Reconstructing the Plant Wall Polysaccharide O-acetylation Mechanism in an Unconventional Yeast Species

• Applicant: Privatdozent Dr.-Ing. Vicente Ramirez, Ph.D.
• Subject Area: Plant Biochemistry and Biophysics ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 517563163

Substitution of plant cell wall polysaccharides with O-acetyl substituents changes the biophysics of the polymers and thus directly impacts plant development, reproduction, and adaptational responses to abiotic stresses such as freezing or drought. In the last decade genetic and biochemical approaches have been instrumental in the identification and characterization of several components of the polysaccharide O-acetylation machinery. Two multigenic families with partially overlapping functions with tissue-specific expression patterns are essential. Moreover, a third component with unknown function is also involved. Unfortunately, the hitherto used approaches have not been able to unambiguously define the function of some of these components, and thus the molecular mechanism of plant cell wall polymer O-acetylation is not understood. Here, we propose a synthetic biology approach by developing a synthetic unicellular model to gain further insights. We have found an unconventional yeast species (Yarrowia lipolytica) that similar to plants O-acetylates cell surface polysaccharides. However, in contrast to plants Yarrowia uses a simplified mechanism composed of one protein combining two key activities: transport of acetyl donor substrates from the cytosol to the Golgi lumen and transfer of the acetyl group to a specific glycan. We took advantage of the endogenous O-acetylation machinery to successfully engineer Yarrowia cells that successfully produce an O-acetylated plant wall polysaccharide (i.e. glucomannan, GM) not present in wildtype yeast walls. We propose utilizing this synthetic model further to define the identity, selectivity, and function of the plant enzymes involved in the decoration of plant polysaccharides with acetyl substituents by pursuing the following objectives: (1) Structural characterization of the AcGM produced in engineered Yarrowia cells; (2) Functional characterization of components of the plant wall O-acetylation machinery; (3) Identification of additional components of the machinery.Given the conservation of the mechanism in other kingdoms, the gained knowledge could be also translated for the better understanding of the O-acetylation of extracellular glycans also in non-plant species such as bacteria, fungi and animal cells.

• DFG Programme: Research Grants