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Unit cell fungus, engineering fungus prepared through motion ferment and application

A technology of Zymomonas and engineering bacteria, which is applied in the field of Zymomonas mobilis engineering bacteria, can solve the problems of ethanol production reduction and achieve the effect of convenient screening

Inactive Publication Date: 2005-03-30
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] When Zymomonas mobilis uses sucrose as a fermentative carbon source, by-product sorbitol leads to reduced ethanol production

Method used

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  • Unit cell fungus, engineering fungus prepared through motion ferment and application
  • Unit cell fungus, engineering fungus prepared through motion ferment and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Preparation of Zymomonas mobilis Engineering Bacteria

[0018] A. Culture of Zymomonas mobilis and DNA extraction

[0019] Zymomonas mobilis was cultured at 30 degrees Celsius for 48 hours in medium X (10% glucose, 0.5% yeast extract, 0.1% ammonium sulfate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, pH7.0), and then used Total DNA was extracted by CTAB method for subsequent genetic manipulation.

[0020] B. Cloning of partial fragments of the glucose-fructose oxidoreductase gene of Zymomonas mobilis XW101

[0021] Using SEQ ID NO: 2 (5'-AAAGCCGGCGAATCAGATAAC-3') and SEQ ID NO: 3 (5'-TGGAAGAACCAATGACGCCT-3') as primers, the glucose-fructose oxidoreductase gene of Zymomonas mobilis was amplified. Obtain the PCR product of XW101 glucose-fructose oxidoreductase gene, its size is 1477bp, sequence is as shown in SEQ ID NO: 1, and cloned on the pGEM-T easy vector, this recombinant plasmid is named pGFO15, after sequencing analysis, The homology ...

Embodiment 2

[0050] Example 2 Engineering strain XW1021 uses sucrose to ferment ethanol

[0051] 1. Culture conditions

[0052] 1.1 Medium composition

[0053] Sucrose 15%, yeast extract 0.5%, ammonium sulfate 0.1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, pH7.0

[0054] 1.2 Culture conditions

[0055] Incubate at 30°C for 70 hours

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Abstract

This invention relates to motion fermentation monocell engineering bacterium, its prepn. method and its application in fermentation for producing ethanol. In the prepn. of said engineering bacterium, the resistance gene of tetracycline is inserted into glucose-fructose oxide-erductase gene of motion fermentation monocell baterium XW101, endo zyme being used for digesting said recombination plasmid to form linear plasmid, after the transformation of motion fermentation monocell bacterium XW101, screening the strain having tetracycline resistance mutation. Carbon source is used as substrate, with this inventive engineering bacterium to produce ethanol with high ethanol prodn. yield and less by-produt-sorbicolan.

Description

technical field [0001] The invention relates to Zymomonas mobilis engineering bacteria, its preparation method, and its application in fermenting and producing ethanol. Background technique [0002] Zymomonas mobilis is the only anaerobic bacterium discovered so far that can ferment glucose, fructose and sucrose to produce ethanol through the Entner-Doudoroff (E-D) pathway. [0003] In the E-D metabolic pathway, glucose-6-phosphate dehydrogenase catalyzes glucose-6-phosphate to generate 6-phosphogluconolactone, and at the same time, reduces NAD to generate NADH. A highly catalytic lactonase rapidly hydrolyzes the lactone, and then 6-phosphogluconate dehydrogenase converts the hydrolyzed product 6-phosphogluconate into 2-keto-3-deoxy-6-phosphogluconate. 2-Keto-3-deoxy-6-phosphogluconate is a unique intermediate in the E-D pathway. Specific aldolases in turn cleave this compound into pyruvate and glyceraldehyde-3-phosphate, which are then converted to a second pyruvate molec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/09C12N15/53C12N15/63C12P7/06C12Q1/68
CPCY02E50/17Y02E50/10
Inventor 徐玉泉武志强陈明平淑珍陆伟林敏
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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