Illuminating Glucomannan Synthases to Explore Cell Wall Synthesis Bottlenecks

Hemicelluloses are important dietary fibers and a key component of lignocellulosic biomass. Despite numerous observations for fluorescently tagged cellulose synthases, the subcellular journeys and biochemical activities of intracellular cellulose synthase-like enzymes such as β-mannan synthases (ManS) remain largely unexplored. This study identifies C-terminal fluorescent protein tags that maintain ManS activity in the yeast Pichia pastoris to accelerate the Design, Build, Test, Learn cycles for polysaccharide biosynthesis. Using the Amorphophallus konjac ManS as a case study, we demonstrate that the enzyme co-localizes with a known yeast marker for the Golgi apparatus despite the toxic effects of plant glucomannan accumulation in yeast. The ManS first transmembrane domain was found to be critical for the punctate localization of the enzyme, its overall expression level and its function. Additionally, we explored how fluorescently tagged ManS is influenced by genetic or chemical perturbations of native yeast cell wall components, such as reducing protein mannosylation and severe disrupting β-1,3-glucans. Finally, we tested alternative promoters and vector backbones for integrative or episomal ManS expression to address outstanding questions regarding hemicellulose production. We propose that expanding the Plant MoClo-compatible plasmid repertoire is essential to swiftly prototype carbohydrate-active enzymes in yeast before proceeding with more time-intensive analyses in plants. By shortening glycan experiments from weeks or months in bioenergy crops to mere hours or days, our yeast strategies provide insights on how to de-risk the biodesign of plant cell walls.