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Recombinant saccharomyces cerevisiae engineering strain and application thereof

A technology of recombinant Saccharomyces cerevisiae and engineering strains, which is applied in the field of recombinant Saccharomyces cerevisiae engineering strains, can solve the problem that Saccharomyces cerevisiae cannot utilize five-carbon sugars, and achieve the effects of high biotransformation ethanol yield, high expression level, and high enzyme activity

Inactive Publication Date: 2011-10-19
新疆农业科学院生物质能源研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to overcome the deficiency that Saccharomyces cerevisiae cannot utilize five-carbon sugars, and provide a novel Saccharomyces cerevisiae strain that can efficiently utilize both five-carbon sugars and six-carbon sugars

Method used

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  • Recombinant saccharomyces cerevisiae engineering strain and application thereof
  • Recombinant saccharomyces cerevisiae engineering strain and application thereof
  • Recombinant saccharomyces cerevisiae engineering strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Construction of recombinant Saccharomyces cerevisiae engineering strain

[0021] 1. Clone the xylose reductase gene (xyl1) fragment (as shown in SEQ ID NO.1) from Candida parapsilosis, and make a Blast comparison with the Candida parapsilosis xylose reductase gene sequence released by Genebank , for homology comparative analysis, the results showed that the homology between the cloned xyl1 gene fragment and the Candida parapsilosis xylose reductase gene released by the gene bank was 100%.

[0022] 2. Clone the xylitol dehydrogenase gene (xyl2) fragment (as shown in SEQ ID NO.2) from Candida tropicalis, and compare the sequence obtained by sequencing with xyl2 in the gene bank (Pubmed+NCBI+Nucleotide) Homology analysis of the gene sequence showed that the sequence was completely consistent with the Genomic DNA sequence of Candida tropicalis xyl2 released by Genebank.

[0023] 3. Cloning the GAP gene fragment from Pichia pastoris (Pichia pastoris) GS115

...

Embodiment 2

[0042] Example 2: Expression identification and activity analysis of xylose reductase gene xyl1 and xylitol dehydrogenase gene xyl2 in Saccharomyces cerevisiae

[0043] Select several robust bacterial strains screened in Example 1 for shake flask culture, break the wall, and analyze the expression situation by SDS-PAGE; select the bacterial strain with the highest expression level, cultivate in large quantities, purify the target protein, and measure the enzyme activity in vitro, wherein xylose reduction The specific enzyme activity of the enzyme gene xyl1 is about 0.521±0.008; the specific activity of the xylitol dehydrogenase gene xyl2 is about 0.401±0.004.

experiment example

[0045] Xinjiang Jimusar Santai Winery Experiment

[0046] The purpose of the experiment: to carry out large-scale fermentation under the same conditions as possible, the amount of fermentation straw is 1 ton, and timely detect the changes of sugar and alcohol in the fermentation liquid during the fermentation process, so as to test the strains in the actual production process of the three wineries The final yield effect of alcohol.

[0047] Experimental equipment and reagents: 722S spectrophotometer, water bath, electric furnace, Ф15mm×180mm test tube, 1.5ml centrifuge tube. Ethanol (analytical pure), glucose (analytical pure), potassium dichromate (analytical pure), pure water, Xinjiang sweet sorghum straw.

[0048] Experimental steps:

[0049] 1. Prepare 15 fermentation vats, divided into 3 groups of 1, 2, and 3, with 5 fermentation vats in each group, numbered: 11, 12, 13, 14, 15; 21, 22, 23, 24, 25; 31 , 32, 33, 34, 35.

[0050] 2. Break up 3 tons of stalks, divide the...

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Abstract

The invention discloses a recombinant saccharomyces cerevisiae engineering strain and application thereof. GAP gene fragments are cloned from Pichiapastoris GS115, and induced promoters of a PYES2 carrier are replaced by constitutive GAP promoters. Then an xylose reductase gene xyl1 fragment and an xylitol dehydrogenase gene xyl2 fragment are connected together and introduced into a PYES2 carrier containing constitutive GAP promoters to constitute a PYES2- GAP-xyl1-xyl2 plasmid. The plasmid is introduced into saccharomyces cerevisiae. The engineering strain of the present invention can co-express xylose reductase and xylitol dehydrogenase, has a high activity and high expression level of xylose reductase and xylitol dehydrogenase. In addition, the invention can efficiently utilize pentose and hexose degraded from cellulose and hemicellulose in straws, so as to increase output of fuel ethanol, reduce production costs and reduce environmental pollution. Therefore, the engineering strain can be used for producing fuel ethanol.

Description

technical field [0001] The invention relates to the construction of genetically engineered bacteria, in particular to a recombinant Saccharomyces cerevisiae engineered strain and its application. Background technique [0002] With the sharp increase in energy demand and the aggravation of the shortage of fossil fuels, research on alternative fuels to petroleum has become an urgent worldwide topic. Fuel alcohol is recognized as a clean and convenient renewable bioenergy. It is the liquid fuel with the most development potential among renewable resources and a "green oil field" for sustainable development in the future. People pay more and more attention to the production of fuel alcohol. Traditional alcohol is fermented from starchy raw materials, but the cost of producing fuel alcohol from lignocellulosic raw materials (such as straw) is lower, which can reduce the volatilization loss of combustion and relieve environmental pressure. Energy crisis and food crisis have consi...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P7/10C12R1/865
CPCY02E50/10
Inventor 叶凯刘敏涂振东
Owner 新疆农业科学院生物质能源研究所
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