Mutant strain producing cellulase, mutant strain highly expressing target protein, construction method and application thereof

A high-efficiency expression and purpose technology, which is applied in the fields of cellulase-producing mutant strains, highly-expressing target protein mutant strains and their construction and application, can solve the problems of time-consuming, difficult, and low transformation efficiency

Active Publication Date: 2015-12-02
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Genetic transformation usually requires genetic transformation, but the transformation efficiency of industrial cellulase fungi is usually low, and it is very time-consuming to mutate two genes at the same time for strains without sexual reproduction (such as Aspergillus niger). More than one gene, extremely difficult

Method used

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  • Mutant strain producing cellulase, mutant strain highly expressing target protein, construction method and application thereof
  • Mutant strain producing cellulase, mutant strain highly expressing target protein, construction method and application thereof
  • Mutant strain producing cellulase, mutant strain highly expressing target protein, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. Construction of double-gene mutant strains BG13 (NCU00130×NCU08755), BG12 (NCU00130×NCU04952), BG23 (NCU04952×NCU08755) and screening of mutant strains for the production of cellulase

[0065] 1. Construction of β-glucosidase double gene mutant strain BG13 (NCU00130×NCU08755)

[0066] The build method includes the following steps:

[0067] 1.1 Hybridization

[0068] 1) β-glucosidase single mutant strain BG1 (NCU00130), FGSC11822 (matching type a) used as the male parent in MM slant medium [50×Vogel's salt 20mL, sucrose 20g, agar 15g, histidine (50mg / mL) 20mL, dilute to 1L, and autoclave. 50 x Vogel’s Salt (1L): Trisodium Citrate (1 / 2H 2 O) 150g, anhydrous KH 2 PO 4 250g, anhydrous NH 4 NO 3 100g, MgSO 4 ·7H 2 O10g, CaCl 2 2H 2 O5g, trace element salt solution 5mL, biotin (0.1mg / mL) 2.5mL, constant volume to 1L. 】Cultivate at 28°C for 7 days before use.

[0069] 2) The β-glucosidase single mutant strain BG3 (NCU08755) and FGSC18388 (matching type...

Embodiment 2

[0109] Example 2. Construction of the β-glucosidase three-gene mutant strain BG123 (NCU00130×NCU04952×NCU08755) for the production of cellulase

[0110] 1.1 Hybridization: BG13 (NCU00130×NCU08755) (matching type a) in Example 1 was used as the female parent, and BG2 (NCU04952), FGSC13732 (matching type A) was used as the male parent for hybridization. For the method, see step 1.1 of Example 1.

[0111] 1.2 Genomic DNA extraction: For the method, see step 1.2 of Example 1.

[0112] 1.3PCR verification and screening of mutants

[0113] The upstream primer hph5f, in order to verify the hygromycin hph gene, the sequence is 5'-TGCAATAGGTCAGGCTCT-3'; the downstream primer NCU00130r, in order to verify that the hygromycin hph gene is at the NCU00130 gene locus, the sequence is 5'-GTAGTGTACAAACCCCAAGC-3'; The downstream primer NCU04952r, in order to verify that the hygromycin hph gene is at the NCU04952 gene locus, the sequence is 5'-AACACACACACACACACTGG-3'; the downstream primer NCU...

Embodiment 3

[0116] Example 3. Functional verification experiments of double-gene mutant strains BG13 (NCU00130×NCU08755), BG12 (NCU00130×NCU04952), BG23 (NCU04952×NCU08755) and triple-gene mutant strain BG123 (NCU00130×NCU04952×NCU08755)

[0117] 1. Cellulase production experiment of mutants

[0118] The double-gene mutant strains BG13 (NCU00130×NCU08755), BG12 (NCU00130×NCU04952), BG23 (NCU04952×NCU08755) and three-gene mutant strain BG123 (NCU00130×NCU04952×NCU08755) constructed above, wild-type N. crassa WT (FGSC2489) and BG1 (NCU00130), BG2 (NCU04952), BG3 (NCU08755) respectively in 2% (2g / 100mL) microcrystalline cellulose (Avicel) medium (recipe: 50 × Vogel's salt 20mL, microcrystalline cellulose 20g , agar 15g, constant volume to 1L, autoclaved.) and 2% (2g / 100mL) cellobiose (CB) (recipe: 50×Vogel's salt 20mL, cellobiose 20g, agar 15g, constant volume to 1L, autoclaved.) Cultivate on the culture medium at 28°C for 3 days, take the supernatant of the culture medium, centrifuge, conc...

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Abstract

The invention discloses gene engineering bacteria for producing cellulose, engineering bacteria capable of performing high-efficiency expression on target proteins and construction methods and application of the gene engineering bacteria and the engineering bacteria. Double-gene mutant strains BG13(NCU00130*NCU08755), BG12(NCU00130*NCU04952) and BG23(NCU04952*NCU08755) and a three-gene mutant strain BG123 (NCU00130*NCU04952*NCU08755) are obtained by modifying three beta-glucosidase genes NCU00130, NCU04952 and NCU08755 of a microorganism; and in a fermentation process, cellobiose serving as a specific inductor is added into the strains to produce the cellulose. Furthermore, according to the gene engineering bacteria, the engineering bacteria and construction methods and application thereof disclosed by the invention, a six-gene mutant strain delta3betaG::delta2cbh::deltahis3 is obtained through hybridization by taking the three-gene mutant strain BG123 as a male parent and a histidine defect type cellobiohydrolase amphimutation deficiency strain delta2cbh::deltahis3 as a female parent; and the target proteins can be produced by the six-gene mutant strain delta3betaG::delta2cbh::deltahis3.

Description

technical field [0001] The invention belongs to the field of genetic engineering, a method for high-efficiency expression and production of cellulase, mainly involving three β-glucosidase genes NCU00130, NCU04952, NCU08755 and a series of mutant strains of these three genes, cellulase inducers and inducers The discovery and application of the conditions, and the construction of the target protein expression system using a series of mutant strains of the three β-glucosidase genes NCU00130, NCU04952, and NCU08755 as the basic strains involve the construction of expression host bacteria, promoter sequences and induction conditions. Background technique [0002] Cellulose is the most widespread type of carbohydrate in nature, and it is also the largest renewable resource on earth. At present, only a small part of cellulose in nature has been utilized, and the vast majority of cellulose is not only wasted, but also causes environmental pollution. Cellulose molecules are chain po...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12N9/42C12N1/15C12N15/80C12R1/66C12R1/885C12R1/80C12R1/77C12R1/645
Inventor 田朝光姜永生刘倩马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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