Method for improving acid stress resistance of candida glabrata

A technology of Toroplasma glabrata and acid stress, which is applied in the field of improving the acid stress resistance of Toroplasma glabrata, and can solve problems such as unclear influence of strains

Active Publication Date: 2015-07-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its effect on the AMP pathway of the strain is unclear

Method used

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  • Method for improving acid stress resistance of candida glabrata
  • Method for improving acid stress resistance of candida glabrata
  • Method for improving acid stress resistance of candida glabrata

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of gene knockout bacteria

[0043] Using the C.glabrataATCC 2001 (wt) genome purchased from the American Type Culture Collection (ATCC) as a template, the gene to be knocked out, CgADE12( The amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2). The left arm (L), histidine gene (M) and right arm (R) were constructed by fusion PCR Deboxing CgADE12-LMR. The starting strain C.glabrataATCC 55 cannot be used in MM screening medium (glucose 20g·L) due to the deletion of histidine gene -1 , urea 7g·L -1 , sodium acetate 3g·L -1 , sodium dihydrogen phosphate 5g·L -1 , magnesium sulfate heptahydrate 0.8g·L -1 , agar powder 20g·L -1 ), and the mutant strains grew on MM medium due to the expression of the 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 P7 / P8 ...

Embodiment 2

[0047] Example 2: Changes in intracellular ATP before and after mutation

[0048] In the YNB medium, the adenylate content in Cgade12Δ cells was determined by HPLC, and the results ( figure 2 ) shows that the ATP level in Cgade12Δ is 72.21, which is 7.69% lower than wt. The intracellular ATP in Cgade12Δ was measured under acetic acid stress, and it was found that the ATP levels of wt and Cgade12Δ were 40.25 and 37.61, which were 48.72% and 48.61% lower than those in YNB medium, respectively. The ATP level of Cgade12Δ was 7.5% lower than wt. The deletion of CgADE12 significantly reduced the ATP level in the strain, and under acetic acid stress, the deletion of CgADE12 increased the difference in ATP level between the strain and wt.

Embodiment 3

[0049] Embodiment 3: Comparison of acid stress resistance before and after bacterial strain mutation

[0050] (1) Acetic acid stress

[0051] The growth of wt and Cgade12Δ were compared under different acetic acid stress. The results showed that under 0.2% acetic acid stress, the OD value of Cgade12Δ was 22.93% higher than that of wt (eg image 3 shown). It shows that the deletion of CgADE12 improves the growth performance of the strain under acetic acid stress.

[0052] At the same time, the cell viability of strains under 0.5% acetic acid stress was compared, and the results showed that Cgade12Δ was increased by 30.27% (8h) and 142.26% (20h) compared with wt. It shows that the cell viability of Cgade12Δ is higher than that of wt under the stress of high concentration of acetic acid.

[0053] The above data indicate that CgADE12 plays a significant role in the maintenance of cell viability.

[0054] (2) Pyruvate stress

[0055] The growth of wt and Cgade12Δ was compared...

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Abstract

The invention discloses a method for improving the acid stress resistance of candida glabrata, and belongs to the field of bioengineering. The method comprises the following steps: improving the candida glabrata, constructing a gene-deleted mutant strain Cgade12Delta, measuring the intracellular ATP level of the constructed strain, and determining the organic acid tolerance of the strain by a plate growth experiment and cellular activity analysis. After the deletion of the gene CgADE12 is found out, the intracellular ATP level of the strain under the stress of 0.2 percent acetic acid is lowered, the growth capability under the stress of acetic acid is improved, and the acetic acid tolerance is improved.

Description

technical field [0001] The invention relates to a method for improving the acid stress resistance of Toruula glabrata, belonging to the field of bioengineering. Background technique [0002] Candida glabrata is the main strain for microbial fermentation to produce pyruvate. In addition, Torulopsis glabrata also has great potential in the production of organic acids such as malic acid, fumaric acid and α-ketoglutaric acid. During the fermentation process, with the accumulation of organic acids, the extracellular pH decreased continuously, and the degree of dissociation of organic acids also continued to decrease, so that they entered the cells in an undissociated state. The higher pH in the cytoplasm dissociates organic acids, releases protons and accumulates, eventually leading to acidification of the cytoplasm and affecting the metabolism and growth of cell metabolism, thereby affecting the fermentation process. In industrial production, usually add a large amount of NaOH...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/65C12N15/52C12N1/19C12P7/40C12R1/72
Inventor 刘立明靳丛丛
Owner JIANGNAN UNIV
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