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Establishment of gene recombination mobile fermentation monad applied to alcoholic fermentation

A Zymomonas and structural gene technology, applied in the field of producing ethanol from pentose sugars by Zymomonas mobilis, can solve the problem that there are not many metabolic pathways to choose from

Inactive Publication Date: 2009-10-28
WUHAN CNZYME BIOTECH
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Compared with other microorganisms, the metabolic pathway of this bacterium is relatively simple, and there are not many alternative metabolic pathways

Method used

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  • Establishment of gene recombination mobile fermentation monad applied to alcoholic fermentation
  • Establishment of gene recombination mobile fermentation monad applied to alcoholic fermentation
  • Establishment of gene recombination mobile fermentation monad applied to alcoholic fermentation

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Embodiment Construction

[0015] In the process of constructing an operon that can be expressed in Zymomonas mobilis, bacterial culture, extraction of bacterial chromosomal DNA, recovery and purification of DNA fragments, extraction of plasmid DNA, PCR, DNA digestion, ligation, transformation, etc. refer to "Molecular Cloning Experiment Guide (Third Edition)" (.J. Sambrook, D.W. Russell, author; Huang Peitang et al. Translated by Beijing, Science Press.).

[0016] Specific implementation method one: cloning of the promoter gene (the schematic diagram of the construction process, see Figure 1)

[0017] 1. Cloning of a promoter that can be recognized by Zymomonas mobilis (taking the promoter gap as an example):

[0018] ①Zymomonas mobilis was treated with 1% yeast extract, 0.2% potassium dihydrogen phosphate, 5% glucose, 30°C, CO 2 environment, shake slowly to mix. Grow 5 ml of the bacterial culture to saturation. Centrifuge 1.5 mL of the culture for 2 minutes. Add 500 μL of TE buffer to the precipit...

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Abstract

The invention relates to a method for applying establishment of gene recombination mobile fermentation monad to alcoholic fermentation. The microorganism is obtained by leading an operon capable of expressing in the mobile fermentation monad, wherein the operon consists of a sequence for coding a promoter and a sequence for coding a structure gene and comprises a sequence coding at least one promoter, and the sequence of the promoter can be identified by the mobile fermentation monad and regulates expression of at least more than one structural gene. The operon comprises a sequence for coding at least one heat shock protein HSP and is used for enhancing the survivability of the mobile fermentation monad in an adverse environment and increasing the temperature of alcoholic fermentation; the operon comprises a sequence for coding at least one lysine anabolism enzyme yfdz so that the mobile fermentation monad can automatically synthesize lysine; the operon comprises a sequence for coding at least one methionine anabolism enzyme metB so that the mobile fermentation monad can automatically synthesize methionine used for lowering the nutrition requirement of the mobile fermentation monad for cultures; and the operon also comprises a sequence for coding a structural gene, which ensures that the mobile fermentation monad can utilize at least one pentaose to generate. The operon enters the mobile fermentation monad in a plasmid transforming way or integrated with the genome of the mobile fermentation monad to obtain a transporton recombinant in a transporton way. The mobile fermentation monad obtained by gene engineering enhances the survivability in an adverse environment, can be fermented to generate the alcohol under higher temperature, obviously lowers the nutrition requirement for the cultures, can utilize the pentaoses to generate the alcohol and still stably retains the characteristics after transfer of culture for 200 times.

Description

technical field [0001] The present invention relates to the biotransformation of cellulosic substances into fuels and chemical substances, in particular to improving the resistance of Zymomonas mobilis to various adversities, increasing its fermentation temperature, and reducing the nutritional requirements for its growth, and at the same time, it also relates to Zymomonas mobilis Bacteria use pentose sugars to produce ethanol. Background technique [0002] The invention relates to a method for genetically engineering Zymomonas mobilis, which is used to increase its resistance to various adversities, increase its fermentation temperature, and reduce its nutritional requirements for growth, while also enabling Zymomonas mobilis to utilize Pentose sugars produce ethanol. [0003] Under high temperature conditions, the catabolism ability of the cells is enhanced, the fermentation time is shortened, and the production capacity is increased; the oxygen solubility is reduced; the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N1/21C12P7/06C12R1/01
CPCY02E50/17Y02E50/10
Inventor 汪浩勇
Owner WUHAN CNZYME BIOTECH
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