Biology subjects

Scheller, S.

Publications and source records attributed to Scheller, S..

5 recordsLinked to original sources

bioRxiv2024

Methane and ethane production rates by methyl-coenzyme M reductase in cell extracts from different methanogens

The enzyme methyl-coenzyme M reductase (Mcr) is responsible for most of the biologically produced methane. Mcr catalyzes the reversible conversion of methyl-coenzyme M and coenzyme B to methane and the corresponding heterodisulfide CoM-S-S-CoB as the last step in all methanogens. In anaerobic methanotrophs, it catalyzes the same reaction to proceed in the reverse direction. While Mcr has been historically considered as an enzyme exclusive to the one-carbon metabolism, recent studies have demonstrated that homologs of Mcr have evolved to catalyze the first step of the archaeal anaerobic oxidation of medium and long-chain alkanes to their corresponding alkyl-coenzyme M thioethers. While ample metagenomic studies and data related to Mcr is available, in vitro experiments are limited, because purifying Mcr in its active nickel-I form is challenging. Fully active enzyme has only been reported for Methanothermobacter marburgensis isoenzyme I, from which most of the enzyme studies have been performed. To get an overview of the substrate scope of different Mcr variants, we tested cell-free lysates from the methanogens Methanosarcina mazei, Methanosarcina acetivorans, Methanococcus maripaludis, Methanothermococcus okinawensis, and M. marburgensis for their rates to convert methyl- and ethyl-coenzyme M to methane and to ethane, respectively. Cell extracts from M. mazei showed an ethane production rate of ca. 8% relative to methane production at 37 {degrees}C, and about 14% at 49 {degrees}C in an assay relying on titanium (III) citrate and cobalamin to regenerate coenzyme B from the CoM-S-S-CoB heterodisulfide. For cell extracts of M. marburgensis, we found an ethane-to-methane production rate of 8%, which is substantially higher than the reported value of ca. 0.3% for the ratio of their maximal catalytic rate for purified isoenzyme I. Since M. marburgensis is known to express two isoenzymes depending on the growth conditions, we hypothesize that isoenzyme II is substantially more promiscuous towards ethane formation than the well-described isoenzyme I. Which it is still challenging to obtain accurate kinetic parameters of the Mcr-catalyzed reaction, our experiments demonstrate that Mcr activity can be quickly and conveniently studied via cell-free lysates, and that substrate promiscuity towards ethane formation is generally larger than anticipated.

biochemistry↗

bioRxiv2024

Nature AND Nurture: Enabling formate-dependent growth in Methanosarcina acetivorans

Methanogenic archaea are crucial in global carbon cycling as around 1 Gt of the potent greenhouse gas, methane, is produced annually. Major contributors belong to the order Methanosarcinales, which contain some of the most versatile methanogens that are capable of acetotrophic, methylotrophic and CO2-reducing methanogenesis. The genetically tractable model methanogen, Methanosarcina acetivorans, by its nature shows versatility in substrate utilization and energy conservation pathways but cannot utilize formate. In this study, we expanded the primary metabolism of M. acetivorans to include formate-dependent methanogenesis. By introducing an exogenous formate dehydrogenase, the two metabolically engineered M. acetivorans strains acquired the capacity for formate-dependent methanogenesis pathways with one capable of formate-dependent methyl-reduction and the other capable of formate-dependent CO2-reduction. Through nurturing the strain capable of CO2-reduction with adaptive laboratory evolution, we were able to enable growth and methanogenesis of M. acetivorans solely on formate, a metabolism only reported in methanogens without cytochromes which are limited by their versatility. M. acetivorans also showed acetogenic potential where the formate-dependent CO2-reducing strain was able to divert {approx} 10% of carbon to acetate instead of methane. Our results show that even though M. acetivorans lacks energy converting hydrogenase and cannot use H2, it has yet-uncharacterized capacity to obtain reduced ferredoxins from oxidizing formate. Our work encourages reevaluation of our understanding of formate utilization in Methanosarcinales. By enabling formate-dependent methanogenesis, we have expanded the substrate spectrum of a versatile model methanogen with cytochromes to include formate as well.

