Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 116, Issue 2, Pages 522-527Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1814003116
Keywords
plant cell wall; polysaccharide; glycosyltransferase; mannan; yeast
Categories
Funding
- Cluster of Excellence on Plant Sciences-Deutsche Forschungsgemeinschaft [EXC1028]
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Heteromannan (HM) is one of the most ancient cell wall polymers in the plant kingdom, consisting of beta-(1-4)-linked backbones of glucose (Glc) and mannose (Man) units. Despite the widespread distribution of HM polysaccharides, their biosynthesis remains mechanistically unclear. HM is elongated by glycosyltransferases (GTs) from the cellulose synthase-like A (CSLA) family. MANNAN-SYNTHESIS RELATED (MSR) putative GTs have also been implicated in (gluco) mannan synthesis, but their roles have been difficult to decipher in planta and in vitro. To further characterize the products of the HM synthases and accessory proteins, we chose a synthetic biology approach to synthesize plant HM in yeast. The expression of a CSLA protein in Pichia pastoris led to the abundant production of plant HM: up to 30% of glycans in the yeast cell wall. Based on sequential chemical and enzymatic extractions, followed by detailed structural analyses, the newly produced HM polymers were unbranched and could be larger than 270 kDa. Using CSLAs from different species, we programmed yeast cells to produce an HM backbone composed exclusively of Man or also incorporating Glc. We demonstrate that specific MSR cofactors were indispensable for mannan synthase activity of a coffee CSLA or modulated a functional CSLA enzyme to produce glucomannan instead of mannan. Therefore, this powerful platform yields functional insight into the molecular machinery required for HM biosynthesis in plants.
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