Expanding the toolbox for genetic manipulations in Methanosarcina acetivorans

Methanosarcina acetivorans is a model methane producer that can utilize many different one-carbon substrates. Genetic tools are needed to investigate its physiology and to extend the product scope beyond methane. The development of the current genetic toolbox in this archaeon, however, is far behind compared with those e.g. for acetogenic bacteria or E. coli. In this study, the genetic toolbox for M. acetivorans has been expanded for efficient genome edition and for controlling gene expression. First, a CRISPR/Cas12a-based genome editing system (Publication I) was introduced for efficient marklerless genome editing in M. acetivorans. Different from the previously developed Cas9-editing system, the Cas12a recognizes distinct PAM sequence and demonstrates high efficiency in large DNA fragments deletion and alleviate the cloning efforts on multiplex genome editing. To facilitate a stable and reliable gene expression, four neutral integration sites in M. acetivorans genome were characterized (Publication II) for heterologous genes integration and metabolic pathway constructions. These neutral sites allow gene expression without disrupting cell viability, providing as non-essential loci for stable long-term genetic engineering. Finally, a promoter-RBS library comparing 33 combinations was developed for fine-tuning gene expression in M. acetivorans (Publication III). By evaluating the native and engineered combinations, the library revealed diverse transcriptional regulation mechanisms mediated by RNA secondary structures. With a broad range of expression strength (ca. 140-fold), the library allows precise control of gene expression levels and enables fine regulation of metabolic flux for various synthetic biology applications. The tools developed in the thesis enhance the capacity for genomic manipulation of M. acetivorans and facilitate the development of metabolic engineering for efficient methane production, carbon flux rewiring, and other biotechnological processes in this organism.