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Method for obtaining biomass conversion related genes from anaerobic fermentation system

A technology for anaerobic microorganisms and biomass, applied in the fields of biotechnology and microorganisms, which can solve the problems of lack of phylogenetic evolution or taxonomic status, inability to obtain target microorganisms, and inability to obtain pure cultures.

Inactive Publication Date: 2011-08-10
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the isolation and culture conditions of most microorganisms in nature are not yet clear, the isolation and culture of pure bacteria is time-consuming and in most cases the target microorganisms cannot be obtained.
Therefore, for most environmental microorganisms, although their existence is known and some of their physiological functions are known, their pure cultures cannot be obtained, especially in anaerobic fermentation systems, more than 90% of microorganisms not only have no Pure cultures, and lack corresponding information on their phylogenetic or taxonomic status
The mining of these uncultivated microbial enzyme resources or gene resources has been unable to use traditional microbiological techniques that rely on pure culture

Method used

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  • Method for obtaining biomass conversion related genes from anaerobic fermentation system
  • Method for obtaining biomass conversion related genes from anaerobic fermentation system
  • Method for obtaining biomass conversion related genes from anaerobic fermentation system

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Experimental program
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Embodiment approach

[0066] As an embodiment of the present invention, the method of the present invention includes: (1) starting from the anaerobic fermentation system, especially starting from the stalk anaerobic fermentation system, extracting and purifying the metagenomic DNA of 30-48kb, after Ligation, packaging and transfection to establish the Fosmid metagenomic library; (2) using the hydrolysis circle color method to screen the Fosmid library for enzymes related to biomass conversion such as endoglucanase, exoglucanase, β- Positive clones of glucosidase and endoxylanase; (3) After the positive clones screened were mixed in equal proportions, the Fosmid DNA was extracted and sequenced by 454 sequencing, and the genes encoding the corresponding enzymes were compared with the existing database Compare to identify whether it is a new gene with less than 85% similarity to the protein sequence encoded by the reported gene, and at the same time obtain the (upstream and downstream) regulatory seque...

Embodiment 1

[0072] Example 1, Construction of Fosmid Metagenomic Library in Biogas Fermentation System

[0073] 1. Genomic DNA extraction

[0074] Take 8mL of biogas slurry (taken from a biogas digester with straw and pig manure as raw materials for anaerobic fermentation at 40°C), centrifuge at 8000g for 5min, take the precipitate and wash it with 20mL of PBS buffer (137mM NaCl, 2.7mMKCl, 1.5mM KH 2 PO 4 , 8.1 mM Na 2 HPO 4 , pH 7.4) and washed 3 times. Resuspend the pellet with 7.68mL DNA extraction buffer (100mM Tris-HCl (pH 8.0), 100mM EDTA, 100mM Na 3 PO 4 , 1.5M NaCl, 1% (w / v) CTAB). Add proteinase K (final concentration 1 mg / mL) and SDS (final concentration 1% (w / v)) and incubate at 55° C. for 20 minutes, and then incubate at 70° C. for 10 minutes. After the crude lysate was centrifuged at 17,000 g for 10 min, the supernatant was collected. The supernatant was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) and then once with chloroform:isoamyl alcohol (24:...

Embodiment 2

[0084] Example 2, Screening of Positive Clones Containing Genes of Enzymes Related to Biomass Conversion

[0085] Fosmid clones were replicated from 384-well plates to LB plates containing 0.5% (w / v) carboxymethylcellulose sodium (CMC-Na) (containing 12.5 μg / mL chloramphenicol) using 384-well plate inoculation pins. After incubation at 37°C for 20 hours, the cells were stained with Congo red. After pouring 0.2% (w / v) Congo red staining solution and staining for 30 minutes, wash with sodium chloride solution (1mol / L) at least twice. The endoglucanase-positive clones have obvious yellow hydrolysis circles on the side of the colonies. From the 281 preserved 384-well plates, select one of them (No. BF009) to screen endoglucanase according to the above method, and screen 2 endoglucanase positive clones with yellow hydrolysis circles, as figure 1 indicated by the middle arrow. Using this method, 341 positive clones were screened from the Fosmid library containing 100,000 clones. ...

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Abstract

The invention relates to a method for obtaining biomass conversion related genes from an anaerobic fermentation system. The method comprises the steps of extracting a metagenomic DNA (Deoxyribonucleic Acid) from an anaerobic microbial community, collecting DNA fragments, connecting the DNA fragments into a carrier, packaging and transfecting into cells to obtain cell clones carrying the DNA fragments; screening cell clones with biomass conversion characters from the obtained cell clones, wherein the clones carry biomass conversion related genes; and identifying or separating the biomass conversion related genes in the obtained clones with the biomass conversion characters. In the invention, without culturing of microbes in the anaerobic microbial community, the metagenomic DNA in the anaerobic microbial community is directly used for screening gene resources of biomass conversion related enzymes. The method is also specific to all microbes in the anaerobic fermentation community and has the advantages of culture deprivation, high pass and low cost.

Description

technical field [0001] The invention belongs to the field of biotechnology and microorganisms, and more specifically, the invention relates to a method for obtaining genes related to biomass transformation from an anaerobic fermentation system. Background technique [0002] In the biomass conversion process of the anaerobic fermentation system, there are mainly four stages: the first stage is the hydrolysis stage, and the macromolecular organic substances mainly composed of lignocellulose in the fermentation raw materials are hydrolyzed into their respective monomer substances; the second stage is the hydrolysis stage. The first stage is the acidification stage, and the monomer substances are further degraded into volatile fatty acids; the third stage is the acetogenic stage, and the fourth stage is the methanogenic stage. These four different stages require the participation of different microbial groups. These microbial groups have genes corresponding to their functions in...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 周志华严兴张珺耿阿蕾程林刘芳华魏勇军
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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