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A Method of Improving Acid Tolerance Ability of Togumin glabrata

A technology of Toroplasma glabrata and amino acids, applied in the field of bioengineering, can solve problems such as heavy workload, increased complexity of product purification, unclear mechanism, etc., and achieve the effect of improving fermentation capacity, cell membrane integrity and fluidity

Active Publication Date: 2020-04-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these strategies have problems such as increasing the complexity of product purification, heavy workload and long cycle.
The study of the mechanism of acid resistance through the regulation of mediators is carried out at the level of gene transcription, which can fundamentally guide and solve this problem, but the mechanism is still unclear

Method used

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  • A Method of Improving Acid Tolerance Ability of Togumin glabrata
  • A Method of Improving Acid Tolerance Ability of Togumin glabrata
  • A Method of Improving Acid Tolerance Ability of Togumin glabrata

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: Construction of overexpression strain

[0034] Using the C.glabrataATCC 2001 (hereinafter abbreviated as wt) genome as a template and P1 / P2 as primers, the amplified fragment CgMED15B ( figure 1 A, 3.3kb). After double enzyme digestion, it was connected to the plasmid pY26 to construct the recombinant plasmid pY26-CgMED15B, and the two fragments of pY26 plasmid (7.2kb) and CgMED15B (3.3kb) were verified by double enzyme digestion ( figure 1 B). Using C.glabrata ATCC 55 (hereinafter abbreviated as HTUΔ) as the starting strain, the recombinant plasmid pY26-CgMED15B was introduced into its competent cells, and MM screening medium was applied to obtain positive transformants. The starting strain cannot grow on MM selection medium due to the deletion of URA3 gene, while the URA3 gene on plasmid pY26 can make the overexpression strain obtain this growth ability. Using P3 / P4 as primers, through colony PCR screening verification, it was found that the starting st...

Embodiment 2

[0039] Example 2: Construction of knockout strains

[0040] Using the wt genome as a template and using P5 / P6, P7 / P8, and P9 / P10 as primers, respectively, the left arm (indicated by L), the right arm (indicated by R) and the HIS3 gene (indicated by M) of CgMED15B were amplified. ), the knockout box CgMED15B-LMR was constructed by fusion PCR ( figure 2 A). The knockout frame was introduced into the starting strain HTUΔ, and a mutant strain in which the marker gene HIS3 was replaced by the target gene CgMED15B was obtained through homologous recombination. The starting strain HTUΔ cannot grow on the screening medium due to the deletion of the HIS3 gene, while the mutant strain can grow on the MM medium due to the expression of the marker gene HIS3. Using P11 / P12 as primers, carry out colony and genome PCR verification on transformants, such as figure 1 As shown in B, it was found that the starting strain HTUΔ produced a gene fragment of about 4.0 kb, and the transformant obt...

Embodiment 3

[0049] Embodiment 3: the growth performance determination of mutant bacteria

[0050] The function of Med15B to improve the acid tolerance of C.glabrata was evaluated by plate growth record, growth curve and growth activity assay:

[0051] Plate growth experiments were used to analyze the effects of different pH conditions on the acid tolerance of the starting strain HTUΔ, the overexpression strain HTUΔ / CgMED15B and the knockout strain med15BΔ, such as image 3 A, found that with pH 6.0 as the control, the growth of C.glabrata will be inhibited when the pH value rises from 6.0 to 8.0, but the growth of the three strains under the same pH conditions is roughly the same; but as the pH decreases from 6.0 to 2.0, Overexpression of Med15B can slightly promote the growth of the strain, and deletion of Med15B can significantly inhibit the growth of the strain. Further measure the growth curve of the thalline under the condition of pH 2.0, such as image 3 B, It was found that the f...

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Abstract

The invention discloses a method for improving acid tolerance of torulopsis glabrata, and belongs to the field of biological engineering. The final biomass of the torulopsis glabrata is increased by 59.2% under the condition of pH2.0 by Med15B overexpression, under the condition of pH2.0, the 12h cell product of the torulopsis glabrata is increased by 73.8% compared with that of original strain, the content of cryptosterol, the content of coprosterol and the content of ergosterol of cell membranes are respectively increased by 970%, 16.6% and 94.5%, the integrity and the liquidity of the cell membrane are enhanced, low pH cell tolerance is increased, growth ability is enhanced, and pyruvic acid fermentation ability is increased by 100% compared with that of the original strain.

Description

technical field [0001] The invention relates to a method for improving the acid tolerance of Togus glabrata, belonging to the field of bioengineering. Background technique [0002] The Mediator Complex (Mediator Complex) is a multi-subunit protein complex that serves as a bridge for information transmission between transcription factors and RNA polymerase II. Different signal transduction pathways in cells stimulate the specific transcription factors controlled by them to interact with specific subunits of mediators, regulate the expression of downstream genes, and then affect cell growth and various physiological functions. [0003] Candida glabrata can be used to produce various organic acids such as pyruvate, fumarate and malate. During organic acid fermentation, C.glabrata will face low pH stress. The low pH environment acidifies the intracellular environment of microorganisms, resulting in impaired bacterial growth and slowing down or even stopping the accumulation of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/40C12R1/72
CPCC07K14/40C12N15/815C12P7/40
Inventor 刘立明齐艳利刘晖
Owner JIANGNAN UNIV
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