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Transgenic wine brewing saccharomycete for degradation cellulose synthetic ethyl alcohol and construction method thereof

A Saccharomyces cerevisiae, a technology for degrading cellulose, applied in the field of microbial fermentation, can solve the problems of incomplete destruction of cellulose structure, serious pollution, complicated operation, etc., and achieve the effects of alleviating tail gas pollution, solving pollution, and ensuring energy security

Inactive Publication Date: 2008-12-10
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The structure of cellulose cannot be completely destroyed by physical methods, and it is very energy-intensive. Although chemical methods can completely degrade cellulose, the operation is complicated, the pollution is serious, and it is not economical.

Method used

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  • Transgenic wine brewing saccharomycete for degradation cellulose synthetic ethyl alcohol and construction method thereof
  • Transgenic wine brewing saccharomycete for degradation cellulose synthetic ethyl alcohol and construction method thereof
  • Transgenic wine brewing saccharomycete for degradation cellulose synthetic ethyl alcohol and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Primer Design of Saccharomyces cerevisiae Multigene Expression Vector and Its Expression Elements

[0051] (1) Specific amplification primers for rDNA

[0052] The rDNA sequence in the Saccharomyces cerevisiae genome is highly repetitive with a length of 9.1kb, and it is the preferred homologous recombination site for constructing a high-copy number integrating vector. In this example, the amplified rDNA fragment of 1.8 kb was selected as the expression element, and at the same time, the position of the fragment in the full-length rDNA sequence of the Saccharomyces cerevisiae genome was clarified.

[0053] With reference to the genome sequence of Saccharomyces cerevisiae (S.cerevisiae) provided by NCBI, the specific primers for amplifying the rDNA 1.8kb fragment were designed using the biological software oligo 6 as follows:

[0054] Upstream primer: 5′-GC TCTAGA CCAGCATCCTTGACTTAC-3';

[0055] ↑

[0056] Xba I

[005...

Embodiment 2

[0081] Embodiment 2 Saccharomyces cerevisiae polygene expression vector and its expression element amplification

[0082] (1) Amplification and purification of rDNA

[0083] Using the genome of Saccharomyces cerevisiae AS2.489 strain (purchased from the Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences) as a template, PCR amplification was performed using the rDNA-specific amplification primers obtained in Example 1.

[0084] PCR reaction conditions:

[0085]

[0086] (2) Amplification and purification of PGK promoter

[0087] The first round of PCR amplification used the genome of Saccharomyces cerevisiae AS 2.489 strain as a template.

[0088] PCR reaction conditions:

[0089]

[0090] The second round of PCR amplification uses the purified first round of PCR product as a template for PCR amplification, and the obtained product is the PGK promoter.

[0091]PCR reaction conditions:

[0092]

[0093] (3) Amplification and pur...

Embodiment 3

[0097] Example 3 Construction of Saccharomyces cerevisiae polygene expression vector

[0098] The detailed construction process of Saccharomyces cerevisiae multigene expression vector is as follows: figure 1 As shown, the specific steps are as follows:

[0099] (1) Connect the G418 resistance gene fragment obtained from the pPIC9K vector (purchased from Invitrogen) into the pBluescript II SK (-) vector with T4 ligase to obtain expression vector 1, and perform PCR-specific primer amplification on expression vector 1 Enrichment, enzyme digestion identification and sequencing to ensure the correctness of the sequence.

[0100] (2) Carry out double digestion (SalI and BamH I) to the PCR product of the PGK promoter that embodiment 2 obtains, after the same double digestion is carried out to the expression vector 1 that step (1) obtains simultaneously, with T4 ligase The enzyme-digested product of the PGK promoter was connected into the expression vector 1 to obtain the expression...

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Abstract

The invention discloses a transgene saccharomy cescerevisiae for degrading cellulose to synthesize ethanol, and a construction method thereof. The transgene saccharomy cescerevisiae is obtained through the following steps that: genes of three enzymes in a cellulose system are introduced into a saccharomy cescerevisiae together through a saccharomy cescerevisiae expression vector; the three enzymes in the cellulose system are endo beta-1, 4-dextranase, exo beta-1, 4- dextranase and cellobiase; the constructed transgene saccharomy cescerevisiae can effectively, massively and simultaneously express the three cellulases, and degrade the cellulose into glucose, combining with the inborn ability of the saccharomy cescerevisiae of synthesizing the ethanol by use of the glucose. Therefore, the transgene saccharomy cescerevisiae is capable of producing the ethanol by fermenting the cellulose, can produce the ethanol by taking agricultural wastes full of cellulose as raw materials, which not only dissolves the pollution caused by the agricultural wastes, but also produces fuel ethanol to form sustainable energy reservation, thereby having inestimable functions to alleviate the shortage of oil resource, guarantee the safety of national energy, and improve the pollution of automobile exhaust.

Description

technical field [0001] The invention relates to the technical field of microbial fermentation, in particular to a transgenic Saccharomyces cerevisiae capable of degrading cellulose into glucose and then synthesizing ethanol and a construction method thereof. 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 produce ethanol from cellulose as much as possible and avoid producing ethanol from starch and sugar. In my country, there are 700 million tons of corn stalks, wheat stalks, rice straws, and sorghum stalks in total every year, but most of the straws are burned at present, causing environmental pollution. It is a key point to use these agricultural and for...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/56C12N15/81C12P7/10C12R1/865
CPCY02E50/10
Inventor 刘泽寰台艳肖文娟王峻梅
Owner JINAN UNIVERSITY
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