Towards universal synthetic heterotrophy using a metabolic coordinator.

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3p), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates.

In-depth Sequence-Function Characterization Reveals Multiple Pathways to Enhance Enzymatic Activity.

Deep mutational scanning (DMS) has recently emerged as a powerful method to study protein sequence-function relationships but it has not been well-explored as a guide to enzyme engineering and identifying pathways by which their catalytic cycle may be improved. We report such a demonstration in this work using a Phenylalanine ammonia-lyase (PAL), which deaminates L-phenylalanine to -cinnamic acid and has widespread application in chemo-enzymatic synthesis, agriculture, and medicine. In particular, the PAL from (AvPAL*) has garnered significant attention as the active ingredient in Pegvaliase, the only FDA-approved drug treating classical Phenylketonuria (PKU).