Genetically engineered algae strain of synechocystisPCC6803 producing cellulase and construction method thereof

A technology of PCC6803 and cellulase, which is applied in the field of industrial microorganisms, can solve problems such as low utilization rate, unfavorable energy conversion, and limitation of bioenergy conversion efficiency and feasibility

Inactive Publication Date: 2020-01-14
TIANJIN UNIV OF SCI & TECH +1
View PDF7 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production methods are mostly produced by solid fermentation, but these enzyme production methods use organic carbon sources such as bran, corn stalks, rice straw and other organic carbon sources as raw materials for production by heterotrophic microorganisms, and a large amount of enzymes are consumed in the enzyme production process. Organic carbon source, which makes the efficiency of biomass conversion into bioenergy process and carbon utilization rate very low, which is unfavorable from the perspective of energy conversion, which seriously limits the efficiency and feasibility of using biomass for bioenergy conversion

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Genetically engineered algae strain of synechocystisPCC6803 producing cellulase and construction method thereof
  • Genetically engineered algae strain of synechocystisPCC6803 producing cellulase and construction method thereof
  • Genetically engineered algae strain of synechocystisPCC6803 producing cellulase and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Construction of homologous recombination plasmid P5ST1T2npt:

[0054] (1) Take SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing as upstream and downstream primers, and take the wild Synechocystis PCC6803 genome as a template; take SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing as the upper and lower primers Downstream primers, with Genbank accession number U02439.1 Escherichia coli as a template; with SEQ ID NO:5 and SEQ ID NO:6 in the sequence table as upstream and downstream primers, with wild Synechocystis PCC6803 as a template, amplified by PCR The technology obtained the photosensitive promoter and upstream arm gene sequence Promoter-up, the terminator T1T2 gene fragment and the downstream arm sequence downstream. The obtained sequence is shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 in the sequence listing;

[0055] (2) Perform ApaI digestion on the photosensitive promoter and upstream arm gene sequence Promoter-up obtained in step (1), and perf...

Embodiment 2

[0063] Construction of recombinant expression vector PSNCⅡ

[0064] Taking SEQ ID NO: 7 and SEQ ID NO: 8 in the sequence listing as upstream and downstream primers, and taking the wild Synechocystis PCC6803 genome as a template, the photosensitive promoter and upstream arm Promoter-up were obtained by PCR technology, and the primers were used in Promoter-up. The restriction site ApaI and homology arms were added at both ends of the up, and the obtained sequence was shown in SEQ ID NO: 5 in the sequence listing; SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing were used as upstream and downstream primers, and the GenBank database was used as the upstream and downstream primers. The corresponding gene sequence of Trichoderma reesei QM9414 is the template, and the cellulose exonuclease CBH II gene fragment is obtained by PCR technology, and the homology arm sequence is added at both ends of the target gene through primers, and the sequence is as SEQ ID in the sequence table. ...

Embodiment 3

[0070] Obtaining of Synechocystis sp. PCC6803 genetically engineered algal strain capable of producing cellulase.

[0071] (1) Plasmid transformation

[0072] The PSNCⅡ plasmid was sterilized by filtration through a 0.22 μm microporous membrane, and then put into a 2 mL sterile centrifuge tube. To this was added an amount of BG11 medium (with HEPES buffer added) so that the final plasmid concentration was about 10 ng / μL. Take 30 mL of wild-type PCC6803 in log phase, centrifuge at 6000 rpm for 7 min, and remove the supernatant. Resuspend the algal sludge with 20 mL of fresh BG11 medium, centrifuge at 6000 rpm for 7 min, and remove the supernatant. The algal slime was resuspended with plasmid-containing medium, and the resuspended algal liquid was cultured at 29°C, 150rpm, and 1400Lux continuous light for 6h. The algal fluid was coated on the solid medium covered with mixed fiber filter membranes for 1 day in light (upright culture), then the membranes were transferred to sol...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a genetically engineered algae strain of synechocystisPCC6803 producing cellulase and a construction method thereof, and belongs to the field of industrial microorganisms. According to the genetically engineered algae strain of synechocystisPCC6803 producing cellulase and the construction method thereof, through the method of homologous recombination, an exogenous cellulaseexcision enzyme CBHII gene is integrated into the genome of the synechocyst is PCC6803 to obtain an algae strain SPSNCII of the synechocystisPCC6803.Under the same culture condition, the activity ofthe produced cellulase is significantly better than that of the wild synechocystis strain. According to the genetically engineered algae strain of the synechocystisPCC6803 producing the cellulase andthe construction method thereof, a novel way of obtaining the cellulase by using inorganic salt and carbon dioxide through photosynthetic organism-synechocystis is opened up, the genetically engineered algae strain of the synechocystisPCC6803 producing the cellulase and the construction method thereof are of great significance to reducing the consumption of an organic carbon source in the processof enzyme production and reducing the release of the greenhouse gases in the process of producing the cellulase, and the application prospect is broad.

Description

technical field [0001] The invention relates to a genetically engineered algal strain of Synechocystis PCC6803 that produces cellulase and a construction method thereof, belonging to the field of industrial microorganisms. Background technique [0002] Cellulose is a polysaccharide composed of glucose with β-1,4 glycosidic bonds. It is an important component of plant cell walls and one of the most abundant renewable resources on the earth. With the depletion of fossil resources such as oil and coal, how to convert and utilize cellulose, a widely distributed, abundant and cheap renewable organic resource in nature, is a key issue restricting the utilization of cellulose and biomass. Using cellulose or biomass for biorefinery, it is first necessary to convert it into water-soluble small molecule oligosaccharides and monosaccharides, which can be further fermented for microbial production of ethanol or biodiesel. The use of biological enzymes to convert cellulose into soluble ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/66C12N9/42C12R1/01
CPCC12N9/2437C12N15/66C12N15/74C12Y302/01C12Y302/01004
Inventor 戴玉杰宁玉林邵强孟庆营赵潇潇陈高吕和鑫贾士儒钟成谭之磊
Owner TIANJIN UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap