yeast glycosylation

CRISPR-Cas9-mediated disruption of a licorice cellulose synthase-derived glycosyltransferase gene, GuCSyGT, demonstrated the in planta role of GuCSyGT as the enzyme catalyzing 3-O-glucuronosylation of triterpenoid aglycones in soyasaponin biosynthesis. Triterpenoid glycosides (saponins) are a large, structurally diverse group of specialized metabolites in plants, including the sweet saponin glycyrrhizin produced by licorice (Glycyrrhiza uralensis) and soyasaponins that occur widely in legumes, with various bioactivities...

Manganese (Mn), an indispensable plant micronutrient, functions as a vital enzyme co-factor in numerous biochemical reactions. In rice, the Golgi-localized PHOTOSYNTHESIS-AFFECTED MUTANT 71-LIKE 3 (OsPML3), a member of the UNCHARACTERIZED PROTEIN FAMILY (UPF0016), plays a pivotal role in Mn homeostasis, particularly in rapidly developing tissues. This study focused on the functional characterization of another UPF0016 family member in rice, OsPML4, to elucidate its involvement in Mn homeostasis. OsPML4 had a 73% sequence identity with OsPML3 and exhibited expression in both shoots and roots, albeit at a lower transcriptional level than OsPML3...

It is known that oligosaccharyltransferase (OST) has hydrolytic activity towards dolichol-linked oligosaccharides, which results in the formation of free N-glycans (FNGs), i.e. unconjugated oligosaccharides with structural features similar to N-glycans. The functional importance of this hydrolytic reaction, however, remains unknown. In this study, the hydrolytic activity of OST was characterized in yeast. It was shown that the hydrolytic activity of OST is enhanced in ubiquitin ligase mutants that are involved in endoplasmic reticulum-associated degradation (ERAD)...

Despite the prevalence and importance of glycoproteins in human biology, methods for isotope labeling suffer significant limitations. Common prokaryotic platforms do not produce mammalian post-translation modifications that are essential to the function of many human glycoproteins, including immunoglobulin G1 (IgG1). Mammalian expression systems require complex media and thus introduce significant costs to achieve uniform labeling. Expression with Pichia is available, though expertise and equipment requirements surpass E...

Chromosome segregation is crucial for the faithful inheritance of DNA to the daughter cells after DNA replication. For this, the kinetochore, a megadalton protein complex, assembles on centromeric chromatin containing the histone H3 variant CENP-A and provides a physical connection to the microtubules. Here, we report an unanticipated role for enzymes required for β-1,6- and β-1,3 glucan biosynthesis in regulating kinetochore function in Saccharomyces cerevisiae. These carbohydrates are the major constituents of the yeast cell wall...

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain (RBD) in P. pastoris (yRBD) with that in human cells...

Eukaryotic dehydrodolichyl diphosphate synthases (DHDDSs), cis-prenyltransferases (cis-PTs) synthesizing precursors of dolichols to mediate glycoprotein biosynthesis require partners, e.g. Nus1 in yeast and NgBR in animals, which are cis-PTs homologues without activity but to boost the DHDDSs activity. Unlike animals, plants have multiple cis-PT homologues to pair or stand alone to produce various chain-length products with less known physiological roles. We chose Cinnamomum kanehirae, a tree that contains two DHDDS-like and three NgBR-like proteins from genome analysis, and found that one DHDDS-like protein acted as a homodimeric cis-PT to make a medium-chain C55 product, while the other formed heterodimeric complexes with either one of two NgBR homologues to produce longer-chain products...

SARS-CoV-2 spike protein is an essential component of numerous protein-based vaccines for COVID-19. The receptor-binding domain of this spike protein is a promising antigen with ease of expression in microbial hosts and scalability at comparatively low production costs. This study describes the production, purification, and characterization of RBD of SARS-CoV-2 protein, which is currently in clinical trials, from a commercialization perspective. The protein was expressed in Pichia pastoris in a large-scale bioreactor of 1200 L capacity...

Continued mutation of the SARS-CoV-2 genome has led to multiple waves of COVID-19 infections, and new variants have continued to emerge and dominate. The emergence of Omicron and its subvariants has substantially increased the infectivity of SARS-CoV-2. RBD genes of the wild-type SARS-CoV-2 strain and the Delta, Omicron BA.1 and Omicron BA.2 variants were used to construct plasmids and express the proteins in glycoengineered Pichia pastoris . A stable 4 L-scale yeast fermentation and purification process was established to obtain high-purity RBD proteins with a complex glycoform N-glycosyl structure that was fucose-free...

Considering the toxicity of conventional therapeutic approaches and the importance of precise mechanistic targets, it is important to explore signaling pathways implicated in fungal pathobiology. Moreover, treatment of paracoccidioidomycosis, a systemic mycosis caused by a dimorphic fungus, requires prolonged therapeutic regimens. Among the numerous factors underpinning the establishment of Paracoccidioides spp. infection, the capacity to transition from the mycelial to the yeast form is of pivotal importance...

