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 yeast 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 colocalizes with a known yeast marker for the Golgi apparatus despite the toxic effects of plant glucomannan accumulation in Pichia pastoris. 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 severely disrupting β-1,3-glucans. Finally, we identified alternative feeding strategies and episomal vectors for Pichia, which were extended to Saccharomyces cerevisiae, to accelerate hemicellulose research. 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. Requiring only hours or days instead of weeks or months for plant transformation/regeneration, our yeast prototyping strategies can derisk the bioengineering of carbohydrate-active enzymes.