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Modular biosynthesis of plant hemicellulose and its impact on yeast cells

Madalen Robert, Julian Waldhauer, Fabian Stritt, View ORCID ProfileBo Yang, View ORCID ProfileMarkus Pauly, View ORCID ProfileCătălin Voiniciuc
doi: https://doi.org/10.1101/2021.04.20.440611
Madalen Robert
1Independent Junior Research Group–Designer Glycans, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany
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Julian Waldhauer
1Independent Junior Research Group–Designer Glycans, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany
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Fabian Stritt
2Institute for Plant Cell Biology and Biotechnology, Heinrich Heine University, Düsseldorf, Germany
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Bo Yang
1Independent Junior Research Group–Designer Glycans, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany
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Markus Pauly
2Institute for Plant Cell Biology and Biotechnology, Heinrich Heine University, Düsseldorf, Germany
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Cătălin Voiniciuc
1Independent Junior Research Group–Designer Glycans, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany
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  • For correspondence: catalin.voiniciuc@ipb-halle.de
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Abstract

Background The carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses. In the past five years, exciting possibilities have emerged in the engineering of polysaccharide-based biomaterials. Despite impressive advances on bacterial cellulose-based hydrogels, comparatively little is known about how plant hemicelluloses can be reconstituted and modulated in cells suitable for biotechnological purposes.

Results Here, we assembled cellulose synthase-like A (CSLA) enzymes using an optimized Pichia pastoris platform to produce tunable heteromannan (HM) polysaccharides in yeast. By swapping the domains of plant mannan and glucomannan synthases, we engineered chimeric CSLA proteins that made β-1,4-linked mannan in quantities surpassing those of the native enzymes while minimizing the burden on yeast growth. Prolonged expression of a glucomannan synthase from Amorphophallus konjac was toxic to yeast cells: reducing biomass accumulation and ultimately leading to compromised cell viability. However, an engineered glucomannan synthase as well as CSLA pure mannan synthases and a CSLC glucan synthase did not inhibit growth. Interestingly, Pichia cell size could be increased or decreased depending on the composition of the CSLA protein sequence. HM yield and glucose incorporation could be further increased by co-expressing chimeric CSLA proteins with a MANNAN-SYNTHESIS-RELATED (MSR) co-factor from Arabidopsis thaliana.

Conclusion The results provide novel routes for the engineering of polysaccharide-based biomaterials that are needed for a sustainable bioeconomy. The characterization of chimeric cellulose synthase-like enzymes in yeast offers an exciting avenue to produce plant polysaccharides in a tunable manner. Furthermore, cells modified with non-toxic plant polysaccharides such as β-mannan offer a modular chassis to produce and encapsulate sensitive cargo such as therapeutic proteins.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • The format and content of the manuscript text were significantly revised. Fig. 2 was expanded.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted May 28, 2021.
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Modular biosynthesis of plant hemicellulose and its impact on yeast cells
Madalen Robert, Julian Waldhauer, Fabian Stritt, Bo Yang, Markus Pauly, Cătălin Voiniciuc
bioRxiv 2021.04.20.440611; doi: https://doi.org/10.1101/2021.04.20.440611
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Modular biosynthesis of plant hemicellulose and its impact on yeast cells
Madalen Robert, Julian Waldhauer, Fabian Stritt, Bo Yang, Markus Pauly, Cătălin Voiniciuc
bioRxiv 2021.04.20.440611; doi: https://doi.org/10.1101/2021.04.20.440611

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