Researchers develop a new method to show that messenger RNA molecules live much shorter lives in the cell than previously thought.

Heteromannan (HM) is one of the most ancient cell wall polymers in the plant kingdom, consisting of β-(1-4)-linked backbones of glucose (Glc) and mannose (Man) units. Despite the widespread distribution of HM polysaccharides, their biosynthesis remains mechanistically unclear. HM is elongated by glycosyltransferases (GTs) from the cellulose synthase-like A (CSLA) family. MANNAN-SYNTHESIS RELATED (MSR) putative GTs have also been implicated in (gluco)mannan synthesis, but their roles have been difficult to decipher in planta and in vitro. To further characterize the products of the HM synthases and accessory proteins, we chose a synthetic biology approach to synthesize plant HM in yeast. The expression of a CSLA protein in Pichia pastoris led to the abundant production of plant HM: up to 30% of glycans in the yeast cell wall. Based on sequential chemical and enzymatic extractions, followed by detailed structural analyses, the newly produced HM polymers were unbranched and could be larger than 270 kDa. Using CSLAs from different species, we programmed yeast cells to produce an HM backbone composed exclusively of Man or also incorporating Glc. We demonstrate that specific MSR cofactors were indispensable for mannan synthase activity of a coffee CSLA or modulated a functional CSLA enzyme to produce glucomannan instead of mannan. Therefore, this powerful platform yields functional insight into the molecular machinery required for HM biosynthesis in plants.plant cell wall | polysaccharide | glycosyltransferase | mannan | yeast P lants thrive in a wide range of aqueous and terrestrial envi-

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. Graphic abstract

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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.