Investigating promoter-RBS strength variations in Methanosarcina acetivorans in response to different growth substrates

Abstract

Methanogenic archaea are strictly anaerobic microorganisms able to convert C1compounds and acetate into methane to derive energy. Methanosarcina acetivorans is considered one of the model organisms for genetic engineering given its metabolic versatility. Despite this advantage, the complete regulatory transcriptional and translational mechanisms that govern its metabolism remain poorly understood. In this study, twelve promoter-RBS combinations from diverse methanogens were selected to test the strength and relevant response in M. acetivorans. Two growth environments were selected (Methanol and Trimethylamine), and the gene expression levels were quantified with the help of a β-glucuronidase reporter system.This activity provided a baseline to engineer the expression level by trying eight promoter-RBS combinations from four candidates: hdr from M. mazei, mcr from Mc.voltae and ech and vht from Mb. Fusaro. Our results reveal alterations in gene expression in response to the promoter-RBS swaps made(up to a 10-times increased activity),which indicates a regulation in the transcriptional and translational level in M. acetivorans. Additionally, eight promoter-RBS combinations were added to the original set of candidates for the UidA gene expression. These insights offer a wider perspective on the available tools for metabolic engineering of methanogens.