Engineering bacteria with motor fermented monad fructosan sucrase gene mutation construction and use thereof
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A technology of fructan sucrase and Zymomonas, which is applied in the direction of fermentation, bacteria, hydrolase, etc., can solve the problems affecting ethanol production and achieve the effect of improving efficiency
Inactive Publication Date: 2006-03-15
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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Problems solved by technology
Therefore, when it is necessary to use sucrose as a fermentation carbon source to prepare ethanol, the production of ethanol is affected due to the generation of by-product fructan
Method used
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Embodiment 1
[0023] The preparation of embodiment 1 Zymomonas mobilis engineering bacteria
[0024] A. Culture of Zymomonas mobilis and DNA extraction
[0025] Zymomonas mobilis in medium RM
[0026] (glucose 20g / l, yeast extract 10g / l, potassium dihydrogen phosphate 2g / l, pH 6.0) after culturing at 30 degrees Celsius for 48 hours, the CTAB method was used to extract the total DNA for future genetic manipulation.
[0027] Extraction of Bacterial Total DNA by CTAB Method
[0028] 1) Take a single colony and inoculate it in 5ml of the corresponding culture medium, and cultivate it at a suitable temperature. Depending on the growth rate of the bacteria, the incubation time can range from a few hours to a few days.
[0029] 2) Take 1.0ml of bacterial liquid in a 1.5ml centrifuge tube, centrifuge at 13,000rpm for 2min, and discard the supernatant.
[0030] 3) The pellet was resuspended in 1.0 ml of 0.85% NaCl.
[0031] 4) Centrifuge at 13,000 rpm for 2 minutes at room temperature, and disc...
Embodiment 2
[0086] Example 2 Engineering strain XW101 (pSST) utilizes sucrose to ferment ethanol
[0091] 30 degrees Celsius static culture for 70 hours
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Abstract
An engineering bacterium with levansucrase gene of kinesthetic zymomonas mutation, its construction and use are disclosed. The engineering bacterium is prepared by inserting tetracin resistance gene into levansucrase gene of kinesthetic zymomonas, inducing the obtained recombinant plasmid into kinesthetic zymomonas, screening mutant strain with tetracin resistance and obtaining target engineering bacterium. It has tetracin resistance and higher alcohol conversion rate.
Description
technical field [0001] The invention relates to an engineering bacterium with a mutated gene of Zymomonas mobilis fructan sucrase; the invention also relates to a construction method of the engineering bacterium and its use in fermentative production of ethanol. Background technique [0002] Zymomonas mobilis (Zymomonas mobilis) is the only Gram-negative facultative anorexia that uses glucose, fructose, and sucrose to produce ethanol through the deoxyketosugar (Entner-Doudoroff, E-D) pathway. oxygen bacteria. [0003] Zymomonas mobilis has three kinds of sucrose hydrolyzing enzymes: SacA (intracellular sucrose-hydrolyzing enzymes), SacC (extracellular sucrose-hydrolyzing enzymes) and SacB (extracellular levansucrase). Since there is no sucrose transporter system on the cell membrane of Zymomonas mobilis, the intracellular sucrose hydrolase SacA actually does not play a role in sucrose hydrolysis and fermentation. SacC can hydrolyze sucrose to generate monosaccharides (gluc...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N9/24C12N1/21C12P7/06
CPCY02E50/17Y02E50/10
Inventor 徐玉泉李淑英林敏陈明
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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