Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in Clostridium cellulovorans [electronic resource]

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Ngôn ngữ: eng

Ký hiệu phân loại: 543.7 Analytical chemistry

Thông tin xuất bản: Washington, D.C. : Oak Ridge, Tenn. : United States. Dept. of Energy. Office of Energy Efficiency and Renewable Energy ; Distributed by the Office of Scientific and Technical Information, U.S. Dept. of Energy, 2019

Mô tả vật lý: Size: p. 5391-5400 : , digital, PDF file.

Bộ sưu tập: Metadata

ID: 262805

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Chủ đề

Clostridium cellulovorans
capable of producing large amounts of acetate and butyrate from cellulose is a promising candidate for biofuels and biochemicals production from lignocellulosic biomass. However, the restriction modification (RM) systems of 
C. cellulovorans
hindered the application of existing shuttle plasmids for metabolic engineering of this organism. To overcome the hurdle of plasmid digestion by host, a new shuttle plasmid (pYL001) was developed to remove all restriction sites of two major RM systems of 
C. cellulovorans
, 
Cce

743
I and 
Cce

743
II. The pYL001 plasmid remained intact after challenge by 
C. cellulovorans
cell extract. Post-electroporation treatments and culturing conditions were also modified to improve cell growth and colony formation on agar plates. With the improvements, the pYL001 plasmid, without in-vivo methylation, was readily transformed into 
C. cellulovorans
with colonies of recombinant cells formed on agar plates within 24 h. Three pYL001-derived recombinant plasmids free of 
Cce

743
I/
Cce

743
II restriction sites, after synonymous mutation of the heterologous genes, were constructed and transformed into 
C. cellulovorans
. Functional expression of these genes was confirmed with butanol and ethanol production from glucose in batch fermentations by the transformants. In conclusion, the pYL001 plasmid and improved transformation method can facilitate further metabolic engineering of 
C. cellulovorans
for cellulosic butanol production.

1. 09 biomass fuels
2. Clostridium cellulovorans
3. Methylation
4. Plasmid
5. Restriction modification system

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Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in <em>Clostridium cellulovorans</em> [electronic resource]