Biology subjects

Rother, M.

Publications and source records attributed to Rother, M..

4 recordsLinked to original sources

bioRxiv2024

Production of the sesquiterpene bisabolene from one-carbon compounds in an engineered Methanosarcina acetivorans

The isoprenoid bisabolene, one of the simplest monocyclic sesquiterpenes, is a natural plant product that, in addition to its biological function, serves as a precursor for many industrial products. Due to the low concentration of bisabolene and the long harvest cycle, industrial production of this isoprenoid in plants is economically challenging. Chemical synthesis of bisabolene also suffers from significant disadvantages, such as low yields, toxic side products and high costs. Archaea appear suitable producers of isoprenoids, as their membrane lipids consist of isoprenoid ethers, which are synthesized via a variant of the mevalonate pathway. Archaeal model species have versatile metabolic capacities, which makes them suitable candidates for biotechnological applications. Here, we engineered Methanosarcina acetivorans for production of -bisabolene from one-carbon substrates by introducing a bisabolene synthase from Abies grandis. Expression of a codon-optimized bisabolene synthase gene in a M. acetivorans wild-type strain resulted in 10.6 mg bisabolene/liter of culture. Overexpressing genes of the mevalonate pathway only slightly increased bisabolene yields, which, however, were reached much earlier during incubations than in the corresponding wild-type strain. The data presented argue for the suitability of M. acetivorans for the biotechnical production of certain isoprenoids.

microbiology↗

bioRxiv2024

Physiological barriers for glucose utilization in Methanosarcina acetivorans

Methanogenesis is a key aspect of anaerobic biomass degradation, and thus of global importance. While methanogenic archaea (methanogens) are ubiquitous in anaerobic habitats, the range of substrates they utilize is very limited. Most methanogens are able to grow chemolithotrophically with H2+CO2, some clades can utilize methylated compounds and/or acetate as well. Organotrophic compounds like amino acids, lipids, nucleotides, or carbohydrates, are not known to support growth of methanogens. This inability to utilize "upstream" intermediates of anaerobic biomass degradation is remarkable considering the presence of cellular metabolism that such intermediates could feed into. Here, we addressed the question why the model methanogen Methanosarcina acetivorans, despite its gluconeogenic and glycolytic capacity, is unable to utilize glucose for methanogenesis and growth. Complementing heterologously with a glucose uptake facilitator allowed a recombinant M. acetivorans strain to convert glucose to methane at a low rate. However, growth with glucose was not observed, neither as energy source nor as carbon source. Instead, methylotrophic growth of the transgenic strain was impaired in a glucose-dependent fashion, which was aggravated when also glucokinase was heterologuously produced. Glucose-dependent growth inhibition coincided with a significant - and microscopically visible - accumulation of intracellular carbohydrate. Since the glucose-utilizing trait is rapidly lost during methylotrophic growth, accumulating growth-inhibiting metabolites probably makes methanogenic and glycolytic catabolism incompatible. Thus, extensive efforts in strain development would be required to enable direct glucose utilization for methanogenesis. ImportanceThe known range of methanogenic growth substrates is very limited and the most reduced carbon compound to support organotrophic growth of M. acetivorans is pyruvate. Yet, gluconeogenesis and glycolysis, i. e., mobilization of stored glycogen, which is known to occur in M. acetivorans makes the capacity of glycolytic methanogenesis in this organism seem feasible. Although this trait could be engineered in M. acetivorans by introducing a single gene, the finding that it could not sustain growth of the organism, not even as an anabolic carbon source, demonstrates that methanogenesis is not easily compatible with carbohydrate utilization, thus probably requiring extensive metabolic rewiring.

microbiology↗

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↗

bioRxiv2017

ALPK1 And TIFA Dependent Innate Immune Response Triggered By The Helicobacter Pylori Type IV Secretion System

Activation of transcription factor NF-{kappa}B is a hallmark of infection with the gastric pathogen Helicobacter pylori and associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous hits involved in H. pylori-, but not IL-1{beta}- and TNF-- dependent NF-{kappa}B regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry and mutant H. pylori strains, identified the H. pylori metabolite D-glycero-{beta}-D-manno-heptose 1,7-bisphosphate ({beta}HBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-{kappa}B activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-{kappa}B activation, TIFA phosphorylation as well as TIFAsome formation depended on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as core of a novel innate immune pathway. ALPK1-TIFA-mediated NF-{kappa}B activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogens T4SS.

molecular biology↗