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Saccharomyces cerevisiae industrial strain for secreting and expressing cellulase and building method thereof

A cellulase, secretory expression technology, applied in the field of bioengineering, can solve problems such as low enzyme diffusion rate

Active Publication Date: 2018-10-09
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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  • Saccharomyces cerevisiae industrial strain for secreting and expressing cellulase and building method thereof
  • Saccharomyces cerevisiae industrial strain for secreting and expressing cellulase and building method thereof
  • Saccharomyces cerevisiae industrial strain for secreting and expressing cellulase and building method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Acquisition of host strain Kα

[0051] Angel Saccharomyces high activity dry yeast (commercially available) was used for sporulation culture, and the haploid strain with mating type 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:

[0052] (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.

[0053] (b) Use an inoculation needle to dip part of the bacterial liquid to streak the YPD solid (1% yeast powder, 2% peptone, 2% glucose, 1.8% agar powder) plate, cultivate at 30°C until the colony is 2-3mm, and pick a single colony for inoculation Into 3mL YPD liquid medium, cultured at 30°C, 150rpm for 20h, then inoculated 50μl of bacterial liquid into 3mL YPD liquid medium, and cultivated at 30°C, 150rpm for 6h.

[0054] (c) Collect the th...

Embodiment 2

[0065] Construction of Cellulase Gene Expression Cassette Site-Directed Integration Plasmid

[0066] The schematic diagram of the cellulase gene expression cassette is shown in figure 1 , where Ptpi is the (Saccharomyces cerevisiae triose phosphate isomerase promoter) promoter, xyn2s (Trichoderma reesei xylanase secreted peptide) is the secreted peptide, cellulase gene is the exon sequence of each cellulase gene, Tadh I (S. cerevisiae alcohol dehydrogenase terminator) is the terminator. The cellulase genes include three cellobiohydrolase genes (Trichoderma reesei cbh1, cbh2 and Aspergillus aculeatus cbh1), two endoglucanase genes (A.aculeatus egl1 and T .reeseiegl2) and a β-glucosidase gene (A.aculeatus bgl1).

[0067] The construction of the above six cellulase gene expression cassettes can be found in our work (Hong Jiefang. Construction of Saccharomyces cerevisiae engineering strains expressing cellulase and research on the production of alcohol by fermentation of lignoce...

Embodiment 3

[0087] Yeast Transformation and Screening

[0088] After the plasmid IP1 was digested with ScaI and the nucleic acid was purified, the host strain Kα was transformed by the lithium acetate method, the transformants were screened on the basic medium plate with default uracil, and the URA3 was removed by reverse selection on the 5-fluoroorotic acid plate. The labeled transformant Kα1 was screened.

[0089] After the plasmid IP2 was digested with ScaI and the nucleic acid was purified, the host strain Kα1 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 Kα2 was screened.

[0090]After the plasmid IP3 was digested with PvuII and the nucleic acid was purified, the host strain Kα2 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 a saccharomyces cerevisiae industrial strain for secreting and expressing cellulase and a building method thereof. The building method comprises the following steps: (1) obtaining host bacteria Kalpha by culturing raw spores by using Angel wine-brewing high-activity dry yeast, screening to obtain a haploid strain with a mating type being an alpha type, and carrying out reverse selection by using a 5-fluororotic acid flat plate to obtain the host bacteria Kalpha; (2) building site-directed integration plasmids of cellulase gene expression boxes; and (3) transforming andscreening the yeasts. The method is capable of achieving site-specific integration, expression and secretion of various cellulase genes in derivative strains of the saccharomyces cerevisiae industrialstrain by repeatedly using the URA3 screening mark; the recombinant strain is capable of effectively improving the efficiency for hydrolyzing cellulose through commercial cellulase; the production cost of the cellulase is reduced; the lignocellulose resource can be efficiently transformed to produce alcohol.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to an industrial strain of Saccharomyces cerevisiae that secretes and expresses various cellulases and a construction method. Background technique [0002] Fossil energy such as oil, natural gas, and coal is currently the main form of energy. Since they are non-renewable, massive consumption will inevitably lead to their rapid depletion. Looking for renewable energy that can (partially) replace fossil energy and is less polluting is a direction for future energy development. The biomass produced by photosynthesis on the earth is as high as 150-200 billion tons every year, and the energy contained in 5% of the biomass is equivalent to the human demand for oil and natural gas. The annual production of lignocellulose in biomass is about 100 billion tons, which is the most abundant carbon source in nature and can provide a potential raw material for the production of ...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/90C12N15/56C12N1/19C12R1/865
CPCC12N9/2437C12N15/81C12N15/905
Inventor 洪解放张敏华邹少兰马媛媛
Owner TIANJIN UNIV
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