Modular biosynthesis of plant hemicellulose and its impact on yeast cells

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

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.

Mechanistic insights from plant heteromannan synthesis in yeast

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.

Insights into the red algae and eukaryotic evolution from the genome ofPorphyra umbilicalis(Bangiophyceae, Rhodophyta)

Porphyra umbilicalis(laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploidPorphyragenome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of thePorphyragenome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins inPorphyraare restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, includingPorphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis

The fungal cell possesses an essential carbohydrate cell wall. The outer layer, mannan, is formed by mannoproteins carrying highly mannosylated O - and N -linked glycans. Yeast mannan biosynthesis is initiated by a Golgi-located complex (M-Pol I) of two GT-62 mannosyltransferases, Mnn9p and Van1p, that are conserved in fungal pathogens. Saccharomyces cerevisiae and Candida albicans mnn9 knockouts show an aberrant cell wall and increased antibiotic sensitivity, suggesting the enzyme is a potential drug target. Here, we present the structure of Sc Mnn9 in complex with GDP and Mn 2+ , defining the fold and catalytic machinery of the GT-62 family. Compared with distantly related GT-78/GT-15 enzymes, Sc Mnn9 carries an unusual extension. Using a novel enzyme assay and site-directed mutagenesis, we identify conserved amino acids essential for Sc Mnn9 ‘priming’ α-1,6-mannosyltransferase activity. Strikingly, both the presence of the Sc Mnn9 protein and its product, but not Sc Mnn9 catalytic activity, are required to activate subsequent Sc Van1 processive α-1,6-mannosyltransferase activity in the M-Pol I complex. These results reveal the molecular basis of mannan synthesis and will aid development of inhibitors targeting this process.

Molecular and Phenotypic Analysis of CaVRG4, Encoding an Essential Golgi Apparatus GDP-Mannose Transporter

ABSTRACT Cell surface mannan is implicated in almost every aspect of pathogenicity of Candida albicans. In Saccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision. The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p. This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatus-specific mannosylation) is a fungus-specific process. To begin to study the role of Golgi mannosylation in C. albicans, we isolated the CaVRG4 gene and analyzed the effects of loss of its function. CaVRG4 encodes a functional homologue of the S. cerevisiae GDP-mannose transporter. CaVrg4p localized to punctate spots within the cytoplasm of C. albicans in a pattern reminiscent of localization of Vrg4p in the Golgi apparatus in S. cerevisiae. Like partial loss of ScVRG4 function, partial loss of CaVRG4 function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes. While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy of CaVRG4 under control of the methionine-repressible MET3 promoter, did not grow in the presence of methionine and cysteine, demonstrating that CaVRG4 is essential for viability. Mutant Candida vrg4 strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth. Because the VRG4 gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies.