Genetic recombinant saccharomyces cerevisiae capable of degrading and utilizing kitchen wastes

A technology for kitchen waste and Saccharomyces cerevisiae, which is applied in the fields of genetic engineering and fermentation engineering, can solve the problems of polluted environment, lack of degradation, rotten and smelly kitchen waste, etc.

Active Publication Date: 2014-04-16
GUANGDONG RECYCLEAN LOW CARBON TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high water content and rich nutrients of kitchen waste, microorganisms will use various organic substances and inorganic salts to rapidly reproduce and metabolize under normal temperature conditions, making kitchen waste rotten and smelly, polluting the environment, and causing trouble for disposal
[0004] Saccharomyces cerevisiae( Saccharomyces cerevisiae ) is the preferred bacterial species for ethanol fermentation in industry. It has the ability to convert glucose into ethanol efficiently, but it lacks enzymes that effectively degrade starch to generate glucose, and lacks enzymes that degrade proteins into polypeptides and amino acids. Under natural conditions, they cannot directly Using starch and protein in food waste as carbon and nitrogen sources to ferment ethanol

Method used

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  • Genetic recombinant saccharomyces cerevisiae capable of degrading and utilizing kitchen wastes
  • Genetic recombinant saccharomyces cerevisiae capable of degrading and utilizing kitchen wastes
  • Genetic recombinant saccharomyces cerevisiae capable of degrading and utilizing kitchen wastes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 α-amylase gene amy , glucoamylase gene ga and acid protease gene ap clone

[0034] Refer to Aspergillus oryzae published on GenBank ( Aspergillus oryzae ) α-amylase gene amy (accession number XM_001821384), Aspergillus niger ( Aspergillus niger ) glucoamylase gene ga (accession number XM_001390493.1) and acid protease gene ap (Accession No. XM_001401056.2), use Oligo 6 primer design software to design primers, and add appropriate restriction sites at the same time:

[0035] amy Gene amplification primers:

[0036]

[0037] The total RNA of Aspergillus oryzae CICC 40344 was extracted, and the reverse transcription PCR amplification reaction was carried out. amy The PCR amplified product of the gene was connected to the pGEM-T Easy vector (purchased from Promega Company) and verified by sequencing.

[0038] in amy The PCR reaction conditions of the gene are:

[0039]

[0040] The total RNA of Aspergillus niger CICC 40179 was extracted,...

Embodiment 2

[0046] Example 2 Three kinds of enzyme gene construction co-expression recombinant vector

[0047] The construction process of the three enzyme gene co-expression recombinant plasmids is as follows: figure 1 shown.

[0048] Obtained by embodiment 1 amy , ga with ap Restriction enzymes for coding sequences Bam H I and Speech I were excised from the pGEM-T Easy vector with double enzymes, respectively, and connected to the vector pScIKP that had been digested with the same double enzymes, to obtain recombinant plasmids pScIKP-amy, pScIKP-ga and pScIKP-ap.

[0049] use Nhe I and Xba I double digested pScIKP-ga, obtained containing PGK promoter and terminator ga Gene expression cassette fragments. use Nhe I single digested pScIKP-amy to linearize it. The two were ligated with T4 DNA ligase (using Nhe I and Xba I is the principle of the same tail enzyme), and the recombinant plasmid pScIKP-amy-ga was obtained. Using the same principle, use Nhe I an...

Embodiment 3

[0050] Example 3 Screening and Validation of Recombinant Yeast Transformants

[0051] Before the electrotransformation of Saccharomyces cerevisiae, the sensitivity of Saccharomyces cerevisiae AS2.489 was tested for the sensitivity of the resistance screening marker G418, and it was found that the yeast had been inhibited and could not grow on the YPD plate with a G418 concentration of 150 μg / ml. When the child can be screened with a concentration of G418 exceeding 150 μg / ml.

[0052] The three gene co-expression recombinant plasmid pScIKP-amy-ga-ap that embodiment 2 obtains uses restriction endonuclease Apa After I was linearized, it was transformed into Saccharomyces cerevisiae AS2.489 by electroporation, and cultured on a YPD agar plate with a G418 concentration of 200 μg / ml for 3 to 4 days, and the colony that could grow normally was picked as the transformation. Transformants of the above recombinant plasmids. Colony PCR was carried out with primers specific to the thr...

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Abstract

The invention relates to the field of genetic engineering and fermentation engineering and in particular discloses genetic recombinant saccharomyces cerevisiae capable of degrading and utilizing kitchen wastes. The genetic recombinant saccharomyces cerevisiae is constructed by transferring alpha-amylase (alpha-amylase) genes, glucoamylase (glucoamylase) genes and acid protease (acid protease) genes through a multi-gene coexpression vector of the saccharomyces cerevisiae and obtaining accurate secretory expression. The previous three enzyme genes are simultaneously transferred into the saccharomyces cerevisiae for realizing the secretory expression, so that the saccharomyces cerevisiae can simultaneously secrete the alpha-amylase, glucoamylase and acid protease. Therefore, main nutritional ingredients such as starch and proteins in the kitchen wastes can be efficiently degraded and become carbon sources and nitrogen sources needed by growth and fermentation of recombinant yeasts, and high-efficiency conversion between the kitchen wastes and ethanol is realized.

Description

technical field [0001] The invention relates to the fields of genetic engineering and fermentation engineering, and more specifically relates to a genetically recombined Saccharomyces cerevisiae capable of degrading and utilizing kitchen waste. Background technique [0002] At present, almost all fuel ethanol used in vehicles in my country is produced with corn and other raw materials. Mass production of fuel ethanol will inevitably lead to the problem of competition between vehicles and people for food, which will directly promote the continuous rise of food prices and cause food shortages. The solution is to use non-food renewable biomass as raw material to produce ethanol as much as possible. Kitchen waste is a huge amount of renewable biomass resources. In my country, the annual output of kitchen waste is more than 60 million tons, most of which are used to feed pigs, landfill, incinerate, and even make "gutter oil" , has caused serious environmental pollution, and a sma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12R1/865
CPCC12N9/2414C12N9/2428C12N9/48C12P7/08C12N9/2417C12Y302/01001C12Y302/01003C12N1/16
Inventor 刘泽寰方龙闫凯康小龙郑阳阳刘人怀林蒋海肖文娟李晶博龚映雪
Owner GUANGDONG RECYCLEAN LOW CARBON TECH CO LTD
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