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Method for inhibiting Pichia kudriavzevii strain cell aggregation, construction and application

A yeast strain and a cell-inhibiting technology, which is applied in the field of yeast, can solve problems affecting the production performance of strains, unfavorable oxygen and nutrient transfer, and achieve benefits for the transfer of nutrients and oxygen, increase biomass, and alleviate cell aggregation effects Effect

Active Publication Date: 2020-09-08
WENZHOU UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Pichia kudriavzevii grows in the form of pseudohyphae and aggregates with each other under acidic conditions with pH less than 2.5 (such as figure 1 shown), this phenomenon is not conducive to the transfer of oxygen and nutrients, which seriously affects the production performance of the strain during low pH fermentation

Method used

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  • Method for inhibiting Pichia kudriavzevii strain cell aggregation, construction and application
  • Method for inhibiting Pichia kudriavzevii strain cell aggregation, construction and application
  • Method for inhibiting Pichia kudriavzevii strain cell aggregation, construction and application

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Embodiment 1

[0020] Example 1: A method for inhibiting cell aggregation of Pichia kurdori strains, preparing 200mM arginine mother liquor, filtering and sterilizing for later use; the Pichia kurdori growth YPD medium includes the following mass concentration components: glucose 20g / L, peptone 20g / L and yeast extract 10g / L; then use HCl or NaOH to adjust the pH of the YPD medium, and add 5mmol / L arginine to it after autoclaving, such as figure 2 As shown, arginine was exogenously added to YPD medium under different pH conditions. The results showed that the addition of 5mmol / L arginine could significantly promote the growth of Pichia pichia under low pH pressure. At the same time, the pHi was significantly increased in the presence of arginine, indicating that arginine played a positive role in protecting Pichia kudori against low pH stress, and the cell aggregation effect was significantly reduced when arginine was added.

Embodiment 2

[0021] Embodiment 2:: the construction method of the recombinant Pichia kudriavzevii that inhibits cell aggregation, in the Pichia kudriavzevii N-X in the Pichia kudriavzevii strain, obtain the recombinant strain that overexpresses the ARG J gene; Wherein, Pichiakudriavzevii N-X is preserved in China Type Culture Collection Center (CCTCC); address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, preservation number: CCTCC M 2017759, gene accession number MT499446 of the ARG J gene; specifically includes the following steps:

[0022] a, using primers Pdc1F, Pdc1R and Pichia kurdori N-X genomic DNA as template amplification to obtain a fragment containing the PDC1 gene promoter, reading frame and terminator, and connecting it into the pMD19T vector; the primer Pdc1F is CGCGGATCCGCGTTTAAGTGGTGGTTGTA, the Primer Pdc1R is CGG GGTACCTGGT GAGATTGATGGATTGT;

[0023] b. Reverse amplification using primers 0-F and 0-R; the primer 0-F is ACAGATAAGTACCTAGGGAGATT,...

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Abstract

The invention discloses a method for inhibiting Pichia kudriavzevii strain cell aggregation, construction and application. By adding arginine, the growth of Pichia kudriavzevii under an extremely acidic condition can be remarkably promoted, the cell aggregation is relieved, the transfer of oxygen and nutrient substances is facilitated, and the production performance of the strain during low-pH fermentation is improved. The invention further constructs Pichia kudriavzevii for inhibiting cell aggregation. The Pichia kudriavzevii is a recombinant bacterium with overexpression of ARGJ, the intracellular arginine content is increased by 26.3%, the biomass is remarkably increased under the condition that the pH value is 2.0, the cell aggregation effect is obviously relieved, and the transfer ofnutrient substances and oxygen is facilitated.

Description

technical field [0001] The invention relates to the field of yeast technology, in particular to a method, construction and application for inhibiting cell aggregation of Pichia kudori strains. Background technique [0002] When organic acids are produced by fermentation, with the accumulation of products, organic acids enter microbial cells by simple diffusion and dissociate into charged acid radical ions and protons, resulting in a decrease in intracellular pH (pHi), affecting the conformation and The progress of the intracellular enzymatic reaction will also interfere with the active transmembrane transport driven by protons, affect the absorption of nutrients by microbial cells, and lead to a sharp decline in fermentation performance. The development of low-pH fermentation technology that relies on acid-tolerant hosts can significantly reduce or even eliminate the use of neutralizers, simplify the production process, reduce the risk of bacterial contamination, and save en...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12N1/38C12N1/19C12R1/84
CPCC07K14/39C12N1/16C12N1/38
Inventor 嵇豪金立波孙达彭仁义林苏娥董夏梦
Owner WENZHOU UNIVERSITY
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