Intracellular Mannanases Sustain Matrix Polysaccharide Biosynthesis

The most ancient and ubiquitous hemicellulosic components in the plant cell wall are the β-1,4-linked mannans. Despite their prominent position in the hydrocolloid market and numerous applications as gelling agents in food, feed, biomedical, and bioenergy sector, how β-mannans are assembled and modified remains poorly understood. This greatly limits our ability to improve their structure in crops or microbial cell factories. Glycosyl hydrolases known as endo-β-mannanases (MANs) are the primary catalysts that mobilize mannans from the plant cell wall by cleaving internal β-1,4 glycosidic bonds. Here, we report that Arabidopsis man2 man5 double mutants produce less β-mannan in the seed coat epidermis, rejecting the current dogma that man mutants should maintain or over accumulate Man-rich polymers. While the reduced mannan content could be partially restored through overexpression of a CELLULOSE SYNTHASE-LIKE A (CSLA) glucomannan synthase in the man double mutant, this impaired other seed mucilage polysaccharides. To characterize the functional relationship between CSLAs and MANs, we applied synthetic biology approaches in a modular yeast platform. MAN2 and MAN5 localized intracellularly and reduced the content of alkaline-insoluble polymers made by plant CSLAs. The beneficial of effects of MAN2 was abolished by mutating a single amino acid in its hydrolytic core. By tracking mannans using a newly developed probe and detecting elevated content of water-soluble polymers, we propose that MAN2/5 sustain hemicellulose production in the Golgi apparatus by cleaving insoluble mannan polymers into more hydrophilic molecules.