Engineering Methanosarcina strains for biofuel production

Biogas presents a constantly overlooked source of renewable energy. Methane, principal component of biogas, can be used for the replacement of fossil fuels. Methanogenic Archaea are to date the only known organisms capable of producing methane as a byproduct of their metabolism. Methanogens use a limited number of carbon sources including acetate, H₂/CO₂ and methanol.  In this work we report our efforts towards engineering the methanogenic archaeon Methanosarcina barkeri Fusaro for biogas production, by creating a strain capable of utilizing a wider substrate range, focusing our efforts in pyruvate utilization. Metabolic models are currently employed to guide our experimental approaches which consist of i) understanding the genetic basis of pyruvate utilization in a previously isolated Methanosarcina barkeri pyruvate utilizing mutant and ii) engineering Methanosarcina strains to use alternative carbon sources as methanogenesis substrates. Whole genome sequence of the previously isolated M. barkeri Fusaro Pyr⁺ allowed us to identify two mutations that could be responsible for the phenotype. Recreation of the mutation present in the pyruvate carboxylase operon (pyc) and deletion of the operon did not render strains capable of pyruvate utilization. Suggesting that the second mutation, present in a putative transcriptional regulator, may be needed for obtaining the phenotype. The Jen1p transporter from Saccharomyces cerevisiae has been cloned into M. barkeri Fusaro in an effort of improving the transport of pyruvate into the cell. Together, these mutations have not render cells with a Pyr⁺ phenotype however, it has giving us an insight of pyruvate utilization in M. barkeri Fusaro strains.