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Crema of the Crop: Producing Designer Heteromannans in Yeast and Plant Cell Walls

Subject Area Plant Biochemistry and Biophysics
Plant Genetics and Genomics
Plant Cell and Developmental Biology
Term from 2018 to 2022
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 414353267
 
Final Report Year 2022

Final Report Abstract

Plant beta-1,4-linked mannans are arguably the most ancient hemicellulosic polysaccharides and are found in algae as well as land plants. However, the biosynthesis and biological roles of β-mannans remained poorly understood compared to other cell wall components in the last decade. Since carbohydrate-based materials with tailored structures and functions remain in short supply, the cellular production of designer glycans represents a major challenge in for the field of plant biology and the broader bioeconomy. Since 2019, I have been leading an independent team that developed a bioengineering platform capable of rapidly producing large amounts of β-mannan polymers in yeast cell walls, which normally lack hemicelluloses. This synthetic biology strategy has already yielded mechanistic insight into the products of different plant cellulose synthase-like (CSL) enzymes and their cofactors, which would have been difficult or tedious to discover otherwise. My independent group is now building modular genetic circuits for yeast and plant metabolic engineering. Yeast offers an advantageous chassis for cell biology and engineering, enabling changes in cell wall content or composition by varying enzyme(s), cultivation strategy (plate format and media) or transcriptional elements used. Recently, we have been transferring the knowledge from the yeast hosts into plant expression systems to validate the top candidates. By heterologously expressing them in the yeast Pichia pastoris and later in Nicotiana leaves, we discovered the activity of the previously uncharacterized CSLK clade of enzymes from red algae, while questioning the long-held assumption that green algal CSLK proteins are functionally analogous to plant CSLA mannan synthases. Although yeast cell walls rapidly accumulate large amounts of β-mannan polymers, the production of (gluco)mannan is unexpectedly toxic in both yeast and plants since transgenic lines with constitutive promoters showed negative selection. Therefore, a more promising strategy to build designer polysaccharide is to apply inducible promoters and/or to engineer enzymes with more desirable products. In addition to microbial surrogate hosts, seed mucilage offers an excellent genetic platform to dissect the biosynthesis, structure and function of different plant polysaccharides. During this project, besides mannan synthesis-related (MSR) factors, several other classes of proteins were found to be required for the content of hemicellulose or cellulose architecture in Arabidopsis seed mucilage. Interestingly, galactoglucomannan mutants were found to enhance the tolerance of germinating seeds and young seedlings to salt stress. Therefore, plant synthetic biology approaches provide promising perspectives for tailor-made polysaccharides and for the modulation of carbohydrate-based agronomic traits.

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