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10/01/2019

Transformation of Clostridium thermocellum via Complete Methylome Analysis and Directed DNA Methylation

The Science

Clostridium thermocellum is an anaerobic, thermophilic bacterium that efficiently deconstructs lignocellulosic biomass, but cannot be genetically manipulated. The objective was to transform C. thermocellum and:

  • Test if directed methylation of DNA will protect it from C. thermocellum restriction-modification systems.
  • Characterize methylome of C. thermocellum genome.

Researchers approach was to:

  • 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.

The Impact

  • 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.

Multiple active systems were identified, and corresponding DNA methyltransferases were expressed from the Escherichia coli chromosome to mimic the C. thermocellum methylome. Plasmid methylation was experimentally validated and successfully electroporated into C. thermocellum ATCC 27405. This combined approach enabled genetic modification of the C. thermocellum-type strain and acts as a blueprint for transformation of other non-model microorganisms.

References

Riley, L. A., L. Ji, R. J. Schmitz, J. Westpheling, and A. M. Guss. 2019. “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), 1435–43. DOI:10.1007/s10295-019-02218-x.