Establishing Formolase Activity in S. cerevisiae

Abstract

One-carbon compounds (C1) such as methanol are an attractive feedstock for biomanufacturing. To make these feedstocks accessible to popular industrial strains, recent efforts have been directed towards establishing C1-assimilation pathways in otherwise heterotrophic strains. In this study, we want to enable methanol-utilization in the yeast S. cerevisiae by expressing the synthetic formolase (FLS) pathway. The FLS pathway consists of three steps which link methanol to the central carbon metabolism of the cell: methanol oxidation, formaldehyde condensation to the three-carbon compound dihydroxyacetone (DHA), and DHA phosphorylation resulting in the glycolytic intermediate DHAP. The condensation reaction is catalyzed by the artificial FLS enzyme, but although the enzyme has been successfully applied in enzymatic cascades for in vitro C1-fixation, its functional expression in S. cerevisiae has not been demonstrated. Our approach is to construct an auxotrophic strain which relies on FLS activity for growth and can serve as a platform to screen FLS variants in vivo. Moreover, such a strain presents a suitable starting point for adaptive laboratory evolution aimed at improving methanol utilization in S. cerevisiae.