a seed solution;
the prepared seed liquid is processed according to the initial OD6000.25 to M9G medium, cultured at 37 ℃ and 200rpm to OD600Centrifugation (4 ℃, 10000g) was performed at 2.5, and 1mL of the culture solution was collected and subjected to cofactor detection.
The extraction and determination process comprises the following steps: determined by the kit method, i.e. NADP+/NADPH Assay Kit with WST-8(S0179,Beyotime),NAD+the/NADH Assay Kit with WST-8(S0175, Beyotime), following Kit instructions, is briefly summarized as follows: the above 1mL of culture solution was immediately and thoroughly removed of supernatant to ensure the accuracy of the measurement, and then immediately frozen with liquid nitrogen to quench the metabolism of the cells. At the same time, ATP standards were also prepared at various concentrations (0 to 10. mu.M). All samples and standards are prepared by using a tandem pipettor to operate simultaneously, so that the consistency and timeliness of the operation of all samples or standards are maintained as much as possible. The reaction was carried out using a 96-well opaque plate, and for the measurement of NADP and NAD, the reaction was carried out using a transparent 96-well plate, and after development for 30 minutes with a Microplate reader, the absorbance value was read at. lambda.450 nm. At the same time, the cellular protein concentration of the extraction mixture was also determined. The concentration of each cofactor is expressed in μmol/g cellular protein. Calculating the cofactor NADH/NAD from the results of the measurement+And NADPH/NADP+The ratio of (a) to (b). The measurement results are shown in FIGS. 3 to 4 and 6.
The results show that Escherichia coli wild type W3110 can synthesize NADH/NAD+In a ratio of 0.0507, synthesizing NADPH/NADP+In a ratio of 0.0347, while strain WJW09 can synthesize NADH/NAD+The ratio is 0.091Synthesis of NADPH/NADP+The ratio of (A) is 0.0840, which is respectively improved by 79.8 percent and 1.42 times compared with the wild strain;
the strain WJW04 can synthesize NADH/NAD+The ratio was 0.453, and NADPH/NADP was synthesized+The ratio of the strain is 0.0580, which is respectively improved by 7.94 times and 67.0 percent compared with the wild strain;
the result shows that the deletion of extracellular polysaccharide gene cluster and LPS gene cluster can improve the cofactor NADH/NAD in the colibacillus+And NADPH/NADP+The ratio of (a) to (b). This shows that the integrity of lipopolysaccharide structure and extracellular polysaccharide structure of Escherichia coli can improve the cofactor NADH/NAD in Escherichia coli+And NADPH/NADP+Plays an important role in the ratio of (a).
Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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 (10)

