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Method for producing ethanol by fermentation of recombinant saccharomyces cerevisiae engineering strain

A technology for recombining Saccharomyces cerevisiae and engineering strains, applied in microorganism-based methods, biochemical equipment and methods, fermentation and other directions, can solve problems such as lack of and inability to utilize xylose, and achieve high expression, reduced unit time, and high activity. Effect

Inactive Publication Date: 2013-09-25
新疆农业科学院生物质能源研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among all fermentation bacteria, Saccharomyces cerevisiae can best meet the above requirements. It has good industrial production traits, its full sequence has been determined, and genetic technology is mature. However, Saccharomyces cerevisiae lacks the initial conversion of xylose Xylulose is an enzyme that cannot utilize xylose

Method used

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  • Method for producing ethanol by fermentation of recombinant saccharomyces cerevisiae engineering strain
  • Method for producing ethanol by fermentation of recombinant saccharomyces cerevisiae engineering strain
  • Method for producing ethanol by fermentation of recombinant saccharomyces cerevisiae engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Construction of recombinant Saccharomyces cerevisiae engineering strain

[0020] 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%.

[0021] 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.

[0022] 3. Cloning of GAP gene fragment from Pichia pastoris GS115

[0023] Primer seque...

Embodiment 2

[0041] Example 2: Expression identification and activity analysis of xylose reductase gene xyl1 and xylitol dehydrogenase gene xyl2 in Saccharomyces cerevisiae

[0042] 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 it 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 1

[0044] Xinjiang Jimusar Santai Winery Experiment

[0045] 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.

[0046] Experimental equipment and reagents: 722S spectrophotometer, water bath, electric furnace, Φ15mm×180mm test tube, 1.5ml centrifuge tube. Ethanol (analytical pure), glucose (analytical pure), potassium dichromate (analytical pure), pure water, Xinjiang sweet sorghum straw.

[0047] Experimental steps:

[0048] 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.

[0049] 2. Break up 3 tons of stalks, divide the...

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Abstract

The invention discloses a method for producing ethanol by fermentation of a recombinant saccharomyces cerevisiae engineering strain which comprises the following steps: (1) pulverizing sweet sorghum straw; (2) adding glucoamylase with a weight of 0.05-0.4% of the straw weight; (3) adding a recombinant saccharomyces cerevisiae engineering strain with a weight of 0.08-0.2% of the straw weight, wherein the engineering strain is a recombinant bacterium containing PYES2-GAP-xyl1-xyl2 plasmid, and the fermentation time is 24-90 hours. The method of the invention decreases the unit time required by fermentation of fuel ethanol, reduces the production cost, minimizes the environment pollution, and can be used without improvement of original production conditions.

Description

technical field [0001] The invention relates to a method for fermenting and producing ethanol by adopting recombinant Saccharomyces cerevisiae engineering strains. Background technique [0002] Lignocellulose is one of the most abundant renewable resources on the earth. Its main components are cellulose, hemicellulose, and lignin. Among them, cellulose is hydrolyzed to obtain glucose, which can be directly used by microorganisms, and its content is second only to cellulose. In the hydrolyzate of hemicellulose, the main components are glucose and D-xylose, and the content of xylose is slightly less. Hemicellulose is one of the main components of plant fiber materials. For example, the content of hemicellulose in straw accounts for 25%-50% of its dry weight, and its main decomposition product is also xylose. Under the action of acid or enzyme, the hydrolysis products of lignocellulose are mainly six-carbon sugars (glucose, mannose and galactose) and five-carbon sugars (xylose...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/10C12P19/14C12N15/53C12N15/63C12N1/19C12R1/865
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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