Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A

Dipti D. Nayak, William W. Metcalf

Research output: Chapter in Book/Report/Conference proceedingChapter


Methanogenic archaea generate methane as a by-product of anaerobic respiration using CO2, C1 compounds (like methanol or methylated amines), or acetate as terminal electron acceptors. Methanogens are an untapped resource for biotechnological advances related to methane production as well as methane consumption. However, key biological features of these organisms remain poorly understood. One such feature is the enzyme methyl-coenzyme M reductase (referred to as MCR), which catalyzes the last step in the methanogenic pathway and results in methane formation. Gene essentiality has limited genetic analyses of MCR thus far. Therefore, studies of this important enzyme have been limited to biochemical and biophysical techniques that are especially laborious and often reliant on sophisticated instrumentation that is not commonly available. In this chapter, we outline our recently developed CRISPR–Cas9-based genome editing tools and describe how these tools have been used for the introduction of a tandem affinity purification tag at the chromosomal mcr locus in the model methanogen, Methanosarcina acetivorans C2A. We also report a protocol for rapid affinity purification of MCR from M. acetivorans C2A that will enable high-throughput studies of this enzyme in the future.

Original languageEnglish (US)
Title of host publicationEnzymes of Energy Technology
EditorsFraser Armstrong
PublisherAcademic Press Inc.
Number of pages23
ISBN (Print)9780128163610
StatePublished - 2018

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988


  • Cas9
  • Genome editing
  • Methane
  • Methanogens
  • Methanosarcina
  • Methyl-coenzyme M reductase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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