Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A.
- 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.
- Research Organization:
- Univ. of Illinois at Urbana-Champaign, IL (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division; Simons Foundation
- Contributing Organization:
- Carl R. Woese Institute
- Grant/Contract Number:
- FG02-02ER15296
- OSTI ID:
- 1596707
- Journal Information:
- Methods in Enzymology, Vol. 613; ISSN 0076-6879
- Publisher:
- ElsevierCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans
|
journal | February 2020 |
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
|
journal | April 2019 |
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