Project description:Our goal is to convert methane efficiently into liquid fuels that may be more readily transported. Since aerobic oxidation of methane is less efficient, we focused on anaerobic processes to capture methane, which are accomplished by anaerobic methanotrophic archaea (ANME) in consortia. However, no pure culture capable of oxidizing and growing on methane anaerobically has been isolated. In this study, Methanosarcina acetivorans, an archaeal methanogen, was metabolically engineered to take up methane, rather than to generate it. To capture methane, we cloned the DNA coding for the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable archaeal organism from a Black Sea mat into M. acetivorans to effectively run methanogenesis in reverse. The engineered strain produces primarily acetate, and our results demonstrate that pure cultures can grow anaerobically on methane. Differential gene analysis of two growth conditions (three biological replicates each) was performed: (i) M. acetivorans/pES1-MATmcr3 grown on methane and (ii) M. acetivorans/pES1-MATmcr3 grown on methanol. All starter cultures (200 mL) were grown on methanol for 5 days, and harvested by centrifugation. Cell pellets were washed three times with HS medium, and resuspended using 5 mL HS medium, 2 Âµg/mL puromycin, and 0.1 mM FeCl3. For condition (i), methane was filled into the headspace of the cultures. For condition (ii), 150 mM methanol was added. All cultures were incubated at 37C for 5 days, followed by rapid centrifugation in the presence of 50 ÂµL RNAlater solution (Ambion, Austin, TX) per mL of culture. Total RNA was isolated using RNeasy Mini kit (Qiagen, Valencia, CA) were then digested with terminator 5â-phosphate-dependent exonuclease (Epicentre, Madison, WI) to partially remove ribosomal RNA. Digested RNA were cleaned up using AgenCourt RNAClean XP beads (AgenCourt Bioscience, Beverly, MA) and used for cDNA library construction using the TruSeq Stranded mRNA Library kit (Illumina). Pooled and barcoded cDNA library was then sequenced on a HiSeq sequencing platform (Illumina). Obtained reads were mapped to the reference genome of M. acetivorans (Genbank accession NC_003552.1) using STAR. The mapped reads were assembled using Cufflink v2.2.1 to identify potential novel transcripts. Assembled, unannotated novel transcripts for all the strains were combined with the list of known genes. Differential expression of genes and potential novel transcripts were determined using Cuffdiff at a significance cutoff at q < 0.07 with a false discovery rate of 0.05. Expression levels of gene transcripts are expressed as fragments per kilobase of transcript per million mapped fragments (FPKM), and expression changes are determined by the ratio of FPKM of culture replicates grown on methane to FPKM of culture replicates grown on methanol.

Project description:Kumar2011 - Genome-scale metabolic network of Methanosarcina acetivorans (iVS941) This model is described in the article: Metabolic reconstruction of the archaeon methanogen Methanosarcina Acetivorans. Satish Kumar V, Ferry JG, Maranas CD. BMC Syst Biol 2011; 5: 28 Abstract: BACKGROUND: Methanogens are ancient organisms that are key players in the carbon cycle accounting for about one billion tones of biological methane produced annually. Methanosarcina acetivorans, with a genome size of ~5.7 mb, is the largest sequenced archaeon methanogen and unique amongst the methanogens in its biochemical characteristics. By following a systematic workflow we reconstruct a genome-scale metabolic model for M. acetivorans. This process relies on previously developed computational tools developed in our group to correct growth prediction inconsistencies with in vivo data sets and rectify topological inconsistencies in the model. RESULTS: The generated model iVS941 accounts for 941 genes, 705 reactions and 708 metabolites. The model achieves 93.3% prediction agreement with in vivo growth data across different substrates and multiple gene deletions. The model also correctly recapitulates metabolic pathway usage patterns of M. acetivorans such as the indispensability of flux through methanogenesis for growth on acetate and methanol and the unique biochemical characteristics under growth on carbon monoxide. CONCLUSIONS: Based on the size of the genome-scale metabolic reconstruction and extent of validated predictions this model represents the most comprehensive up-to-date effort to catalogue methanogenic metabolism. The reconstructed model is available in spreadsheet and SBML formats to enable dissemination. This model is hosted on BioModels Database and identified by: MODEL1507180035. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Methanosarcina acetivorans

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