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

Abstract

Methanogenic archaea generate methane as a by-product of anaerobic respiration using CO 2 , C 1 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 publicationMethods in Enzymology
EditorsFraser Armstrong
PublisherAcademic Press Inc.
Pages325-347
Number of pages23
ISBN (Print)9780128163610
DOIs
StatePublished - 2018

Publication series

NameMethods in Enzymology
Volume613
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Fingerprint

Methanosarcina
Genetic Techniques
Methane
Methanogens
Purification
Enzymes
Genes
Enzymes and Coenzymes
Archaea
Carbon Monoxide
Amines
Byproducts
Methanol
Respiration
Acetates
Throughput
Electrons
methyl coenzyme M reductase

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Nayak, D. D., & Metcalf, W. W. (2018). Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A. In F. Armstrong (Ed.), Methods in Enzymology (pp. 325-347). (Methods in Enzymology; Vol. 613). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2018.10.012

Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A. / Nayak, Dipti D.; Metcalf, William W.

Methods in Enzymology. ed. / Fraser Armstrong. Academic Press Inc., 2018. p. 325-347 (Methods in Enzymology; Vol. 613).

Research output: Chapter in Book/Report/Conference proceedingChapter

Nayak, DD & Metcalf, WW 2018, Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A. in F Armstrong (ed.), Methods in Enzymology. Methods in Enzymology, vol. 613, Academic Press Inc., pp. 325-347. https://doi.org/10.1016/bs.mie.2018.10.012
Nayak DD, Metcalf WW. Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A. In Armstrong F, editor, Methods in Enzymology. Academic Press Inc. 2018. p. 325-347. (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2018.10.012
Nayak, Dipti D. ; Metcalf, William W. / Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A. Methods in Enzymology. editor / Fraser Armstrong. Academic Press Inc., 2018. pp. 325-347 (Methods in Enzymology).
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