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Aureobasidium pullulans alb1 gene knockout mutant strain and its application

A technique of Aureobasidium pullulans and gene knockout, applied in the field of genetic engineering, can solve the problems that the synthetic pathway of Aureobasidium pullulans melanin and related genes have not yet been reported, and achieve the effect of improving the purification yield and product quality

Active Publication Date: 2019-03-26
SHANDONG ACADEMY OF PHARMACEUTICAL SCIENCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aureobasidium pullulans melanin belongs to dihydroxynaphthalene (DHN) melanin, but the synthesis pathway and related genes of Aureobasidium pullulans melanin in vivo have not been reported yet

Method used

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  • Aureobasidium pullulans alb1 gene knockout mutant strain and its application
  • Aureobasidium pullulans alb1 gene knockout mutant strain and its application

Examples

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Effect test

Embodiment 1

[0037] Example 1: Construction of Aureobasidium pullulans alb1 gene knockout mutant

[0038] (1) Synthesis of gene knockout fragments

[0039] A recombinant fragment containing alb1 left homology arm-hygromycin B resistance gene cassette-alb1 right homology arm was synthesized by whole gene synthesis method. The nucleotide sequence is shown in SEQ ID NO.4, and the recombinant fragment was connected to pUC18 The EcoRI site on the plasmid was used to construct the alb1 gene knockout plasmid.

[0040] The above recombinant fragments were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0041] (2) Preparation of Aureobasidium pullulans As3.3984 electroporation competent

[0042] Pick a single colony of As3.3984 (purchased from the General Microbiology Center of China Microbiological Culture Collection Management Committee, the strain number is 3.3984) and inoculate it into 3mL YPD liquid medium, and culture it with shaking at 28°C for 48 hours; transfer 100μL culture to...

Embodiment 2

[0056] Embodiment 2: microbial polysaccharide fermentation and purification experiment

[0057] 1. Fermentation and purification of microbial polysaccharides:

[0058] (1) Strain fermentation: Aureobasidium pullulans originator As3.3984 and Aureobasidium pullulans mutant strain As3.3984Δalb1 obtained in Example 1 were respectively inoculated in the polysaccharide fermentation medium, and shake cultured at 28° C. for 72 hours.

[0059] Components of the above polysaccharide fermentation medium: sucrose 100.0g / L, yeast extract 2.0g / L, NaCl 1.0g / L, MgSO 4 .7H 2 O0.2g / L, K 2 HPO 4 5.0g / L, (NH 4 ) 2 SO 4 0.6g / L, pH6.5, sterilized at 115°C for 30 minutes.

[0060] (2) Precipitate crude polysaccharide:

[0061] The fermentation broth was centrifuged at 8000rpm for 10 minutes to collect the supernatant; 3 times the volume of industrial ethanol was added to the supernatant to precipitate the polysaccharide, the precipitate was collected by centrifugation at 3000rpm for 5 minu...

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Abstract

The invention discloses aureobasidium pullulan alb1 gene knockout mutant strain. According to the mutant strain, all or part of coding genes in alb1 gene in aureobasidium pullulan strain are knocked out and replaced by a hygromycin B resistance gene cassette. The hygromycin B resistance gene cassette comprises aureobasidium pullulan transcription elongation factor promoter PTEF and hygromycin B resistance gene HrgR; the sequence of the aureobasidium pullulan transcription elongation factor promoter PTEF is as shown in SEQ ID NO.2 in a sequence table; the sequence of the hygromycin B resistance gene HrgR is as shown in SEQ ID NO.3 in the sequence table. The mutant strain is used for the fermentation production of microbial polysaccharide, does not produce melanin, and can obviously the yield and quality of polysaccharide during the purification process.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a mutant strain of Aureobasidium pullulans alb1 gene knockout and application thereof. Background technique [0002] Aureobacidium pullulans is a kind of yeast-like fungus, which has two forms of yeast-like and fungal mycelium. Because it produces a large amount of melanin, it is also called black yeast. Fermentation of Aureobasidium pullulans can produce various products such as exopolysaccharides, enzymes, antibiotics, and single-cell proteins, especially the produced pullulan has excellent film-forming properties, oxygen barrier properties, plasticity, stability, and viscosity. It has many unique physical and biological characteristics such as solidity and easy natural degradation. It is non-toxic and harmless, and has no side effects on the human body. It can be widely used in many fields such as medicine, food, cosmetics, tobacco, oil extraction, etc. It is an ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C12P19/04C12R1/645
CPCC07K14/37C12P19/04
Inventor 张金华刘飞朱希强张林军郝荣华袁超张祥奎侯重文张秀华凌沛学
Owner SHANDONG ACADEMY OF PHARMACEUTICAL SCIENCES
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