1. A drug for reducing the resistance of escherichia coli to vancomycin, wherein the drug is capable of inhibiting or down-regulating the expression of a gene cluster of the composition; the composition gene cluster is as follows: core saccharide gene cluster gmhD-waaQ of lipopolysaccharide, O-antigen gene cluster wbbL-rmlB, extracellular polysaccharide gene cluster galF-wza and type 4 capsular polysaccharide synthesis gene cluster yccC-ymcD;
or the composition gene cluster is: the extracellular polysaccharide gene cluster galF-wza.
2. The medicament of claim 1, wherein the core carbohydrate cluster gmhD-waaQ gene IS waaQ, waaG, waaP, waaS, waaB, waaO, waaR, waaY, waaZ, waaU, waaL, waaC, waaF, gmdD, and the O-antigen cluster wbbL-rmlB gene IS wbbL:: IS5, wbbK, wbbJ, wbbI, wzy, glf, wzx, rmlC, rmlA, rmlD, rmlB, and the extracellular polysaccharide cluster galF-wza gene IS wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, gmg, wcaH, wmaC, manwyJ, wmyJ, wcaC, myc etp, myc, and myc.
3. A drug action target point for reducing the drug resistance of escherichia coli to vancomycin is characterized in that the action target point is a composition gene cluster; the composition gene cluster is as follows: core saccharide gene cluster gmhD-waaQ of lipopolysaccharide, O-antigen gene cluster wbbL-rmlB, extracellular polysaccharide gene cluster galF-wza and type 4 capsular polysaccharide synthesis gene cluster yccC-ymcD; or the composition gene cluster is: the extracellular polysaccharide gene cluster galF-wza.
4. The application of a deletion composition gene cluster in reducing the drug resistance of escherichia coli to vancomycin is characterized in that the composition gene cluster is as follows: core saccharide gene cluster gmhD-waaQ of lipopolysaccharide, O-antigen gene cluster wbbL-rmlB, extracellular polysaccharide gene cluster galF-wza and type 4 capsular polysaccharide synthesis gene cluster yccC-ymcD; or the composition gene cluster is: the extracellular polysaccharide gene cluster galF-wza.
5. The use according to claim 4, wherein the core carbohydrate gene cluster gmhD-waaQ is waaQ, waaG, waaP, waaS, waaB, waaO, waaR, waaY, waaZ, waaU, waaL, waaC, waaF, gmdD, NCBI accession numbers of the gene sequences are BAE77660.1, BAE77661.1, BAE77662.1, BAE77663.1, BAE77664.1, BAE77665.1, BAE77666.1, BAE77667.1, BAE77668.1, BAE77669.1, BAE77670.1, BAE77671.1, BAE77672.1, BAE 77673.1;
IS5, wbbK, wbbJ, wbbI, wzy, glf, wzx, rmlC, rmlA, rmlD, rmlB, NCBI accession numbers of gene sequences are BAA15873.1, BAA15874.1, BAA15875.1, BAA15876.1, BAA15877.1, BAA15878.1, BAA15879.1, BAA15880.1, BAA15881.1, BAA15882.1, BAA15883.1 and BAA15884.1 in sequence;
the extracellular polysaccharide gene cluster galF-wza is wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, wcaG, wcaH, wcaI, manC, manB, wcaJ, wzx, wcaK, wcaL, wcaM, and galF respectively, NCBI accession numbers of gene sequences are BAE76576.1, BAE76575.1, BAA15913.1, BAA15912.1, BAA15911.1, BAE76574.1, BAE76573.1, BAE76572.1, BAA15910.1, BAA15909.1, BAA15908.1, BAA15907.1, BAA15906.1, BAA15905.1, BAA15901.1, BAA15900.1, BAA15899.1, BAE76571.1, BAA15898.1, BAA15897.1 and BAA15896.1 respectively;
the type 4 capsular polysaccharide synthetic gene cluster yccC-ymcD is yccC, etp, yccZ, ymcA, ymcB, ymcC and ymcD respectively, and NCBI accession numbers of gene sequences are BAA35746.1, BAA35747.2, BAA35748.1, BAA35749.1, BAA35750.2, BAA35751.1 and BAA35752.2 in sequence.
6. The use according to claim 5, wherein the E.coli is E.coli K-12W 3110.
7. The use of claim 6, wherein the deletion is knock-out or non-expression.
8. The application of the composition gene cluster as a drug action target point in preparing drugs for reducing the antibiotic resistance of escherichia coli is characterized in that the composition gene cluster is as follows: core saccharide gene cluster gmhD-waaQ of lipopolysaccharide, O-antigen gene cluster wbbL-rmlB, extracellular polysaccharide gene cluster galF-wza and type 4 capsular polysaccharide synthesis gene cluster yccC-ymcD; or the composition gene cluster is: the extracellular polysaccharide gene cluster galF-wza.
9. The use of claim 8, wherein the medicament is capable of inhibiting or down-regulating the expression of a gene cluster of the composition.
10. Method for improving preparation of acetyl coenzyme A and cofactor NADH/NAD by escherichia coli+And NADPH/NADP+Method for the production of a combination of genes or exopolysaccharide gene cluster galF-wza in the E.coli genome, wherein the combination of genes is: the composition gene cluster is as follows: fat and oilThe core saccharide gene cluster gmhD-waaQ of the polysaccharide, the O-antigen gene cluster wbbL-rmlB, the extracellular polysaccharide gene cluster galF-wza and the type 4 capsular polysaccharide synthesis gene cluster yccC-ymcD.
CN202110469210.5A 2021-04-28 2021-04-28 Method for reducing drug resistance of vancomycin and application of method in cofactor production Active CN113106050B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110469210.5A CN113106050B (en) 2021-04-28 2021-04-28 Method for reducing drug resistance of vancomycin and application of method in cofactor production

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110469210.5A CN113106050B (en) 2021-04-28 2021-04-28 Method for reducing drug resistance of vancomycin and application of method in cofactor production

Publications (2)

Publication Number Publication Date
CN113106050A true CN113106050A (en) 2021-07-13
CN113106050B CN113106050B (en) 2023-09-08

Family

ID=76720290

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110469210.5A Active CN113106050B (en) 2021-04-28 2021-04-28 Method for reducing drug resistance of vancomycin and application of method in cofactor production

Country Status (1)

Country Link
CN (1) CN113106050B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112575041A (en) * 2019-09-30 2021-03-30 江南大学 Engineering bacterium for producing PHB (polyhydroxybutyrate) by high-efficiency fermentation of mixed carbon source and application of engineering bacterium

