Integrated genomics and phenotype microarray analysis of Saccharomyces cerevisiae industrial strains for rice wine fermentation and recombinant protein production.

The industrial potential of Saccharomyces cerevisiae has extended beyond its traditional use in fermentation to various applications, including recombinant protein production. Herein, comparative genomics was performed with three industrial S. cerevisiae strains and revealed a heterozygous diploid genome for the 98-5 and KSD-YC strains (exploited for rice wine fermentation) and a haploid genome for strain Y2805 (used for recombinant protein production). Phylogenomic analysis indicated that Y2805 was closely associated with the reference strain S288C, whereas KSD-YC and 98-5 were grouped with Asian and European wine strains, respectively. Particularly, a single nucleotide polymorphism (SNP) in FDC1, involved in the biosynthesis of 4-vinylguaiacol (4-VG, a phenolic compound with a clove-like aroma), was found in KSD-YC, consistent with its lack of 4-VG production. Phenotype microarray (PM) analysis showed that KSD-YC and 98-5 displayed broader substrate utilization than S288C and Y2805. The SNPs detected by genome comparison were mapped to the genes responsible for the observed phenotypic differences. In addition, detailed information on the structural organization of Y2805 selection markers was validated by Sanger sequencing. Integrated genomics and PM analysis elucidated the evolutionary history and genetic diversity of industrial S. cerevisiae strains, providing a platform to improve fermentation processes and genetic manipulation.

Effects of altered N-glycan structures of Cryptococcus neoformans mannoproteins, MP98 (Cda2) and MP84 (Cda3), on interaction with host cells.

Cryptococcus neoformans is an opportunistic human fungal pathogen causing lethal meningoencephalitis. It has several cell wall mannoproteins (MPs) identified as immunoreactive antigens. To investigate the structure and function of N-glycans assembled on cryptococcal cell wall MPs in host cell interactions, we purified MP98 (Cda2) and MP84 (Cda3) expressed in wild-type (WT) and N-glycosylation-defective alg3 mutant (alg3Δ) strains. HPLC and MALDI-TOF analysis of the MP proteins from the WT revealed protein-specific glycan structures with different extents of hypermannosylation and xylose/xylose phosphate addition. In alg3Δ, MP98 and MP84 had truncated core N-glycans, containing mostly five and seven mannoses (M5 and M7 forms), respectively. In vitro adhesion and uptake assays indicated that the altered core N-glycans did not affect adhesion affinities to host cells although the capacity to induce the immune response of bone-marrow derived dendritic cells (BMDCs) decreased. Intriguingly, the removal of all N-glycosylation sites on MP84 increased adhesion to host cells and enhanced the induction of cytokine secretion from BMDCs compared with that on MP84 carrying WT N-glycans. Therefore, the structure-dependent effects of N-glycans suggested their complex roles in modulating the interaction of MPs with host cells to avoid nonspecific adherence to host cells and host immune response hyperactivation.

Extension of O-Linked Mannosylation in the Golgi Apparatus Is Critical for Cell Wall Integrity Signaling and Interaction with Host Cells in Cryptococcus neoformans Pathogenesis.