microbiology↗

bioRxiv2023

CRISPR/Cas12a toolbox for genomic manipulation in Methanosarcina acetivorans

Methanogenic archaea play an important role in the global carbon cycle and are regarded as promising host organisms for the biotechnological generation of fuels and chemicals from one-carbon substrates. Methanosarcina acetivorans is extensively studied as a model methanogen due to the availability of genetic tools and its versatile substrate range. Although genome editing in M. acetivorans via CRISPR/Cas9 has already been demonstrated, we now describe a user-friendly CRISPR/Cas12a toolbox that recognizes a T-rich (5'-TTTV) PAM sequence. This new system can manage deletions of 3500 bp (i.e., knockout of the entire frhADGB operon) and heterologous gene insertions with 80% efficiency observed in ten PurR transformants. Our CRISPR/Cas12a system also enables multiplex genome editing at high efficiency, which helps speed up genetic engineering. Deletions of 100 bp generated on two separate sites of the genome yielded 8/8 correctly edited transformants. Simultaneous gene deletion (100 bp) and replacement (100-bp region replaced by the 2400-bp uidA expression cassette) at a separate site was achieved, with 3/6 of transformants being edited correctly. In combination with the Cas9-based system, our CRISPR/Cas12a toolbox enables targeted genome editing at two sites (guanine-rich and thymine-rich, respectively) and, in so doing, hastens the overall genetic engineering of the Methanosarcinales species.

molecular biology↗

bioRxiv2022

Application of the fluorescence-activating and absorption-shifting tag (FAST) for flow cytometry in methanogenic archaea.

Methane-producing archaea play a crucial role in the global carbon cycle and are used for biotechnological fuel production. Methanogenic model organisms such as Methanococcus maripaludis and Methanosarcina acetivorans are biochemically characterized and can be genetically engineered using a variety of molecular tools. Methanogens anaerobic lifestyle and autofluorescence, however, restrict the use of common fluorescent reporter proteins (e.g., GFP and derivatives) which require oxygen for chromophore maturation. Here, we employ the tandem activation and absorption-shifting tag protein 2 (tdFAST2) which is fluorescent when the cell-permeable fluorescent ligand (fluorogen) 4-hydroxy-3,5-dimethoxybenzylidene rhodanine (HBR-3,5DOM) is present. tdFAST2 expression in M. acetivorans and M. maripaludis is not cytotoxic and tdFAST2:HBR-3,5DOM fluorescence can be clearly distinguished from the autofluorescence. In flow cytometry experiments, mixed methanogen cultures can be clearly distinguished which allows high-throughput investigations of dynamics within single and mixed cultures. ImportanceMethane-producing archaea play an essential role in the global carbon cycle and have a high potential for biotechnological applications such as biofuel production, carbon dioxide capture, and in electrochemical systems. The oxygen sensitivity and high autofluorescence hinder the use of common fluorescent proteins to study methanogens. By using the tdFAST2:HBR-3,5DOM fluorescence, which is functional also under anaerobic conditions and distinguishable from the autofluorescence, real-time reporter studies and high-throughput investigation of dynamics within (mixed) cultures via flow cytometry are possible. This will accelerate the exploitation of the methanogens biotechnological potential.

molecular biology↗

bioRxiv2021

Efficient CRISPR/Cas12a-based genome editing toolbox for metabolic engineering in Methanococcus maripaludis

The rapid-growing and genetically tractable methanogen Methanococcus maripaludis is a promising host organism for the biotechnological conversion of carbon dioxide and renewable hydrogen to fuels and value-added products. Expansion of its product scope through metabolic engineering necessitates reliable and efficient genetic tools, particularly for genome edits to the primary metabolism that affect cell growth. Here, we have designed a genome editing toolbox by utilizing Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery endogenously present in M. maripaludis. Remarkably, this toolbox can knock out target genes with a success rate of up to 95%, despite the hyper-polyploidy of M. maripaludis. For the purposes of demonstrating a large-sized deletion, we have replaced the flagellum operon (ca. 8.9 kbp) by the {beta}-glucuronidase gene. To facilitate metabolic engineering and flux balancing in M. maripaludis, the relative strength of 15 different promoters were quantified in the presence of the two common growth substrates, formate or carbon dioxide and hydrogen. This CRISPR/LbCas12a toolbox can be regarded as a reliable and fast method for genome editing in a methanogen.

bioengineering↗