Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A method for regulating acid stress resistance of T. glabrata using the transcription factor crz1p

A technology of acid stress of Toroplasma glabrata, which is applied in the field of regulating acid stress resistance of Toroplasma glabrata, and can solve problems that have not been widely carried out

Active Publication Date: 2018-01-16
JIANGNAN UNIV
View PDF0 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the research on acid stress of P. glabrata has not been widely carried out. Proteomic analysis found that P. glabrata has a stronger tolerance to low pH environment than high pH environment.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for regulating acid stress resistance of T. glabrata using the transcription factor crz1p
  • A method for regulating acid stress resistance of T. glabrata using the transcription factor crz1p
  • A method for regulating acid stress resistance of T. glabrata using the transcription factor crz1p

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction of Deletion Mutants

[0026] With the C.glabrataATCC 2001 (wt) genome as a template, P1 / P2 (sequences are shown in SEQ ID NO:1, SEQ ID NO:2), P3 / P4 (sequences are respectively shown in SEQ ID NO:3, SEQ ID NO: 4), P5 / P6 (sequence shown in SEQ ID NO: 5, SEQ ID NO: 6) are primers, amplify the left arm (L) of the gene to be knocked out, the histidine gene ( M) and the right arm (R), the knockout box CgCRZ1-LMR was constructed by fusion PCR ( figure 1 ). The starting strain C.glabrataATCC 55 cannot grow on MM screening medium due to the deletion of histidine gene, while the mutant strain can grow on MM medium due to the expression of marker gene histidine after homologous recombination. Perform PCR verification on transformants, such as figure 1 As shown, the genome of the transformant was extracted, and it was found that when using P7 / P8 (sequences shown in SEQ ID NO:7 and SEQ ID NO:8) as primers, the wild-type strain wt produced a gene fragment o...

Embodiment 2

[0035] Embodiment 2: Determination of growth performance of deletion mutant bacteria

[0036] Functional characterization of CgCRZ1 by plate growth assays and cell growth assays:

[0037] 1) Plate growth experiment:

[0038] Inoculate a single colony of the strain to be tested in 20 mL of YNB (0.67% Yeast Nitrogen Base without Amino Acids, 2% Glucose, pH 5.7) liquid medium, culture on a shaker at 30°C for 12 hours to the logarithmic growth phase, measure the concentration of the bacteria and Adjust the bacterial suspension to OD 600 = 1.0, using this as the initial concentration, carry out 5 times of 10-fold gradient dilution, inoculate 4 μ L of the bacterial liquid on the solid medium to be tested in turn, cultivate at 30°C for 2 days, observe the growth of the bacterial cells and take pictures.

[0039] 2) Cell growth assay:

[0040] Inoculate the strain to be tested in the logarithmic phase into 20 mL of YNB (0.67% Yeast Nitrogen Base without Amino Acids, 2% Glucose, pH ...

Embodiment 3

[0042] Example 3: Determination of cell membrane properties of deletion mutant bacteria

[0043] The cell membrane properties of Toruula glabrata were evaluated by measuring cell membrane fatty acid and cell membrane permeability.

[0044] (1) Cell membrane fatty acid determination

[0045] Cell culture: Yeast cells treated with YNB-5.7 and YNB-2.3 for 12 hours were collected, washed three times with PBS buffer, and then freeze-dried before use. Lipid extraction and treatment: Weigh 30 mg of freeze-dried cells, place in a test tube, add 1 ml of solution I, and conduct a water bath at 100°C for 30 minutes. Use an ice bath to rapidly cool down, then add 2mL of solution II, mix well and treat in a water bath at 80°C for 5min. Cool rapidly, and add 1.25 mL of solution III. Shake well for 5 minutes, then keep the upper layer solution. Then add 3ml of solution IV and a few drops of saturated sodium chloride solution to the upper layer solution, and shake for 5 minutes. Stand st...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for using the transcription factor Crz1p to regulate acid stress resistance of Toruula glabrata, belonging to the field of bioengineering. In the present invention, by deleting or overexpressing the Cgcrz1 gene of Torulopsis glabrata, the acid stress resistance of the strain is correspondingly reduced or improved. The present invention also compares the cell membrane fatty acid, sterol composition, ratio and permeability of the deletion mutant strain Cgcrz1Δ under acid stress. Acid stress resistance.

Description

technical field [0001] The invention relates to a method for using transcription factor Crz1p to regulate the acid stress resistance of Toruula glabrata, belonging to the field of bioengineering. Background technique [0002] Candida glabrata is the only microorganism that produces pyruvate industrially. In addition, C.glabrata can also be used for industrial production of fumaric acid, malic acid, α-ketoglutaric acid and other organic acids. During the organic acid fermentation process, with the accumulation of products, the pH of the medium decreased rapidly, resulting in slowing down or even stopping the growth of bacteria and the accumulation of products. In recent years, strategies such as adding exogenous auxiliary substrates, mutation breeding, genetic engineering and adaptive evolution have been used to improve the acid tolerance and organic acid production of bacteria at home and abroad. Research on the acid resistance mechanism of C.glabrata can fundamentally sol...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N1/19C12P7/64C12P7/40C12P7/46C12P7/50C12R1/72
Inventor 刘立明闫冬妮刘晖
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com