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110387347A (en) * 2019-07-31 2019-10-29 江南大学 One plant of Escherichia coli membranous wall simplifies the application of chassis bacterial strain and its high yield PHB
CN110387346A (en) * 2019-07-31 2019-10-29 江南大学 Lack the genetic engineering bacterium and its application of 21 coding transposon mutagenesis genes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110387347A (en) * 2019-07-31 2019-10-29 江南大学 One plant of Escherichia coli membranous wall simplifies the application of chassis bacterial strain and its high yield PHB
CN110387346A (en) * 2019-07-31 2019-10-29 江南大学 Lack the genetic engineering bacterium and its application of 21 coding transposon mutagenesis genes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
王建莉: "革兰氏阴性细菌外膜关键分子对细胞全局调控及聚羟基脂肪酸酯合成效率的影响机制", 《中国优秀博硕士学位论文全文数据库(博士)基础科学辑》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112575041A (en) * 2019-09-30 2021-03-30 江南大学 Engineering bacterium for producing PHB (polyhydroxybutyrate) by high-efficiency fermentation of mixed carbon source and application of engineering bacterium
CN112575041B (en) * 2019-09-30 2022-12-13 江南大学 Engineering bacterium for producing PHB (polyhydroxybutyrate) by high-efficiency fermentation of mixed carbon source and application of engineering bacterium

Also Published As

Publication number Publication date
CN113106050B (en) 2023-09-08

Similar Documents

Publication Publication Date Title
Jing et al. Newly isolated Streptomyces sp. JBS5-6 as a potential biocontrol agent to control banana Fusarium wilt: genome sequencing and secondary metabolite cluster profiles
KR20130021764A (en) A intestinal microbial enzyme mixture and it's preparation thereof
Günther et al. The transcriptomic profile of Pseudozyma aphidis during production of mannosylerythritol lipids
Huang et al. Mitochondrial complex I bridges a connection between regulation of carbon flexibility and gastrointestinal commensalism in the human fungal pathogen Candida albicans
Kozuma et al. Identification and biological activity of ogipeptins, novel LPS inhibitors produced by marine bacterium
Sabag-Daigle et al. A metabolic intermediate of the fructose-asparagine utilization pathway inhibits growth of a Salmonella fraB mutant
Wu et al. A novel strategy to regulate 1-deoxynojirimycin production based on its biosynthetic pathway in Streptomyces lavendulae
Hu et al. Deletion of Atg22 gene contributes to reduce programmed cell death induced by acetic acid stress in Saccharomyces cerevisiae
Jones-Dias et al. Quantitative proteome analysis of an antibiotic resistant Escherichia coli exposed to tetracycline reveals multiple affected metabolic and peptidoglycan processes
Wu et al. A new perspective on the antimicrobial mechanism of berberine hydrochloride against Staphylococcus aureus revealed by untargeted metabolomic studies
CN113106050A (en) Method for reducing vancomycin resistance and application of method in cofactor production
Tragni et al. Targeting mitochondrial metabolite transporters in Penicillium expansum for reducing patulin production
Jin et al. Non-targeted metabolomic profiling of filamentous cyanobacteria Aphanizomenon flos-aquae exposed to a concentrated culture filtrate of Microcystis aeruginosa
Liu et al. Transmission and stable inheritance of carbapenemase gene (blaKPC-2 or blaNDM-1)-encoding and mcr-1-encoding plasmids in clinical Enterobacteriaceae strains
Bastos et al. Secondary metabolites produced during Aspergillus fumigatus and Pseudomonas aeruginosa biofilm formation
Su et al. The transcriptome analysis on urea response mechanism in the process of ergosterol synthesis by Cordyceps cicadae
Yao et al. Impact of Weissella cibaria BYL4. 2 and its supernatants on Penicillium chrysogenum metabolism
Weng et al. Comparative proteome analysis of Actinoplanes utahensis grown on various saccharides based on 2D-DIGE and MALDI-TOF/TOF-MS
Sun et al. Identification of a Novel Coumarins Biosynthetic Pathway in the Endophytic Fungus Fusarium oxysporum GU-7 with Antioxidant Activity
Smajdor et al. Fast and reliable voltammetric determination of menaquinone (vitamin K2) produced in vitro by Bacillus subtilis cultures
US20240082165A1 (en) Methods and compositions for precision release of probiotics to improve human health
Perlatti et al. Identification of the antifungal metabolite chaetoglobosin P from Discosia rubi using a Cryptococcus neoformans inhibition assay: Insights into mode of action and biosynthesis
Sun et al. Antimicrobial activity and mechanism of Magnolia officinalis root extract against methicillin-resistant Staphylococcus aureus based on mannose transporter
Minaeva et al. Responses triggered in chloroplast of Chlorella variabilis NC64A by long-term association with Paramecium bursaria
CN113174393B (en) Method for improving drug resistance of escherichia coli and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information

Inventor after: Wang Jianli

Inventor after: Wang Xiaoyuan

Inventor after: Ma Wenjian

Inventor after: Chen Shanshan

Inventor before: Wang Xiaoyuan

Inventor before: Wang Jianli

Inventor before: Ma Wenjian

Inventor before: Chen Shanshan

CB03 Change of inventor or designer information
GR01 Patent grant
GR01 Patent grant