A Shuttle-Vector System Allows Heterologous Gene Expression in the Thermophilic Methanogen Methanothermobacter thermautotrophicus ΔH

Fink, Christian; Beblawy, Sebastian; Enkerlin, Andreas M; Mühling, Lucas; Angenent, Largus T.; Molitor, Bastian

doi:10.1128/mbio.02766-21

Cited by 29 publications

(57 citation statements)

References 51 publications

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“…Thus, developing genetic manipulation tools for these organisms has been a major area of interest. Very recently, the first reliable system for M. thermautotrophicus ΔH was reported ( Fink et al, 2021 ). Methanothermobacter thermautotrophicus ΔH was found to be amenable to plating on solid medium with good efficiencies, and neomycin was shown to be effective for selection.…”

Section: Methanogens As Hosts For the Heterologous Production Of Reco...mentioning

confidence: 99%

“…Methanothermobacter thermautotrophicus ΔH was found to be amenable to plating on solid medium with good efficiencies, and neomycin was shown to be effective for selection. This allowed for the construction of a shuttle vector containing a full array of cloning sites, selections markers for E. coli and M. thermautotrophicus ΔH, and the β-galactosidase-encoding gene bgaB, which can be used as a reporter ( Fink et al, 2021 ). Transformation of M. thermautotrophicus ΔH is possible via interdomain conjugation with E. coli S17-1.…”

Section: Methanogens As Hosts For the Heterologous Production Of Reco...mentioning

confidence: 99%

“…Transformation of M. thermautotrophicus ΔH is possible via interdomain conjugation with E. coli S17-1. As a proof-of-concept, this system was utilized to heterologously express formate dehydrogenase, thus enabling M. thermautotrophicus ΔH to grow with formate as a substrate ( Fink et al, 2021 ). This genetic system could potentially be very useful for the future heterologous expression of MCRs since established methods for MCR activation in M. marburgensis (see section below) would likely also be effective for MCRs isolated from M. thermautotrophicus ΔH.…”

Section: Methanogens As Hosts For the Heterologous Production Of Reco...mentioning

confidence: 99%

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Overview of Diverse Methyl/Alkyl-Coenzyme M Reductases and Considerations for Their Potential Heterologous Expression

Gendron

Allen

2022

Front. Microbiol.

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Methyl-coenzyme M reductase (MCR) is an archaeal enzyme that catalyzes the final step of methanogenesis and the first step in the anaerobic oxidation of methane, the energy metabolisms of methanogens and anaerobic methanotrophs (ANME), respectively. Variants of MCR, known as alkyl-coenzyme M reductases, are involved in the anaerobic oxidation of short-chain alkanes including ethane, propane, and butane as well as the catabolism of long-chain alkanes from oil reservoirs. MCR is a dimer of heterotrimers (encoded by mcrABG) and requires the nickel-containing tetrapyrrole prosthetic group known as coenzyme F430. MCR houses a series of unusual post-translational modifications within its active site whose identities vary depending on the organism and whose functions remain unclear. Methanogenic MCRs are encoded in a highly conserved mcrBDCGA gene cluster, which encodes two accessory proteins, McrD and McrC, that are believed to be involved in the assembly and activation of MCR, respectively. The requirement of a unique and complex coenzyme, various unusual post-translational modifications, and many remaining questions surrounding assembly and activation of MCR largely limit in vitro experiments to native enzymes with recombinant methods only recently appearing. Production of MCRs in a heterologous host is an important step toward developing optimized biocatalytic systems for methane production as well as for bioconversion of methane and other alkanes into value-added compounds. This review will first summarize MCR catalysis and structure, followed by a discussion of advances and challenges related to the production of diverse MCRs in a heterologous host.

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Section: Methanogens As Hosts For the Heterologous Production Of Reco...mentioning

confidence: 99%

Section: Methanogens As Hosts For the Heterologous Production Of Reco...mentioning

confidence: 99%

Section: Methanogens As Hosts For the Heterologous Production Of Reco...mentioning

confidence: 99%

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Overview of Diverse Methyl/Alkyl-Coenzyme M Reductases and Considerations for Their Potential Heterologous Expression

Gendron

Allen

2022

Front. Microbiol.

