Bacillus coli alcoholic fermentation engineering bacterium and uses thereof

A technology of Escherichia coli and ethanol fermentation, applied in the direction of fermentation, application, genetic engineering, etc., can solve problems such as inappropriateness, achieve the effect of reducing investment and increasing ethanol yield

Inactive Publication Date: 2009-05-13
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Bacillus coli alcoholic fermentation engineering bacterium and uses thereof

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Embodiment 1

[0023] Example 1 Preparation of ethanol-resistant Escherichia coli ethanol fermentation engineering bacteria

[0024] A. Screening of Escherichia coli ethanol-tolerant mutants

[0025] Escherichia coli JM109 was cultivated in LB medium, and after growing to the logarithmic phase, it was transferred to the LB medium culture containing 10g / L, 20g / L, 30g / L, 40g / L and 50g / L ethanol. um of filter membrane and join in the LB substratum), the ethanol concentration of 30g / L has obviously suppressed the growth of escherichia coli JM109. The JM109 grown in LB containing 30g / L ethanol was transferred through successive generations, and after each transfer three generations, it was applied to a solid LB medium containing isopropanol (which is not volatile and can maintain the selective pressure of ethanol) On the top, select a single colony that grows rapidly and continue to transfer.

[0026] Taking JM109 grown in ethanol-free LB medium as a control, calculate the number of colonies an...

Embodiment 2

[0071] Example 2 Fermentation of engineering strain ET1bc to produce ethanol

[0072] 1. Culture conditions

[0073] 1.1 Medium composition

[0074] Glucose 10%, yeast extract 0.5%, sodium chloride 1.0%, peptone 1.0%, phosphate buffer pH7.0;

[0075] Xylose 10%, yeast extract 0.5%, sodium chloride 1.0%, peptone 1.0%, phosphate buffer pH 7.0;

[0076] 1.2 Culture and fermentation conditions

[0077] Inoculate ET1bc into LB medium and culture to OD 600 =When around 0.2, add 1.0mM IPTG to induce, when ET1bc grows to OD 600 = 1.0, the bacteria were collected by centrifugation, inoculated into the fermentation medium described in 1.1, and made OD after inoculation 600 The value is about 1.0, anaerobic culture at 37 degrees Celsius.

[0078] 2. Determination of residual sugar in fermentation broth

[0079] Use high-performance liquid chromatography to detect the residual sugar content in the fermentation broth, and the measurement conditions are as follows, detector: Waters41...

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Abstract

The invention relates to a colon bacillus alcohol fermentation engineering bacterium resisting alcohol. A colon bacillus mutant strain highly resisting the alcohol is obtained through a plurality of generations of directional screening; and an alcohol dehydrogenase gene II and a pyruvate decarboxylase gene of zymomonas mobilis are transferred into the mutant strain to obtain the novel colon bacillus alcohol fermentation engineering bacterium. When the engineering bacterium uses pentose and hexose as substrates to ferment for producing the alcohol, the engineering bacterium can still maintain higher fermentation rate under high-concentration alcohol, and has higher transformation rate of the alcohol.

Description

technical field [0001] The invention relates to an ethanol-resistant Escherichia coli ethanol fermentation engineering bacterium and a preparation method thereof, and also relates to the application of the Escherichia coli ethanol fermentation engineering bacterium in fermentation production of ethanol. Background technique [0002] In crop straw and residues, the content of cellulose and hemicellulose can account for more than 75% of the dry weight. Fermenting crop straw to produce ethanol is a good way to obtain ethanol. [0003] Cellulose is mainly a straight-chain molecule composed of glucose molecules connected by β-1,4 glycosidic bonds; hemicellulose is an amorphous heterogeneous polysaccharide including xylose, arabinose, galactose, mannose, and glucose. polymer, and the content of xylose is the highest. [0004] Escherichia coli contains all the necessary enzymes to utilize six-carbon sugars and five-carbon sugars, and can ferment six-carbon sugars such as glucose a...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/60C12P7/06C12R1/19
CPCY02E50/17Y02E50/10
Inventor 陈明王占朝林敏张维陆伟平淑珍
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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