Abstract
BackgroundThe 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.ResultsHere, 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.ConclusionThe 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.Graphic abstract
Highlights
The carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses
Fluorescent reporters can speed up colony screening, but an N-terminal superfolder green fluorescent protein fusion inhibited AkCSLA3 activity in Pichia [18]
While the AkCSLA3-superfolder green fluorescent protein (sfGFP) produced alkaline-insoluble polymers rich in Man, fluorescently tagged AtCSLA2 did not increase the relative content of Man compared to sfGFP and empty vector controls (Fig. 2b)
Summary
The carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses. 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. GTs from the Cellulose Synthase-Like (CSL) superfamily produce the backbones of xyloglucans, heteromannans (HM), as well as β-1,3-1,4linked-glucans [6, 7], which can account for one-third or more of the cell wall biomass. Of these polymers, HMs have the simplest structures since they can be found as linear β-1,4-linked mannans (defined as containing > 90% mannose, Man), or as glucomannans that contain β-1,4-linked glucose (Glc) units [8]. Since HMs are excellent thickening and gelling agents, locust bean gum (extracted from Ceratonia siliqua, known as carob) and guar gum (from Cyamopsis tetragonoloba) are commonly used hydrocolloids in the food (approved as E410 and E412 stabilizers within the European Union), cosmetic and pharmaceutical industries
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