Post‐translational modifications in the active site region of methyl‐coenzyme M reductase from methanogenic and methanotrophic archaea

Kahnt, Jörg; Buchenau, Bärbel; Mahlert, Felix; Krüger, Martin; Shima, Seigo; Thauer, Rudolf K.

doi:10.1111/j.1742-4658.2007.06016.x

Cited by 66 publications

(102 citation statements)

References 41 publications

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“…Most notably, multiplexed gene-editing plasmids will enable generation of strains with multiple mutations, ranging from SNPs to large indels, in a matter of weeks versus years. These tools will enable researchers to swiftly tag genes at their native loci on the host chromosome, allowing the study of context-specific gene expression, "pull-down" experiments to establish protein-protein and protein-DNA interaction networks, and purification of proteins that contain unique amino acids (27) or novel posttranslational modifications (28). Furthermore, deleting a gene of interest using the NHEJ-based technique is very cost-effective, as it simply requires the insertion of a commercially synthesized DNA fragment containing the appropriate sgRNAs into pDN243, the vector containing Cas9 and the NHEJ machinery.…”

Section: Discussionmentioning

confidence: 99%

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Nayak

Metcalf

2017

Proc. Natl. Acad. Sci. U.S.A.

121

112

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Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methanogenic archaeon Methanosarcina acetivorans. Introduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the nonhomologous end-joining (NHEJ) machinery from the closely related archaeon, Methanocella paludicola, allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJdependent mutations included deletions ranging from 75 to 2.7 kb in length, most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homologydirected repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.he CRISPR (clustered regularly interspaced palindromic repeats) array and associated cas genes are widespread in microbial genomes (1), where they confer acquired immunity to phage and foreign DNA elements (2). The type IIA system from Streptococcus pyogenes is especially well characterized and has been widely applied as a remarkably effective genome-editing tool (3). During genome editing, heterologous expression of the RNA-guided DNA endonuclease Cas9 and a chimeric singleguide (sg) RNA, comprised of a 20-bp spacer that targets the chromosome and an 80-bp scaffold that binds Cas9, leads to a lethal double-strand break (DSB) at all target sites within the genome that are flanked by a 3′ NGG protospacer adjacent motif (PAM) (4) (Fig. S1). In eukaryotes, the nonhomologous endjoining (NHEJ) repair pathway can mend the DSB by generating simple insertions or deletions at the sgRNA target site, thus preventing additional rounds of Cas9-mediated cleavage (3, 5). Alternatively, the native homology-dependent repair (HDR) pathway can repair the fatal DSB, so long as a repair template that modifies or removes the sgRNA target site is provided, again preventing additional rounds of Cas9-mediated cleavage (3, 5) (Fig. S1). Appropriately ...

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Section: Discussionmentioning

confidence: 99%

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Nayak

Metcalf

2017

Proc. Natl. Acad. Sci. U.S.A.

121

112

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“…However, this approach for analysis of in situ function must be used with caution. MR specific activity is strongly temperature dependent (15), regulation of process rate may require transcription of several genes (25,58), and ratios for natural ecosystems may not be directly comparable with those for pure cultures. Also, the approximation of in situ physiological MR activity and cell content requires detailed information, from pure-culture studies of representative acidophilic temperate peat methanogens (4), on mRNA transcript abundances and related enzyme activities under natural conditions.…”

Section: Discussionmentioning

confidence: 99%

Correlation of Methane Production and Functional Gene Transcriptional Activity in a Peat Soil

Freitag

Prosser

2009

Appl Environ Microbiol

150

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The transcription dynamics of subunit A of the key gene in methanogenesis (methyl coenzyme M reductase; mcrA) was studied to evaluate the relationship between process rate (methanogenesis) and gene transcription dynamics in a peat soil ecosystem. Soil methanogen process rates were determined during incubation of peat slurries at temperatures from 4 to 37°C, and real-time quantitative PCR was applied to quantify the abundances of mcrA genes and transcripts; corresponding transcriptional dynamics were calculated from mcrA transcript/gene ratios. Internal standards suggested unbiased recovery of mRNA abundances in comparison to DNA levels. In comparison to those in pure-culture studies, mcrA transcript/gene ratios indicated underestimation by 1 order of magnitude, possibly due to high proportions of inactive or dead methanogens. Methane production rates were temperature dependent, with maxima at 25°C, but changes in abundance and transcription of the mcrA gene showed no correlation with temperature. However, mcrA transcript/gene ratios correlated weakly (regression coefficient ‫؍‬ 0.76) with rates of methanogenesis. Methanogen process rates increased over 3 orders of magnitude, while the corresponding maximum transcript/gene ratio increase was only 18-fold. mcrA transcript dynamics suggested steady-state expression in peat soil after incubation for 24 and 48 h, similar to that in stationary-phase cultures. mcrA transcript/gene ratios are therefore potential in situ indicators of methanogen process rate changes in complex soil systems.

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“…43 Additionally, the crystal structure of MCR from ANME group 1 has been determined (Figure 6) and differs from the methanogen MCR in changes to the F 430 cofactor, the presence of a cysteine-rich patch near the F 430 cofactor, and an altered pattern of post-translational modifications. 156,157 It is unclear which, if any, of these modifications are critical for reversing MCR chemistry or even how well these modifications are conserved across ANME MCR. Despite the many lines of evidence pointing to ANME MCR as the key enzyme for activating methane in AOM, it has yet to be isolated in an active form.…”

Section: Anaerobic Methane Oxidation and Methyl-coenzyme M Reductasementioning

confidence: 99%

Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion

2016

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Biological conversion of natural gas to liquids (Bio-GTL) represents an immense economic opportunity. In nature, aerobic methanotrophic bacteria and anaerobic archaea are able to selectively oxidize methane using methane monooxygenase (MMO) and methyl coenzyme M reductase (MCR) enzymes. Although significant progress has been made toward genetically manipulating these organisms for biotechnological applications, the enzymes themselves are slow, complex, and not recombinantly tractable in traditional industrial hosts. With turnover numbers of 0.16–13 s−1, these enzymes pose a considerable upstream problem in the biological production of fuels or chemicals from methane. Methane oxidation enzymes will need to be engineered to be faster to enable high volumetric productivities; however, efforts to do so and to engineer simpler enzymes have been minimally successful. Moreover, known methane-oxidizing enzymes have different expression levels, carbon and energy efficiencies, require auxiliary systems for biosynthesis and function, and vary considerably in terms of complexity and reductant requirements. The pros and cons of using each methane-oxidizing enzyme for Bio-GTL are considered in detail. The future for these enzymes is bright, but a renewed focus on studying them will be critical to the successful development of biological processes that utilize methane as a feedstock.

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Post‐translational modifications in the active site region of methyl‐coenzyme M reductase from methanogenic and methanotrophic archaea

Cited by 66 publications

References 41 publications

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Correlation of Methane Production and Functional Gene Transcriptional Activity in a Peat Soil

Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion

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