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UDPG pyrophosphatase produced from aureobasidium pullulans, coding gene, carrier and application

A technology of pyrophosphatase encoding and Aureobasidium pullulans, which is applied in the fields of application, genetic engineering, and plant genetic improvement, and can solve the problems of no research results being retrieved

Inactive Publication Date: 2015-11-11
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above pathways are only hypothetical inferences, so far no research results on the synthesis pathway of pullulan at the molecular level have been retrieved

Method used

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  • UDPG pyrophosphatase produced from aureobasidium pullulans, coding gene, carrier and application
  • UDPG pyrophosphatase produced from aureobasidium pullulans, coding gene, carrier and application
  • UDPG pyrophosphatase produced from aureobasidium pullulans, coding gene, carrier and application

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Experimental program
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Embodiment 1

[0031] Cloning of embodiment 1, UDPG pyrophosphatase gene

[0032] (1) UDPG pyrophosphatase gene core fragment

[0033]Bioinformatics analysis of fungal UDPG pyrophosphatase gene sequences reported in NCBI (TrichodermareeseiQM6a, XM_006960998.1; AspergillusnigerCBS513.88, XM_001395134.2; Fusarium graminearumPH-1, XM_011317898.1; Emericellanidulans, AY850189.inado609.1; 1) After comparing the amino acid sequences using DNAMAN software, find out the conserved region and design degenerate primers. The upstream primer is: Ap190F: 5'-CCNTTYGTNGCNGAYGC-3' (SEQ ID NO.5); the downstream primer is: AP440R: 5'-NCCNGCNCCNCCRTGRTG-3' (SEQ ID NO. 6). Then take Aureobasidium pullulans NRRL12974 (source of open literature: JournalofIndustrialMicrobiology&Biotechnology.2011Sep; 38(9):1211-8.doi:10.1007 / s10295-010-0899-y.) genome as template, carry out PCR amplification, amplification condition It is: pre-denaturation at 94°C for 5 minutes; 32 cycles of denaturation at 94°C for 30 sec, annea...

Embodiment 2

[0039] Embodiment 2 constructs the Escherichia coli recombinant expression vector of UDPG pyrophosphatase

[0040] (1) Extract the total RNA of Aureobasidium pullulans NRRL12974 with the Yeast Total RNA Extraction Kit (purchased from BIOSCI Company, article number RT001), and then synthesize cDNA with the reverse transcriptase kit (purchased from BIOSCI Company, article number RT002) The first strand is used as a template for PCR amplification,

[0041] Upstream primer: 5'-ATGTCCTCCGAGATGGCAA-3' (SEQ ID NO.9);

[0042] Downstream primer: 5'-CTAGTGCTCGAGAAGTC-3' (SEQ ID NO.10).

[0043] PCR amplification conditions were: 94°C pre-denaturation for 5 min; 94°C denaturation for 50 s, 59°C annealing for 50 s, 68°C extension for 2 min, 32 cycles; 68°C extension for 5 min; 25°C for 10 min. The amplified product was connected to PMD19-TVector, transformed into E.coliJM109 competent cells, screened to obtain positive recombinants, and then sequenced to obtain a 1436bp sequence, as sh...

Embodiment 3

[0048] Expression and purification of embodiment 3UDPG pyrophosphatase

[0049] Transfer the recombinant expression vector PET28a-UGPPase constructed in Example 2 into E.coliBL21(DE3), culture the recombinant bacteria at 37°C and 180rpm until the OD600nm is about 0.6, add 0.1.mmol / L IPTG to induce After continuing to cultivate for 6 hours, collect the cells, resuspend the cells in 20mM Tris-HCl (pH7.0) buffer, lyse the cells by ultrasonication, and centrifuge to obtain the cell supernatant; pass the cell supernatant through Ni-NTA affinity chromatography (the affinity chromatography column is Ni-NTAAgarose from Qiagen Company, catnumber30210), and eluted with 150 mM imidazole aqueous solution to obtain purified protein. Then use SDS-PAGE to detect the protein, the results are as follows figure 1 shown. Depend on figure 1 It can be seen that a single band appears at about 58kDa on lane 6, which is in line with the predicted molecular weight of UDPG pyrophosphatase at 57.5K...

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Abstract

The invention discloses UDPG pyrophosphatase produced from aureobasidium pullulans, a coding gene, a carrier and application to improvement of the yield of exopolysaccharides of the aureobasidium pullulans. An amino acid sequence of the UDPG pyrophosphatase is as shown in SEQ ID NO.1. The gene engineering technology is utilized for over-expression of the UDPG pyrophosphatase in an original fungus of the aureobasidium pullulans, the yield of pulullan in recombined aureobasidium pullulans can be improved by 32%, the yield of beta-1,3 glucan in the recombined aureobasidium pullulans can be improved by 55%. The UDPG pyrophosphatase is proofed to participate in the synthesis of two exopolysaccharides from the molecular aspect, so as to provide a technological base and methodological guidance to modify a synthetic pathway of the exopolysaccharides of the aureobasidium pullulans according to the metabolic engineering technology.

Description

(1) Technical field [0001] The invention relates to an Aureobasidium pullulans UDPG pyrophosphatase, a coded gene, a carrier and an application in increasing the output of Aureobasidium pullulans exopolysaccharide. (2) Background technology [0002] Aureobasidium pullulans is an important industrial microorganism capable of producing a variety of exopolysaccharides. Includes polymalic acid, beta-1,3 glucan and pullulan. [0003] Pullulan has good biocompatibility and degradability, and can be widely used in the fields of biomaterials, food packaging, pharmaceutical manufacturing, cosmetics and agricultural environmental protection, and has broad application prospects. [0004] Polymalic acid is a class of polymers with biodegradability, bioabsorbability and compatibility, and can be widely used as biomedical materials in the fields of drug delivery systems and tissue engineering. [0005] β-1,3 glucan is a biological polysaccharide with anti-tumor activity. It can inhibit ...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N15/80C12P19/10C12P19/08C12R1/645
CPCC12N9/14C12P19/08C12P19/10C12Y306/01045
Inventor 李海峰黄黎锋蓝袁洋高允允
Owner HANGZHOU NORMAL UNIVERSITY
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