Rapid, modular biosynthesis of plant hemicellulose and its impact on yeast cells

Interest in the engineering of polysaccharide-based biomaterials has emerged in recent years. Despite impressive advances on bacterial cellulose, comparatively little is known about how plant hemicelluloses can be reconstituted and modulated in cells suitable for biotechnological purposes. Here, we optimized the cultivation of the yeast Pichia pastoris for the orthogonal production of plant polysaccharides, and enhanced heteromannan (HM) production by assembling modular cellulose synthase-like A (CSLA) enzymes. Chimeric proteins swapping the domains of a plant mannan synthase and a glucomannan synthase led, in three cases, to higher yields or improved growth compared to the parental CSLA enzymes. Prolonged expression of a glucomannan synthase from Amorphophallus konjac (AkCSLA3) was toxic to yeast cells, as demonstrated by reduced biomass accumulation and elevated uptake of dyes that are normally restricted to the extracellular matrix. However, no growth inhibition was observed for CSLA variants producing relatively pure mannan or a CSLC glucan synthase. The toxicity of AkCSLA3 was reduced by swapping its C-terminal region with that of a mannan synthase. HM production was further boosted by co-expressing chimeric CSLA proteins with the MANNAN-SYNTHESIS-RELATED1 (MSR1) putative glycosyltransferase. Interestingly, Pichia cells either increased or decreased in size depending on the CSLA variant expressed, and most of them remained viable even producing copious amounts of hemicellulose. Therefore, yeast modified with non-toxic plant polysaccharides could represent a modular chassis to produce and protect sensitive cargo such as therapeutic proteins.