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Recombination gene and method for increasing aspergillus niger expressed saccharifying enzyme

A recombinant gene, Aspergillus niger technology, applied in the field of genetic engineering, can solve the problems of unpredictable results, a lot of time and labor costs, time-consuming and laborious Aspergillus screening, etc., and achieves transformation efficiency and expression of saccharification enzyme activity. Eliminate the need for screening and transformation effect of subprocess

Active Publication Date: 2014-07-23
NANJING BESTZYME BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Regardless of the above-mentioned methods, their common feature is that the transformation efficiency is extremely low, and the transformation efficiency per microgram of DNA is only a few to several hundred transformants, which is higher than the transformation efficiency of bacteria (such as E. coli) and yeast (such as Saccharomyces cerevisiae). orders of magnitude lower
In addition, the screening of Aspergillus is time-consuming and laborious. Therefore, like traditional mutagenesis-screening breeding, the random integration DNA breeding process also requires a lot of time and labor costs, and the results are unpredictable.

Method used

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  • Recombination gene and method for increasing aspergillus niger expressed saccharifying enzyme
  • Recombination gene and method for increasing aspergillus niger expressed saccharifying enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Randomly integrated GA expression cassette construction

[0038] In order to compare with the site-specific integration results, a random integration plasmid was constructed, which contains the following parts:

[0039] (1) Fragments obtained after EcoRI-HindIII double digestion of pUC57 plasmid;

[0040] (2) Glucoamylase (TeGA) expression cassette, wherein the glucoamylase is a Talaromyces variant, synthesized by GenScript, and its sequence is shown in SEQ ID NO.1;

[0041] (3) Selection marker Hyg expression cassette, synthesized by GenScript Company, the sequence is shown in SEQ ID NO.2;

[0042] First, use primers GA_F and GA_R, hyg_F0 and hyg_R0 to amplify the GA gene and hyg gene with recombination arms by PCR, and then use PCR to assemble the GA gene and hyg gene into a straight chain through the overlapping sequences at both ends of the primers GA_F and hyg_R0 The fragment was then recombined with the pUC57EcoRI-HindIII digested linear recovery fragm...

Embodiment 2

[0045] Embodiment 2 Transformation and screening of randomly integrated GA

[0046] The preparation of protoplast: cultivate Aspergillus niger mycelia (Aspergillus niger CBS513.88, purchased from the Netherlands Fungal Culture Collection (CBS-KNAW Fungal Biodiversity Centre). The mycelium was filtered from the culture medium through mira-cloth (Calbiochem Company) and washed with 0.7M NaCl (pH 5.8). Sigma) and lysozyme (Sigma) 0.2% enzymatic solution. 30°C, 65rpm enzymatic hydrolysis for 2.5-3h. The enzymatic hydrolysis containing protoplasts was then placed on ice and filtered through four layers of lens tissue. The obtained filtrate was centrifuged at 3000rpm at 4°C for 10min, and the supernatant was discarded; the protoplasts attached to the tube wall were treated with STC solution (1M D-sorbitol, 50mM CaCl 2 , 10mM Tris-HCl, pH7.5) washed once, and finally the protoplasts were resuspended in an appropriate amount of STC solution.

[0047] Protoplast transformation: Ad...

Embodiment 3

[0048] Example 3 Construction of site-specific integration of GA expression cassettes

[0049] Site-specific integration was performed according to the method described by Kim (Kim et al., Biochem. Biophys. Res. Commun390(3):983-988, 2009) et al. (Double-Joint PCR with Split Dominant Selectable Markers). Two exogenous DNA fragments are required for co-transformation into Aspergillus niger:

[0050] Fragment 1: An12g08830 locus 5' flanking sequence (SEQ ID NO.3)+hyg2 (SEQ ID NO.4)

[0051] Fragment 2: An12g08830 locus 3' flanking sequence (SEQ ID NO.5)+TeGA expression cassette (SEQ ID NO.1)+hyg1 (SEQ ID NO.6)

[0052] Among them, hyg1 contains a promoter and a 680bp part of the hyg CDS sequence, and hyg2 contains another part of the hyg CDS sequence (616bp of which is homologous to the hyg part of the CDS sequence in hyg1) and a terminator.

[0053] Construction of Fragment 1: Genomic DNA of Aspergillus niger CBS513.88 (purchased from CBS-KNAW Fungal Biodiversity Centre) was ...

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Abstract

The invention belongs to the field of gene engineering, and discloses a recombination gene and a method for increasing aspergillus niger expressed saccharifying enzyme. The method for increasing aspergillus niger expressed saccharifying enzyme disclosed by the invention is as follows: by means of a homologous recombination method, a saccharifying enzyme expression cassette and selectable marker genes are integrated in an aspergillus niger An12g08830 gene locus in a targeted manner; then, saccharifying enzyme is obtained by recombining aspergillus niger through conventional resistance screening and fermentation cultivating. According to the invention, after the saccharifying enzyme expression cassette is integrated in the aspergillus niger An12g08830 gene locus in a targeted manner through the improved homologous recombination method, the conversion efficiency and the saccharifying enzyme expressing activity are obviously increased; furthermore, a lot of processes for screening converters are omitted.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a recombinant gene and a method for improving the expression of glucoamylase in Aspergillus niger. Background technique [0002] Glucoamylase (1,4-α-D-glucan glucohydrolase, EC3.2.1.3) is an enzyme that catalyzes the release of D-glucose from the non-reducing ends of polysaccharide molecules such as starch or oligosaccharides. Commercially, glucoamylase is produced by several filamentous fungi and yeasts including Aspergillus niger and Aspergillus oryzae. Filamentous fungi are well known as cell factories to produce valuable products (such as enzymes). Among them, Aspergillus niger and Aspergillus oryzae are widely used because of their characteristics as generally recognized as safe bacteria (Generally Recognized As Safe, GRAS). Expression hosts for the production of food additives. [0003] Initially, scientists and commercial companies carried out breeding through traditional...

Claims

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Application Information

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IPC IPC(8): C12N9/34C12N15/52C12N1/15C12R1/685
CPCC12N9/2428C12N15/80C12Y302/01003
Inventor 白挨玺孙健卞芙蓉张垠李峰徐凤徐红潘金龙章方良
Owner NANJING BESTZYME BIO ENG CO LTD
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