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

Nayak, Dipti; Metcalf, William W.

doi:10.1016/bs.mie.2018.10.012

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

Journal Article · Fri Nov 23 00:00:00 EST 2018 · Methods in Enzymology

DOI:https://doi.org/10.1016/bs.mie.2018.10.012· OSTI ID:1596707

Nayak, Dipti ^[1]; Metcalf, William W. ^[2]

Univ. of Illinois at Urbana-Champaign, IL (United States). Carl R. Woese Inst. for Genomic Biology and Dept. of Microbiology; University of Illinois at Urbana-Champaign
Univ. of Illinois at Urbana-Champaign, IL (United States). Carl R. Woese Inst. for Genomic Biology and Dept. of Microbiology

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. Yet, 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. Moreover, we 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.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of Illinois at Urbana-Champaign, IL (United States)

Sponsoring Organization:: Simons Foundation; USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). Chemical Sciences, Geosciences & Biosciences Division

Contributing Organization:: Carl R. Woese Institute

Grant/Contract Number:: FG02-02ER15296

OSTI ID:: 1596707

Journal Information:: Methods in Enzymology, Journal Name: Methods in Enzymology Vol. 613; ISSN 0076-6879

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Methyl (Alkyl)-Coenzyme M Reductases: Nickel F-430-Containing Enzymes Involved in Anaerobic Methane Formation and in Anaerobic Oxidation of Methane or of Short Chain Alkanes Thauer, Rudolf K. Biochemistry, Vol. 58, Issue 52 https://doi.org/10.1021/acs.biochem.9b00164	journal	April 2019
Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans Nayak, Dipti D.; Liu, Andi; Agrawal, Neha PLOS Biology, Vol. 18, Issue 2 https://doi.org/10.1371/journal.pbio.3000507	journal	February 2020

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59 BASIC BIOLOGICAL SCIENCES
Cas9
Genome editing
Methane
Methanogens
Methanosarcina
Methyl-coenzyme M reductase

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

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