Illuminating Glucomannan Synthases To Explore Cell Wall Synthesis Bottlenecks

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Hemicelluloses are vital dietary fibers and major components of lignocellulosic biomass.
• Subcellular localization and biochemical functions of intracellular cellulose synthase-like enzymes, such as beta-mannan synthases (ManS), are underexplored.
• Understanding ManS is crucial for advancing polysaccharide biosynthesis research.

Purpose of the Study:

• To identify functional C-terminal fluorescent protein tags for ManS in yeast.
• To accelerate Design, Build, Test, Learn cycles for polysaccharide biosynthesis.
• To facilitate the bioengineering of carbohydrate-active enzymes.

Main Methods:

• Utilized fluorescent protein tagging of Amorphophallus konjac ManS in Pichia pastoris.
• Investigated enzyme colocalization with yeast Golgi markers.
• Assessed the role of the ManS first transmembrane domain in localization, expression, and function.
• Examined the impact of genetic and chemical perturbations on ManS activity.
• Developed and tested alternative feeding strategies and episomal vectors in Pichia and Saccharomyces cerevisiae.

Main Results:

• Identified functional fluorescent protein tags that maintain ManS activity in yeast.
• Demonstrated ManS colocalization with the yeast Golgi apparatus, despite toxic glucomannan accumulation.
• The first transmembrane domain of ManS was critical for its localization, expression, and function.
• Observed effects of yeast cell wall component perturbations on fluorescently tagged ManS.
• Validated yeast prototyping strategies for accelerating hemicellulose research.

Conclusions:

• Fluorescently tagged ManS in yeast provides a rapid prototyping system for studying hemicellulose biosynthesis.
• Yeast prototyping accelerates enzyme engineering, derisking subsequent plant-based studies.
• Expanding Plant MoClo-compatible plasmids is key for efficient carbohydrate-active enzyme prototyping.