Expanding the Dynamic Range of Methanosarcina Acetivorans Through Recombinant Expression of an RNA Polymerase

Methanogens are anaerobic archaea that grow and conserve energy through the production of methane from acetate or C1 carbon substrates such as methanol and carbon dioxide. They play an important role in the global carbon cycle, contributing between 350 and 420 Tg of methane to the atmosphere annually. When used in anaerobic digesters, methanogens ferment organic waste to produce biogas containing methane, which can be used as a renewable fuel source. Due to their use of low-cost feedstocks and their unique metabolism, methanogens can also be a cheap and efficient host organism for the synthesis of organic molecules such as bioplastics and isoprene, a precursor to rubber. One limiting factor for the industrial use of methanogens is a limited understanding of archaeal transcription and translation, and limited genetic tools available for their regulation. This study aims to expand the dynamic range of gene expression of Methanosarcina acetivorans through the recombinant in vivo expression of an RNA Polymerase. Using in vitro transcription, we show that the RNA Polymerase effectively transcribes genes in the presence of M. acetivorans lysate. We then demonstrate that the RNA Polymerase produces high levels of transcription in M. acetivorans in vivo by expressing the reporter gene YFAST under the polymerase’s specific promoter. Additionally, we demonstrate a new tool for understanding transcriptional and translational regulation in methanogens. Going forward, a new inducible expression system will be introduced in M. acetivorans as well to push its dynamic transcriptional capabilities further.