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Transformation of Clostridium thermocellum via Complete Methylome Analysis and Directed DNA Methylation
Published: October 01, 2019
Posted: August 03, 2021

Objectives

  • Transform Clostridium thermocellum.
  • C. thermocellum is an anaerobic, thermophilic bacterium that efficiently deconstructs lignocellulosic biomass, but cannot be genetically manipulated.
  • Test if directed methylation of DNA will protect it from C. thermocellum restriction-modification systems.
  • Characterize methylome of C. thermocellum genome.

Approach

  • Sequence C. thermocellum ATCC 27405 using PacBio single-molecule real-time (SMRT) sequencing to detect 6-methyladenine and 4-methylcytosine, and the rarely used whole-genome bisulfite sequencing to detect 5-methylcytosine.
  • Clone and express C. thermocellum ATCC 27405 DNA methyltransferases genes in E. coli.
  • Pass transformation DNA through Escherichia coli expressing methyltransferases.

Results/Impacts

  • No transformation was detected without methylation of DNA, but up to 80 transformants per μg DNA with methylation.
  • Specifically provides for transformation and genetic manipulation of this important microbe.
  • More broadly provides tools for genetic transformation and engineering of other non-model microbes.

Riley, L.A. et al. “Rational development of transformation in Clostridium thermocellum ATCC 27405 via complete methylome analysis and evasion of native restriction–modification systems.” Journal of Industrial Microbiology & Biotechnology 46(9–10), 1435–1443 (2019). [DOI:10.1007/s10295-019-02218-x]

Topic Areas:

  • Research Area: Genomic Analysis and Systems Biology
  • Research Area: Microbes and Communities
  • Research Area: DOE Bioenergy Research Centers (BRC)

Division: SC-33.2 Biological Systems Science Division, BER

 

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