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Recombination saccharomyces cerevisiae strain secreting and expressing tricoderma reesei exoglucanase I and application thereof

A technology of exo-glucanase and Saccharomyces cerevisiae strain, which can be used in recombinant DNA technology, fermentation, fungi, etc., and can solve many problems.

Active Publication Date: 2012-07-11
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] At present, there are few reports on the successful realization of heterologous secretory expression of Tr-Cel7A in Saccharomyces cerevisiae. ME etc. ( ME et al., Gene 1988, 63(1): 103-112.) realized the heterologous secretory expression of Tr-Cel7A in Saccharomyces cerevisiae, but the recombinant Tr-Cel7A was modified by excessive glycosylation; the same Den Haan R et al. (Den Haan R et al., Enzyme Microb Tech 2007, 40:1291-1299.) The Tr-Cel7A expressed heterologously in Saccharomyces cerevisiae is also modified by excessive glycosylation, and the enzyme activity of Tr-Cel7A is only 10mU g -1 DW

Method used

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  • Recombination saccharomyces cerevisiae strain secreting and expressing tricoderma reesei exoglucanase I and application thereof
  • Recombination saccharomyces cerevisiae strain secreting and expressing tricoderma reesei exoglucanase I and application thereof
  • Recombination saccharomyces cerevisiae strain secreting and expressing tricoderma reesei exoglucanase I and application thereof

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Embodiment 1

[0048] Example 1: Preparation of Saccharomyces cerevisiae strain with improved ability to secrete heterologous proteins with large molecular weight and rich disulfide bonds: Saccharomyces cerevisiae B SX010

[0049] 〖1〗Knockout of protein missorting pathway receptor gene Sc-VPS10 involved in protein transport

[0050] Methods as below:

[0051] (1) PCR amplification of the recombination arm of the Sc-VPS10 gene: using the Saccharomyces cerevisiae CEN.PK102-3A genome as a template, using primer VPSL1 and primer VPSL2 to amplify the 200bp recombination arm 1 ( figure 1 ), with primer VPSL3 and primer VPSL4, amplified to obtain the recombinant arm 2 of 200bp ( figure 1 );

[0052] Wherein, the above-mentioned VPSL1, VPSL2, VPSL3 and VPSL4 primer sequences are:

[0053] VPSL1: 5'-ACAGCATGATGCAACAGCT-3'

[0054] VPSL2: 5'-TATTAAGGGTTGTCGACCTGCACTTCAGGGCTTTTCCA-3'

[0055] VPSL3: 5'-TGATATCAGATCCACTAGTGTAGATAATTCCATATACTTTTT-3'

[0056] VPSL4: 5'-CCGTAACATATGATATATCC-3'

[00...

Embodiment 2

[0145] Example 2: Secreted expression of Tr-Cel7A in Saccharomyces cerevisiae

[0146] Methods as below:

[0147] (1) Construction of secretion expression vector pJCF

[0148] ①The Tr-cel7A gene fragment with Flag tag was obtained by whole gene synthesis ( Figure 12 );

[0149] ②Construction of the recombinant plasmid pJCF: pass the Tr-cel7A gene fragment (nucleotide as shown in SEQ ID NO.1) with the Flag tag obtained in step ① through Xba I, and pass the empty plasmid pJFE2 (Peng et al., Metab Eng 2012, 14 (1): 9-18.) Digested with Xba I ( Figure 13 ), purified the digested product with a PCR product recovery kit, treated pJFE2 digested with Xba I with CIAP, ligated with T4 ligase, reacted at 16°C for 12 hours, and obtained the recombinant plasmid pJCF( Figure 14 );

[0150] (2) Recombinant strain construction:

[0151] ① transformation of Saccharomyces cerevisiae with recombinant plasmids: respectively transform 25 microliters (1) Partial steps ② Gained recombinant ...

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Abstract

The invention provides a recombination saccharomyces cerevisiae strain secreting expressing tricoderma reesei exoglucanase I Tr-Cel7A, the strain is named saccharomyces cerevisiae BSXC10, and is preserved in General Microbiology Center of Chinese Microorganism Strain Preservation Committee on December 27, 2011 with the preserving number being CGMCC No.5657. Comparing the saccharomyces cerevisiae BSXC10 with the common saccharomyces cerevisiae strain BSXC01, the enzyme activity of the extracellular Tr-Cel7A is improved by 2.56 times, and compared with former document reports, the enzyme activity of the extracellular Tr-Cel7A is improved by 8.06 times. According to the saccharomyces cerevisiae strain, a new idea is provided for the research of the CBP yeast, and the probability utilizing the saccharomyces cerevisiae to produce chemical products taking cellulose as raw material is provided at the same time.

Description

technical field [0001] The invention relates to a recombinant Saccharomyces cerevisiae strain secreting and expressing Trichoderma reesei exoglucanase I Tr-Cel7A and its application in the research of CBP yeast and the production of chemical products with cellulose as raw material. Background technique [0002] Cellulosic ethanol is a potential future fuel, and the production of cellulosic ethanol requires the cooperation of cellulosic hydrolyzing microorganisms and sugar fermenting microorganisms. CBP (Consolidated bioprocessing) is a combination or partial combination of cellulase and hemicellulase production, cellulose hydrolysis and ethanol fermentation, and is completed by a microorganism. The CBP process is beneficial to reduce the cost of the biotransformation process, and has received more and more attention from researchers. A successful CBP strain requires not only strong ethanol fermentation ability, but also cellulose hydrolysis ability. As a traditional ethano...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P7/10C12R1/865
CPCY02E50/16Y02E50/10
Inventor 鲍晓明沈煜徐丽丽
Owner SHANDONG UNIV
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