The human-pathogenic yeast Cryptococcus neoformans assembles two types of O-linked glycans on its proteins. In this study, we identified and functionally characterized the C. neoformans CAP6 gene, encoding an α1,3-mannosyltransferase responsible for the second mannose addition to minor O-glycans containing xylose in the Golgi apparatus. Two cell surface sensor proteins, Wml1 (WSC/Mid2-like) and Wml2, were found to be independent substrates of Cap6-mediated minor or Ktr3-mediated major O-mannosylation, respectively. The double deletion of KTR3 and CAP6 (ktr3Δ cap6Δ) completely blocked the mannose addition at the second position of O-glycans, resulting in the accumulation of proteins with O-glycans carrying only a single mannose. Tunicamycin (TM)-induced phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) was greatly decreased in both ktr3Δ cap6Δ and wml1Δ wml2Δ strains. Transcriptome profiling of the ktr3Δ cap6Δ strain upon TM treatment revealed decreased expression of genes involved in the Mpk1-dependent cell wall integrity (CWI) pathway. Consistent with its defective growth under several stress conditions, the ktr3Δ cap6Δ strain was avirulent in a mouse model of cryptococcosis. Associated with this virulence defect, the ktr3Δ cap6Δ strain showed decreased adhesion to lung epithelial cells, decreased proliferation within macrophages, and reduced transcytosis of the blood-brain barrier (BBB). Notably, the ktr3Δ cap6Δ strain showed reduced induction of the host immune response and defective trafficking of ergosterol, an immunoreactive fungal molecule. In conclusion, O-glycan extension in the Golgi apparatus plays critical roles in various pathobiological processes, such as CWI signaling and stress resistance and interaction with host cells in C. neoformans. IMPORTANCE Cryptococcus neoformans assembles two types of O-linked glycans on its surface proteins, the more abundant major O-glycans that do not contain xylose residues and minor O-glycans containing xylose. Here, we demonstrate the role of the Cap6 α1,3-mannosyltransferase in the synthesis of minor O-glycans. Previously proposed to be involved in capsule biosynthesis, Cap6 works with the related Ktr3 α1,2-mannosyltransferase to synthesize O-glycans on their target proteins. We also identified two novel C. neoformans stress sensors that require Ktr3- and Cap6-mediated posttranslational modification for full function. Accordingly, the ktr3Δ cap6Δ double O-glycan mutant strain displays defects in stress signaling pathways, CWI, and ergosterol trafficking. Furthermore, the ktr3Δ cap6Δ strain is completely avirulent in a mouse infection model. Together, these results demonstrate critical roles for O-glycosylation in fungal pathogenesis. As there are no human homologs for Cap6 or Ktr3, these fungus-specific mannosyltransferases are novel targets for antifungal therapy.

Selective Vapor Permeation Behavior of Crosslinked PAMPS Membranes.

The effect of crosslinking on vapor permeation behavior of polyelectrolyte membranes was studied. Poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) membranes were crosslinked by using crosslinkers with different lengths between the reactive ends. Crosslinked membranes with a longer crosslinking length showed lower water vapor permeability due to the lower sorption coefficient. It was also shown that the permeation behavior of PAMPS membranes was more affected by sorption than diffusion. For chemical protection applications, the ratio of water over chemical warfare agent permeability (i.e., selectivity) was measured. Due to the high water solubility of polyelectrolytes, crosslinked PAMPS allowed for the selective permeation of water over harmful chemical vapor, showing a selectivity of 20. The addition of electrospun nylon nanofibers in the membranes significantly improved the selectivity to 80, since the embedded nanofibers effectively reduced both diffusion and sorption coefficients of chemical warfare agents.

Surface crosslinking of 6FDA-durene nanofibers for porous carbon nanofiber electrodes in electrochemical double layer capacitors.

Tailoring the chemical structures of a precursor polymer for carbon nanofibers (CNFs) produced by thermal treatment of electrospun nanofibers was studied to prepare the electrodes for electrochemical double layer capacitors (EDLCs). To improve energy storage performance of CNF electrodes, 6FDA-durene nanofibers were crosslinked by a vapor crosslinking method, and subsequently carbonized. Chemical modification via crosslinking was confirmed by FTIR spectra while the conversion of crosslinked 6FDA-durene into carbon was done by Raman spectroscopy. Electrochemical performance of these CNF electrodes was evaluated by assembling coin cells, and the CNFs derived from crosslinked 6FDA-durene nanofibers showed higher specific capacitances, energy densities and cycling stability than those from non-crosslinked ones. It was also shown that CNFs prepared using 1 min crosslinking exhibit the highest energy storage performances, a specific capacitance of 301 F g-1 (at 10 mV s-1), and the maximum energy density of 11.1 Wh kg-1 (at 0.5 A g-1) and power density of 1.8 kW kg-1 (at 6 A g-1). Surface area and porosity of CNFs, which is critical for the performance of EDLC electrodes, were studied by nitrogen adsorption/desorption measurements, and it was clearly seen that surface crosslinking of precursor polymers improved surface properties of the resultant CNFs.