δ integration and secretion of industrial strains of Saccharomyces cerevisiae expressing cellulase and its application

A technology of cellulase and Saccharomyces cerevisiae, which is applied in the field of bioengineering, can solve the problems of low enzyme diffusion rate, achieve high-efficiency degradation, and increase the effect of ethanol yield

Active Publication Date: 2022-05-13
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another disadvantage of cell surface display is that cellulases immobilized on the cell surface have a lower diffusivity than secreted enzymes

Method used

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  • δ integration and secretion of industrial strains of Saccharomyces cerevisiae expressing cellulase and its application
  • δ integration and secretion of industrial strains of Saccharomyces cerevisiae expressing cellulase and its application
  • δ integration and secretion of industrial strains of Saccharomyces cerevisiae expressing cellulase and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Acquisition of host strain Kα

[0019] Sporulation culture was carried out on the highly active dry yeast of Angel Saccharomyces cerevisiae, and the haploid strain whose mating type was α was obtained by screening, and the host strain Kα was obtained by reverse selection with 5-fluoroorotic acid plate. Specific steps are as follows:

[0020] (a) Take 0.01g Angel Saccharomyces cerevisiae high active dry yeast, inoculate it in 5ml YPD liquid medium (1% yeast powder, 2% peptone, 2% glucose), and culture it at 30°C and 150rpm for 20h.

[0021] (b) Use an inoculation needle to dip a part of the bacterial liquid and streak the YPD solid medium (1% yeast powder, 2% peptone, 2% glucose, 1.8% agar powder) plate, cultivate at 30°C until the colony is 2-3mm, pick a single The colonies were inoculated into 3 mL of YPD liquid medium and cultured at 30°C and 150 rpm for 20 h, then 50 μl of bacterial liquid was inoculated into 3 mL of YPD liquid medium and cultured at 30°C and 150 rp...

Embodiment 2

[0035] Construction of Cellulase Gene Expression Cassette δ Integration Plasmid

[0036] The schematic diagram of the cellulase gene expression cassette is shown in figure 1 , wherein Ptpi is (Saccharomyces cerevisiae triose phosphate isomerase promoter) promoter, xyn2s (Trichoderma reesei xylanase secreted peptide) is secreted peptide, cellulase gene is cellulase gene exon sequence, Tadh I (Saccharomyces cerevisiae alcohol dehydrogenase terminator) is the terminator. The cellulase genes include cbh1, cbh2, egl2 derived from Trichoderma reesei and bgl1, cbh1 derived from Aspergillus aculeatus.

[0037] The construction of the above five cellulase gene expression cassettes can be found in our work (Hong Jiefang. Construction of Saccharomyces cerevisiae engineering strains expressing cellulase and their research on the production of alcohol by fermentation of lignocellulose. 2014 Tianjin University doctoral dissertation; Zhang Weina. Expression Construction of Saccharomyces ce...

Embodiment 3

[0051] Yeast Transformation and Screening

[0052] After the plasmid δP1 was digested with NotI and the nucleic acid was purified, the host strain Kα was transformed by the lithium acetate method, and the transformants were screened on the basic medium plate with default uracil, and then reverse-selected on the 5-fluoroorotic acid plate to obtain URA3 The labeled transformant Kαδ1 was screened.

[0053] After the plasmid δP2 was digested with NotI and the nucleic acid was purified, the host strain Kαδ1 was transformed by the lithium acetate method, and the transformants were screened on the basic medium plate with default uracil, and then reverse-selected on the 5-fluoroorotic acid plate to obtain URA3 The labeled transformant Kαδ2 was screened.

[0054] After the plasmid IP3 was digested with NotI and the nucleic acid was purified, the host strain Kαδ2 was transformed by the lithium acetate method, the transformants were screened on the basic medium plate with default uracil...

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Abstract

The invention discloses an industrial strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) with delta integration, secretion and expression of cellulase and its application. A Saccharomyces cerevisiae industrial strain RδBEC with delta integration, secretion and expression of cellulase has a preservation number of CGMCC NO.16825. Application of the aforementioned industrial strain RδBEC, deposit number CGMCC NO.16825, in ethanol production. The bacterial strain RδBEC of the present invention, with the preservation number CGMCC NO.16825, secretes and expresses five kinds of cellulase: Trichoderma reesei CBH1, CBH2 and EG2; Aspergillus aculeatus BGL1 and CBH1. The bacteria can cooperate with existing commercialized cellulase to efficiently degrade cellulose and increase the yield of ethanol.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an industrial strain of Saccharomyces cerevisiae that secretes and expresses various cellulases and its application. Background technique [0002] The development and utilization of renewable energy is an active measure taken by human beings to face the problems of energy, environment and food. As the main bio-energy, fuel ethanol has attracted extensive attention due to its unique properties for vehicles. From 2005 to 2016, the global consumption of biofuel ethanol increased from 36.28 million tons to 79.15 million tons, with an average annual growth rate of 7.3%. At present, more than 40 countries and regions promote biofuel ethanol and ethanol gasoline for vehicles. The annual consumption of ethanol gasoline is about 600 million tons, accounting for about 60% of the total gasoline consumption in the world. In my country, in order to meet the growing energy ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12P7/10C12R1/865
CPCC12N9/2437C12P7/10Y02E50/10
Inventor 洪解放张敏华马媛媛邹少兰
Owner TIANJIN UNIV
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