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Genetically engineered bacterium and applications thereof

A technology of genetically engineered bacteria and genes, applied in genetic engineering, application, plant genetic improvement, etc., can solve the problems of low endogenous expression of soluble polysaccharide monooxygenase and secretion capacity of soluble polysaccharide monooxygenase, etc. Achieve the effect of increasing production, increasing degradation speed and degree, and high glucose release

Inactive Publication Date: 2015-05-06
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first object of the present invention is to overcome the defect of low endogenous expression of soluble polysaccharide monooxygenase with CBM module in the prior art and provide a soluble polysaccharide monooxygenase with CBM module secretion ability High genetically engineered bacteria

Method used

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  • Genetically engineered bacterium and applications thereof
  • Genetically engineered bacterium and applications thereof
  • Genetically engineered bacterium and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] This example is used to illustrate the method for constructing genetically engineered bacteria provided by the present invention.

[0077] Take Phanerochaetechrysosporium hyphae, absorb water on filter paper, grind with liquid nitrogen, add GenStar's RNA extraction reagent Trizol by volume: mass ratio 1:1, shake for 5 minutes with a shaker, and stand at room temperature for 1 minute; Volume: mass ratio 0.2:1 add chloroform, shake for 15s, let stand for 2min, centrifuge at 4℃, 12000rpm for 15min; aspirate the supernatant, add isopropanol according to volume ratio 1:1, precipitate at -20℃ for 30min; at 4℃, Centrifuge at 12000rpm for 15min, discard the supernatant, wash the pellet with 1ml of 75% ethanol, centrifuge at 4℃, 7500rpm for 5min, discard the supernatant, and dry for 10min; add DEPC water to dissolve to obtain total RNA, use reverse transcription kit to remove the total RNA Reverse transcription into cDNA; use the sequence shown in SEQ ID NO: 3 as the upstream prime...

Embodiment 2

[0085] This example is used to illustrate the secretion ability of soluble polysaccharide monooxygenase with CBM module obtained by the method for constructing genetically engineered bacteria provided by the present invention.

[0086] The genetically engineered single colony of Pichia pastoris obtained in Example 1 was selected and cultured in BMGY medium at 28°C and 150 rpm to OD 600 =2-6; collect the bacteria, resuspend the bacteria with BMMY to make OD 600 =1.0, add 100% methanol to the culture medium to a final concentration of 1.0%; add methanol every 24 hours to a final concentration of 1.0%, and end the induction after 24 hours. SDS-PAGE electrophoresis detects the fermentation broth supernatant after 72 hours of induction.

[0087] BMGY medium formula: 10g / L yeast powder, 20g / L peptone, 100mM pH 6.0 potassium phosphate, 13.4g / L YNB, 4×10 6 g / L biotin, l0g / L glycerol.

[0088] BMMY medium formula: 10g / L yeast powder, 20g / L peptone, 100mM pH 6.0 potassium phosphate, 13.4g / L YNB...

Embodiment 3

[0091] This example is used to illustrate the application of the genetically engineered bacteria provided by the present invention in the degradation of cellulose materials.

[0092] Add 2g of cellulase and 0.1g of the soluble polysaccharide monooxygenase 10320 obtained in Example 2 to 20g of microcrystalline cellulose as a substrate, react at 50°C for 72 hours, and release glucose up to 11.5g / L. Compared with a single addition of cellulase (Cellulase "Onozuka" R-10), the glucose release increased by 30% ( image 3 ).

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Abstract

The invention relates to a genetically engineered bacterium which comprises a host cell and a target gene transferred into the host cell, and the genetically engineered bacterium is characterized in that the target gene is an encoding gene of phanerochaete chrysosporium LPMO (lytic polysaccharide monooxygenases). The invention also discloses a construction method of the genetically engineered bacterium and an application of the genetically engineered bacterium in degrading raw materials such as celluloses. The genetically engineered bacterium provided by the invention has a high CBM module containing LPMO (LPMO10320) secretion capacity, and the secretion capacity can reach 1.39mg / ml; and when the genetically engineered bacterium is applied to the degradation treatment of raw materials such as celluloses, the degradation velocity and degree of the celluloses can be improved, the generation amount of glucose can be increased, and compared with endogenously expressed phanerochaete chrysosporium, the generation amount is increased by nearly 100 times.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a genetically engineered bacteria and its application. Background technique [0002] In most cases, to make high-value utilization of cellulose, it must first be effectively degraded by a variety of different cellulase enzymes. Most of the current commercial operations are combining 15-20 different enzymes to make enzyme preparations, including three main cellulase enzymes: endoglucanase, exoglucanase (cellulase two Glycohydrolase) and β-glucosidase. The endoglucanase first randomly decomposes glycosidic bonds from the inside of the cellulose molecule to produce non-reducing ends, then cellobiohydrolase binds to them, and hydrolyzes the non-reducing ends to produce cellobiose, which then becomes β -Glucosidase decomposes the substrate to be degraded into fermentable glucose. In addition to the main cellulase, commercial enzyme preparations will also include some auxiliary enzymes to en...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/53C12P19/14C12P19/02C12R1/865C12R1/84C12R1/78
CPCC12N9/0071C12P19/02C12P19/14
Inventor 袁红莉朱宁杨金水
Owner CHINA AGRI UNIV
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