Method of producing stalk ethanol by means of mixed fermentation of transgenetic yeast

A mixed fermentation and genetically modified technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, fermentation, etc., can solve problems such as limiting competitiveness, high cost of straw ethanol, and unutilized xylose, so as to reduce production costs and reduce The cost of detoxification and the effect of increasing the yield of ethanol

Inactive Publication Date: 2014-05-21
QINGDAO UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Chinese patent application 200880119451.X discloses bacteria capable of simultaneously fermenting glucose, mannose and xylose and a method for producing bioethanol using the bacteria, however, the process cannot Direct use of straw can only use glucose, xylose, and mannose, and the cost of converting straw into glucose is much higher than that of converting glucose into ethanol
[0004] Chinese patent application 201110135607.7 discloses a process for producing ethanol by synchronous saccharification and fermentation using low-temperature cellulase. However, due to the use of cellulase, the production cost is greatly reduced. Moreover, xylose cannot be utilized, resulting in a low conversion rate of straw into ethanol and a lack of market competitiveness
greatly increase the cost of production
[0007] Chinese patent application 201110460248.2 discloses the fermentation method of cellulosic yeast, double strain Sac...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Screening and domestication of Saccharomyces cerevisiae and Pichia stipitis (or Candida shohata)

[0049] Saccharomyces cerevisiae and Pichia stipitis (or Candida shohata) were screened and domesticated with steam-exploded straw pretreatment solution, and the strains with high ethanol tolerance were screened at the same time. Through screening and domestication, strains of Saccharomyces cerevisiae and Pichia stipitis (or Candida shohata) resistant to acetic acid, furfural and ethanol were obtained.

[0050] The specific experimental conditions are as follows:

[0051] Steam explosion conditions, temperature 160-200°C, time, 3-20 minutes, pressure, 1.1-1.8MP, because steam explosion conditions affect the degradation and conversion rate of cellulose and xylan, as well as the production of acetic acid and furfural, the used , was optimized by response surface method, and the optimal steam explosion conditions were obtained, time: 5-8 minutes; pressure: 1....

Embodiment 2

[0053] Example 2 Construction of transgenic cellulase Saccharomyces cerevisiae 1

[0054] Vector pYEX-BX (7.1kb) (containing three selection markers Ampr, URA3 and leu2), 3-phosphate glyceraldehyde dehydrogenase gene promoter (GAPDH P ) and terminator (GAPDH T ), the signal peptide coding sequence adopts the signal peptide sequence (XYNSEC) of the xylanase gene in T. reesei to construct the recombinant expression vector Ⅰ, and its expression sequence frame is GAPDH P -XYNSEC-cbh1-GAPDH T -GAPDH P -XYNSEC-eg1-GAPDH T -GAPDH P –GLUSEC-bglc-GAPDH T , named pYEX-BX-GAPDH-EchBl. The constructed recombinant expression vector was transformed into Saccharomyces cerevisiae kdn-6 by electric shock transformation method; according to the selectable marker on the transferred recombinant expression vector, the transformed yeast cells were coated with ampicillin or kana If the transformation is successful, the yeast cells will acquire antibiotic resistance or auxotrophic resistanc...

Embodiment 3

[0055] Example 3 Cellulase transgenic Saccharomyces cerevisiae 2

[0056] Vector pYEX-BX (7.1kb) (contains three selection markers Ampr, URA3 and leu2), phosphoglycerate kinase gene promoter (PGK P ) and terminator (PGK T ), the signal peptide coding sequence adopts the signal peptide sequence (XYNSEC) of the xylanase gene in T. reesei to construct the recombinant expression vector II, and its expression sequence frame is PGK P -XYNSEC-cbh1-PGK T -PGK P -XYNSEC-eg1-PGK T -PGK P -PGK P -GLUSEC-bglc-PGK T , named as pYEX-BX-PGK-EchBl; the constructed recombinant expression vector was transformed into Saccharomyces cerevisiae kdn-59 by electric shock transformation method; Cells are plated on medium containing ampicillin or kanamycin or on an auxotrophic solid medium plate. If the transformation is successful, the yeast cells will acquire antibiotic resistance or auxotrophic resistance. A single colony containing the recombinant will grow on the corresponding selective...

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PUM

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Abstract

The invention discloses a method of producing stalk ethanol by means of mixed fermentation of transgenetic yeast. The transgenetic yeast can respectively degrade cellulose and xylan of stalks to glucose and xylose and ferment glucose and xylose to ethanol without additionally providing cellulase and xylanase. Degradation of cellulose and xylan and fermentation are synchronously carried out. The method disclosed by the invention has the advantages that by means of transgenetic saccharomyces cerevisiae, transgenetic pichia stipitis and transgenetic candida shehatae, synchronous enzyme hydrolysis-fermentation of a stalk cellulose raw material is realized to produce ethanol without additionally adding cellulase and xylanase, thereby greatly lowering the production cost of stalk cellulose ethanol and promoting the progress of replacing petroleum fuel by the stalk cellulose ethanol.

Description

technical field [0001] The invention relates to the fields of fermentation engineering and genetic engineering, especially using transgenic Saccharomyces cerevisiae and transgenic Pichia stipitis and other mixed fermentations to degrade fibrous raw materials such as straw into glucose and xylose and then convert them into ethanol through a synchronous enzymatic hydrolysis-fermentation process According to the method, the recombinant Saccharomyces cerevisiae can secrete three kinds of cellulase, the transgenic Pichia stipitis and the transgenic Candida shohata can secrete three kinds of xylanase, and can produce ethanol by fermenting xylose. Moreover, the above-mentioned strains have been screened and domesticated, and have good tolerance to ethanol, acetic acid, and furfural. Background technique [0002] As fossil fuels turn from shortage to depletion, the energy crisis is a common problem faced by mankind. In June 2006, my country promulgated the "Interim Measures for the...

Claims

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Application Information

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IPC IPC(8): C12P39/00C12P7/06C12R1/84C12R1/865C12R1/72
CPCY02E50/10
Inventor 张媛媛刘均洪宿烽
Owner QINGDAO UNIV OF SCI & TECH
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