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Trichoderma reesei strains for heterologous expression of xylanase/cellulase CbXyn10c genes and application

A technology of Trichoderma reesei and heterologous expression, applied in the field of genetic engineering

Pending Publication Date: 2022-01-18
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Trichoderma reesei is an important industrial production strain with strong ability to express and secrete proteins. So far, the successful expression of bacterial-derived genes in Trichoderma reesei has not been reported

Method used

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  • Trichoderma reesei strains for heterologous expression of xylanase/cellulase CbXyn10c genes and application
  • Trichoderma reesei strains for heterologous expression of xylanase/cellulase CbXyn10c genes and application
  • Trichoderma reesei strains for heterologous expression of xylanase/cellulase CbXyn10c genes and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Main reagents

[0038] In this experiment, rTaq enzyme, PrimeSTAR high-fidelity enzyme, restriction endonuclease EcoR I. Pst I. T4 ligase, RNase and the DL10000 DNA Marker and DL5000 DNA Marker used were purchased from TaKaRa Company; ClonExpressMultiS One Step Cloning Kit and 2×Rapid Taq Master Mix were purchased from Nanjing Novizym Biotechnology Co., Ltd.; the Marker used in this experiment III. Plasmid mini-extraction kit and ordinary agarose gel DNA recovery kit were purchased from Tiangen Biochemical Technology Co., Ltd.; high-efficiency competent cell preparation kit was purchased from Shanghai Jierui Bioengineering Co., Ltd. All the synthetic sequences and primers in this paper were synthesized by Jinweizhi Biotechnology Co., Ltd., where the forward primer is F and the reverse primer is R. The primers used in the experiment are shown in Table 1-2:

[0039] Table 1 The strains and plasmids used in this experiment

[0040]

[0041] Table 2 Primers used...

Embodiment 2

[0069] SDS-PAGE verification and mass spectrometry identification analysis of CbXyn10C expression

[0070] Take 1 ml of the fermented broth of the transformant on day 1 for SDS-PAGE verification (such as figure 1 ) and protein profile validation (eg figure 1 ), confirming the successful expression of CbXyn10C. Analyzing the SDS-PAGE results, in addition to the transformants of lanes 10 and 11, lanes 4, 6, and 7 (i.e. transformants successfully expressing CbXyn10C) were less than the transformants of the other lanes and the control TU-6 indicated by the red arrow "→". The indicated band was identified as aspartic protease (XM_006961706.1) by mass spectrometry.

[0071] 2. Determination of xylanase activity of positive transformants and TU-6 fermentation broth

[0072] Positive transformants and TU-6 fermented broth xylanase activity assay results are as follows figure 2 Shown: Transformants CbXyn10C-7, -10, -12 can detect different degrees of Xylan activity, and the enzy...

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Abstract

The invention discloses trichoderma reesei strains for heterologous expression of xylanase / cellulase CbXyn10c genes and an application. Three trichoderma reesei strains successfully expressing CbXyn10C are prepared by carrying out codon optimization on bifunctional xylanase / cellulase CbXyn10c genes derived from bacterium Caldicellosiruber bescin, and then integrating into the position of an aspartic protease gene in trichoderma reesei. The strains successfully overexpress CbXyn10c genes optimized by a codon, and are named as CbXyn10C-7, -10 and -12 trichoderma reesei strains. The invention aims to determine that the CbXyn10c genes can be subjected to successful heterologous expression through codon optimization and integration into a key gene locus of trichoderma reesei. The trichoderma reesei strains can be applied to simultaneously improving the activity of trichoderma reesei cellulase / xylanase.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to heterologous expression of bacterial-sourced bifunctional cellulase / xylanase CbXyn10c in a Trichoderma reesei strain and an application thereof. Background technique [0002] Plant biomass is widely distributed and rich in content, and it is the most used feed raw material, accounting for about 52% of the total raw materials. Plant cell wall polysaccharides include cellulose, hemicellulose (xylan, xyloglucan, mannan, etc.) and pectin, which are the three main components of non-starch polysaccharides (NSP). For plant-derived feed, non-starch polysaccharides are one of the main anti-nutritional factors in feed materials, which increase the viscosity of chyme and hinder the release of nutrients such as starch and protein, thereby reducing the nutritional value of plant-derived feed. palatability, digestibility, etc. In plant cell wall NSP, xylan and cellulo...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12N15/66C12N15/90C12N15/56C12R1/885
CPCC12N9/2437C12N9/2482C12Y302/01091C12Y302/01004C12Y302/01008C12N15/80C12N15/902C12N2800/22Y02E50/10
Inventor 薛鲜丽王德培王静然毕杭杭
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY