(390b) A Synthetic Biosensor to Determine Peroxisomal Acetyl-CoA Concentration for Compartmentalized Metabolic Engineering

Feng, X., Virginia Polytechnic Institute and State University
Sheng, J., Virginia Polytechnic Institute and State University
Sub-cellular compartmentalization is used by all eukaryotes and some prokaryotes as a means to create favorable microenvironments for various metabolic reactions. These compartments can concentrate enzymes, separate competing metabolic reactions, and isolate toxic intermediates in metabolic pathways. Such advantages have been recently harnessed by metabolic engineers to improve the production of various high-value chemicals via compartmentalized metabolic engineering. However, one challenge in compartmentalized metabolic engineering is to determine key metabolite level in these compartments. Conventional techniques such as metabolomics analysis and transcription-based biosensors could only reflect cytosolic metabolite concentration instead of compartmental metabolite concentration. To this end, we developed a synthetic biosensor to determine a key metabolite, i.e., acetyl-CoA, in a representative compartment of yeast, i.e., peroxisome. This synthetic biosensor used highly efficient enzyme re-localization via PTS1 signal peptides to construct a metabolic pathway in peroxisome, which solely converted peroxisomal acetyl-CoA to polyhydroxybutyrate (PHB) via three enzymes, phaA phaB and phaC. By quantifying the PHB level in yeast, we successfully determined peroxisomal acetyl-CoA level under various culture conditions. We next demonstrated our biosensor by screening a library of single knockout yeast mutants and identified one yeast mutant, ΔRPD3, which had elevated level of peroxisomal acetyl-CoA compared to wild type yeast. We expect our synthetic biosensors can be widely used to deepen our understanding of sub-cellular compartmental metabolism and facilitate the “design-build-test” cycle of compartmentalized metabolic engineering.