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High fermentation rate type recombinant alcoholic fermentation yeast, the building and expression carrier thereof

A technology of alcohol yeast and fermentation rate, which is applied in the direction of introducing foreign genetic material, fermentation, and recombinant DNA technology by using carriers, and can solve problems such as increasing the complexity of alcohol fermentation process operations

Inactive Publication Date: 2008-10-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Although the application of acid protease in the alcoholic fermentation process has the above advantages, it is still unacceptable to add acidic protease in the alcoholic fermentation process. Complexity of Alcoholic Fermentation Process Operation

Method used

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  • High fermentation rate type recombinant alcoholic fermentation yeast, the building and expression carrier thereof
  • High fermentation rate type recombinant alcoholic fermentation yeast, the building and expression carrier thereof
  • High fermentation rate type recombinant alcoholic fermentation yeast, the building and expression carrier thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Embodiment 1: Construction of yeast expression vector

[0130] The primers used in the construction of yeast expression vectors were synthesized by a DNA synthesizer. The primers used are as follows:

[0131] Primer P1: cg agatct ct attttcgagg accttgtcac

[0132] Primer P2: tttgtttgtt tatgtgtgtt

[0133] Primer P3: ttataagacg ggcaaaata

[0134] Primer P4: taaccaa ttctgattag aaa

[0135] Primer P5: ttt agatct a acgggaaacg tcttgct

[0136] Primer P6: aac agatct c tgcctcgcgc gtttcggtga t

[0137] Primer P7: tcg agatct c gaataataac tgttattttt ca

[0138] The single underlined part is the artificially introduced restriction enzyme cutting site; the double underlined part is the sequence artificially introduced to facilitate splicing of fragments.

[0139] The construction process of the expression vector is as follows:

[0140] Synthetic S MFα And in the process of synthesis, multiple cloning sites and transcription termination sequences T art And a p...

Embodiment 2

[0141] Embodiment 2: the construction of alcohol yeast expression acid protease gene ap recombinant plasmid

[0142] The acid protease gene ap was cloned by using the PCR technique, using the chromosomal DNA or total RNA of the filamentous fungus as a template, and mediated by a pair of specific primers. The artificially designed and synthesized primers for acid protease gene cloning and expression, its characteristic sequence is:

[0143] An-Ap1:acat gaattc atggtcgtct tcagcaaaac cgctgccctc

[0144] An-Ap2: gtga gaattc ttttttttttttttttttttt

[0145] Af-Ap1:acat gaattc atggctccat tcacgtttct ggtagggata

[0146] Af-Ap2: gtga gaattc tcatggggta cttgaaacac tcgtggccaa t

[0147] Ao-Ap1:acat gaattc atgatgcggg gcgcatctct cctaccagtt

[0148] Ao-Ap2: gtga gaattc ttacaaagca agaagaagac cagcacccgc a

[0149] Mp-Ap1:acat gaattc atggtcgtct tcagcaagat caccgccgtt

[0150] Mp-Ap2: gtga gaattc ttacaaagca agaagaagac cagcacccg

[0151] Nc-Ap1:acat gaattc atgtcttcga...

Embodiment 3

[0157] Example 3: Construction of recombinant industrial alcohol production yeast CICIM MMY0034 (CCTCC M206009)

[0158] The activated industrial alcohol-producing strain Saccharomyces cerevisiae 825 was cultured in 500mL YPD (1% yeast extract, 2% peptone, 2% glucose) at 30°C for 18h until OD 600 =1.7, 5000r / min centrifugation collects the thalline, successively washes the thalline with 500mL, 250mL precooled sterile water, centrifuges to remove the supernatant, and suspends the thalline with 20mL precooled 1mol / L sorbitol. After centrifugation, the bacteria were suspended in 0.5 mL pre-cooled sorbitol, and used as competent cells for electroshock transformation.

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Abstract

The invention relates to recombination alcohol yeast and construction and the expression carrier it used. The invention supplies expression carrier of exogenous acid protease gene pYEI-222 and recombination particle of exogenous acid protease gene pYEI-ap. And the construction method of recombination alcohol yeast CCTCC M206009 and recombination alcohol yeast CCTCC M206009 that has high alcohol fermenting speed. It could be used to improve the alcohol yeast and alcohol industry.

Description

technical field [0001] A high-fermentation-rate recombinant alcoholic yeast, its construction and the expression vector used therein, involve the transformation of the acid protease gene into industrial alcoholic yeast and its application in the industrial alcoholic yeast Saccharomyces cerevisiae through the integrated expression vector of yeast, and obtain a high The invention relates to a recombinant alcohol yeast with a fermentation rate, which belongs to the field of microbial fermentation engineering. Background technique [0002] In industrial production, alcohol can be produced by fermentation or synthesis, and more than 95% of factories in my country use fermentation to produce alcohol. The starchy raw materials commonly used in alcohol production include potatoes, grains, wild plants and by-products of agricultural product processing. The chemical composition of various starchy raw materials is shown in Table 1 below. [0003] Table 1 Reference Table of Chemical C...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/57C12N15/81C12N9/50
Inventor 王正祥牛丹丹石贵阳
Owner JIANGNAN UNIV
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