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“…We dried the plates for two hours in the anaerobic chamber. Afterwards, M. thermautotrophicus ΔH cell suspension could be plated on the surface 23 . We incubated solidified media plates in pressurized stainless-steel jars (Raff und Grund, Freiberg, Germany) with an H 2 /CO 2 (80/20 volume%) headspace at 60°C.…”

Section: Microbial Strains Media and Cultivation Conditionsmentioning

confidence: 99%

“…We performed the transformation of M. thermautotrophicus ΔH with the suicide-vector construct as described before 23 . However, for plating of M. thermautotrophicus ΔH deletion mutants we applied 100 µg/mL of neomycin instead of 250 µg/mL, which we used for liquid selective media.…”

Section: Thermautotrophicus δHmentioning

confidence: 99%

The targeted deletion of genes responsible for expression of the Mth60 fimbriae leads to loss of cell-cell connections in M. thermautotrophicus ΔH

Fink

Martinez-Cano

Shuster

et al. 2022

Preprint

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This study was continued by the Environmental Biotechnology Group of the University of Tübingen in memoriam to Reinhard Wirth, who initiated the work on Mth60 fimbriae at the University of Regensburg.Growth in biofilms or biofilm-like structures is the prevailing lifestyle for most microbes in nature. The first step to initiate biofilms is the adherence of microbes to biotic and abiotic surfaces. Therefore, it is important to elucidate the initial step of biofilm formation, which is generally established through cell-surface structures (i.e., cell appendages), such as fimbriae or pili, that adhere to surfaces. The Mth60 fimbriae of Methanothermobacter thermautotrophicus ΔH are one of only few known archaeal cell appendages that do not assemble via the type-IV assembly mechanism. Here, we report the constitutive expression of Mth60 fimbriae-encoding genes from a shuttle-vector construct, as well as the deletion of the Mth60 fimbriae-encoding genes from the genomic DNA of M. thermautotrophicus ΔH. We expanded our system for genetic modification of M. thermautotrophicus ΔH by an allelic-exchange method. While overexpression of the respective genes resulted in an increase of the Mth60 fimbriae, deletion of the Mth60 fimbriae-encoding genes led to a loss of Mth60 fimbriae in planktonic cells of M. thermautotrophicus ΔH. This either increased or decreased number of Mth60 fimbriae correlated with a significant increase or decrease of biotic cell-cell connections in the respective M. thermautotrophicus ΔH strains compared to the wild-type strain.Originality-Significance StatementMethanothermobacter spp. have been studied for the biochemistry of hydrogenotrophic methanogenesis for many years. However, due to the lack of genetic tools, the detailed investigation of certain aspects, such as regulatory processes, was not possible. Here, we amend our genetic toolbox for M. thermautotrophicus ΔH with an allelic exchange method. We report the deletion of genes that encode for the Mth60 fimbriae. Our findings provide a first insight into the regulation of the expression of these genes and reveal a role of the Mth60 fimbriae in the formation of cell-cell connections of M. thermautotrophicus ΔH.

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Methanothermobacter thermautotrophicus and Alternative Methanogens: Archaea-Based Production

Mühling,

Baur,

Molitor

2024

Advances in Biochemical Engineering/Biotechnology

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A Shuttle-Vector System Allows Heterologous Gene Expression in the Thermophilic Methanogen Methanothermobacter thermautotrophicus ΔH

Abstract: The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change.

Cited by 29 publications

References 51 publications

Overview of Diverse Methyl/Alkyl-Coenzyme M Reductases and Considerations for Their Potential Heterologous Expression

Overview of Diverse Methyl/Alkyl-Coenzyme M Reductases and Considerations for Their Potential Heterologous Expression

The targeted deletion of genes responsible for expression of the Mth60 fimbriae leads to loss of cell-cell connections in M. thermautotrophicus ΔH

Methanothermobacter thermautotrophicus and Alternative Methanogens: Archaea-Based Production

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