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

Bao, Teng; Zhao, Jingbo; Zhang, Qianxia; Yang, Shang-Tian

doi:10.1007/s00253-019-09882-0

Title: Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in Clostridium cellulovorans

Journal Article · Tue May 21 00:00:00 EDT 2019 · Applied Microbiology and Biotechnology

DOI:https://doi.org/10.1007/s00253-019-09882-0· OSTI ID:1572189

Bao, Teng ^[1]; Zhao, Jingbo ^[1]; Zhang, Qianxia ^[1];

^[1]

The Ohio State Univ., Columbus, OH (United States)

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, Cce₇₄₃I and Cce₇₄₃II. 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 Cce₇₄₃I/Cce₇₄₃II 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.

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Research Organization:: The Ohio State Univ., Columbus, OH (United States)

Sponsoring Organization:: USDOE Office of Energy Efficiency and Renewable Energy (EERE)

Grant/Contract Number:: EE0007005

OSTI ID:: 1572189

Journal Information:: Applied Microbiology and Biotechnology, Vol. 103, Issue 13; ISSN 0175-7598

Publisher:: SpringerCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 3 works

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Title: Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in Clostridium cellulovorans

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