Madalen Robert1, Julian Waldhauer1, Fabian Stritt2, Bo Yang1, Markus Pauly2, Cătălin Voiniciuc3. 1. Independent Junior Research Group - Designer Glycans, Leibniz Institute of Plant Biochemistry, 06120, Halle, Germany. 2. Institute for Plant Cell Biology and Biotechnology, Heinrich Heine University, 40225, Düsseldorf, Germany. 3. Independent Junior Research Group - Designer Glycans, Leibniz Institute of Plant Biochemistry, 06120, Halle, Germany. catalin.voiniciuc@ipb-halle.de.
Abstract
BACKGROUND: The carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses. In the past 5 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.
BACKGROUND: The carbohydrate polymers that encapsulate plants cells have benefitedhumans for centuries and have valuable biotechnological uses. In the past 5 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 optimizedPichia pastoris platform to produce tunable heteromannan (HM) polysaccharides in yeast. By swapping the domains of plant mannan andglucomannan synthases, we engineered chimeric CSLA proteins that made β-1,4-linkedmannan 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 engineeredglucomannan 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 decreaseddepending on the composition of the CSLA protein sequence. HM yield andglucose 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.
Authors: Charlie Gilbert; Tzu-Chieh Tang; Wolfgang Ott; Brandon A Dorr; William M Shaw; George L Sun; Timothy K Lu; Tom Ellis Journal: Nat Mater Date: 2021-01-11 Impact factor: 43.841
Authors: Cătălin Voiniciuc; Maximilian Heinrich-Wilhelm Schmidt; Adeline Berger; Bo Yang; Berit Ebert; Henrik V Scheller; Helen M North; Björn Usadel; Markus Günl Journal: Plant Physiol Date: 2015-07-28 Impact factor: 8.340
Authors: Li Yu; Jan J Lyczakowski; Caroline S Pereira; Toshihisa Kotake; Xiaolan Yu; An Li; Soren Mogelsvang; Munir S Skaf; Paul Dupree Journal: Plant Physiol Date: 2018-09-05 Impact factor: 8.340
Authors: Thomas Gassler; Michael Sauer; Brigitte Gasser; Michael Egermeier; Christina Troyer; Tim Causon; Stephan Hann; Diethard Mattanovich; Matthias G Steiger Journal: Nat Biotechnol Date: 2019-12-16 Impact factor: 54.908