Icariin (ICA) has wide applications in nutraceuticals and medicine with strong anticancer activities. However, the structural complexity and low abundance in plants of ICA lead to the unsustainable and high-cost supply from chemical synthesis and plant extraction. Here, the whole biosynthesis pathway of ICA was elucidated, then was constructed in Saccharomyces cerevisiae, including a 13-step heterologous ICA pathway from eleven kinds of plants as well as deletions or overexpression of ten yeast endogenous genes...

In recent years, the popularity of fermented foods has strongly increased based on their proven health benefits and the adoption of new trends among consumers. One of these health-promoting products is water kefir, which is a fermented sugary beverage based on kefir grains (symbiotic colonies of yeast, lactic acid and acetic acid bacteria). According to previous knowledge and the uniqueness of each water kefir fermentation, the following project aimed to explore the microbial and chemical composition of a water kefir fermentation and its microbial consortium, through the integration of culture-dependent methods, compositional metagenomics, and untargeted metabolomics...

The chitin synthase Chs3 is a multipass membrane protein whose trafficking is tightly controlled. Accordingly, its exit from the endoplasmic reticulum (ER) depends on several complementary mechanisms that ensure its correct folding. Despite its potential failure on its exit, Chs3 is very stable in this compartment, which suggests its poor recognition by ER quality control mechanisms such as endoplasmic reticulum-associated degradation (ERAD). Here we show that proper N-glycosylation of its luminal domain is essential to prevent the aggregation of the protein and its subsequent recognition by the Hrd1-dependent ERAD-L machinery...

Coupling cell wall expansion with cell growth is a universal challenge faced by walled organisms. Mutations in Schizosaccharomyces pombe css1, which encodes a PM inositol phosphosphingolipid phospholipase C, prevent cell wall expansion but not synthesis of cell wall material. To probe how Css1 modulates cell wall formation we used classical and chemical genetics coupled with quantitative mass spectrometry. We found that elevated levels of the sphingolipid biosynthetic pathway's final product, mannosylinositol phosphorylceramide (MIPC), specifically correlated with the css1-3 phenotype...

Tunicamycins (TUNs) are Streptomyces -derived natural products, widely used to block protein N -glycosylation in eukaryotes or cell wall biosynthesis in bacteria. Modified or synthetic TUN analogues that uncouple these activities have considerable potential as novel mode-of-action antibacterial agents. Chemically modified TUNs reported previously with attenuated activity on yeast have pinpointed eukaryotic-specific chemophores in the uridyl group and the N -acyl chain length and terminal branching pattern. A small molecule screen of fatty acid biosynthetic primers identified several novel alicyclic- and neo -branched TUN N -acyl variants, with primer incorporation at the terminal omega -acyl position...

As one of the most important human fungal pathogens, Candida albicans senses and adapts to host niches with different pH values through the pH-responsive Rim101 pathway. Its transcription factor Rim101 activates the expression of alkaline pH-induced genes including PHR1 that encodes a glycosylphosphatidylinsitol-anchored β(1,3)-glucanosyltransferase critical for hyphal wall formation. The calcium/calcineurin signaling pathway is mediated by the transcription factor Crz1 in yeasts and other lower eukaryotes...

Stressors such as lack of access to feed, hot temperatures, transportation, and pen changes can cause impairment of ruminal and intestinal barrier function, also known as "leaky gut". Despite the known benefits of some nutritional approaches during periods of stress, little is understood regarding the underlying mechanisms, especially in dairy cows. We evaluated the effect of feeding a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek®, Diamond V, Cedar Rapids, IA) on the ileal transcriptome in response to feed restriction (FR), an established model to induce intestinal barrier dysfunction...

In order to produce therapeutic glycoproteins modified with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. In this study, a Pichia pastoris mutant Glyco4, which has a humanized N-glycosylation pathway and could successfully generate the human-like N-glycans, was carefully characterized. Glyco4 displays a significant growth delay and cell wall defects. Comparative transcriptomics reveals that manipulating the N-glycosylation pathway could notably affect the expression pattern of numerous biological pathways in Glyco4...

Escin is a mixture of over 30 glycosylated triterpenoid (saponin) structures, extracted from the dried fruit of horse chestnuts. Escin is currently used as an anti-inflammatory, and has potential applications in the treatment of arthritis and cancer. Engineered yeast would enable production of specific bioactive components of escin at industrial scale, however many saponins have been shown to be toxic to yeast. Here we report that a Saccharomyces cerevisiae strain specifically lacking the sterol C-5 desaturase gene ERG3, exhibits striking enhanced tolerance to escin treatment...

Recent research has unveiled numerous important functions of protein glycosylation in development, homeostasis, and diseases. A type of glycosylation taking center stage is protein O-mannosylation (POM), a posttranslational modification conserved in a wide range of organisms, from yeast to humans. In animals, POM plays a crucial role in the nervous system, while POM defects cause severe neurological abnormalities and congenital muscular dystrophies. However, the molecular and cellular mechanisms underlying POM functions and biosynthesis remain not well understood...