Molecular cloning and expression of fungal cellobiose transporters and ß-glucosidases conferring efficient cellobiose fermentation in Saccharomyces cerevisiae.

Cellobiose was once regarded as a byproduct that should be removed from biomass hydrolysates because of its inhibitory activity to cellulases. It was revealed, however, that cellobiose could serve as a co-substrate for xylose fermentation by engineered Saccharomyces cerevisiae. Despite its advantages, to date, little is known about cellodextrin transporters that endow S. cerevisiae with cellobiose transporting ability. In this study, engineered S. cerevisiae strains capable of fermenting cellobiose were constructed by expressing various fungal cellobiose transporters and intracellular ß-glucosidases. Among them, the strain expressing a putative sugar transporter from Penicillium chrysogenum (Pc_ST) and ß-glucosidase from Thielavia terrestris (Tt_BG) showed an improved cellobiose fermentation performance compared to the strain expressing a cellodextrin transporter from Neurospora crassa (Nc_CDT-1) and ß-glucosidase from N. crassa (Nc_GH1-1). Cellobiose fermentation by S. cerevisiae Pc_ST/Tt_BG under microaerobic conditions resulted in 14.5±0.5g/L of final ethanol concentration with a yield of 0.37±0.01g ethanol/g cellobiose, which are 22% and 26% higher than the corresponding values of S. cerevisiae Nc_CDT-1/Nc_GH1-1. These results suggest that the yield and rate of cellobiose fermentation can be improved by adopting optimal pairs of cellobiose transporters and ß-glucosidase.

Enhanced immune response of T-cell independent or dependent antigens in SIGN-R1 knock-out mice.

Dextran was used to explore a novel method of enhancing an immune response against T-cell independent type 2 (TI-2) polysaccharide antigens, because of its suitability as a model for the immunogenecity of many TI-2 polysaccharide antigens and its high affinity to SIGN-R1. Here we showed that the primary immune response of IgM, IgG3, and IgG2b was enhanced by dextran in SIGN-R1 knock-out (KO) mice, further evoking the induction of a secondary immune response to IgG2b in parallel. On the other hand, an immune response of IgG1 and IgG2b against T-cell dependent (TD) antigen was strongly enhanced by the administration of ovalbumin (OVA) in SIGN-R1 KO mice. These results indicate that SIGN-R1 is critical in the regulation of immune responses. Therefore, our study suggests that inhibition of TI-2 polysaccharide antigen uptake in SIGN-R1(+) macrophages contributes to the development of novel vaccination strategies against TI-2 polysaccharide antigens.

SIGN-R1, a C-type lectin, binds to Bip/GRP78 and this interaction mediates the regurgitation of T-cell-independent type 2 antigen dextran through the endoplasmic reticulum.

Capsular polysaccharides of Streptococcus pneumoniae are representative T-cell-independent type 2 (TI-2) antigens, frequently causing serious infections in children, the elderly, and immunocompromised patients. However, the detailed mechanism of this immune escape by CPSs is poorly understood. To pursue this question, polysaccharide dextran, ligand of SIGN-R1 as well as an appropriate model of the immunogenicity of many TI-2 polysaccharide antigens was used. SIGN-R1 bound to binding immunoglobulin protein (BiP), a well-characterized endoplasmic reticulum (ER) chaperone, primarily in non-ER compartments. Interestingly, SIGN-R1(+) macrophages in the MZ showed high expression of BiP, implying an important role of SIGN-R1 binding to BiP in vivo. To our surprise, dextran is rapidly transported into the ER and subsequently regurgitated out of cells in vitro or in vivo. BiP down-regulation in SIGN-R1 transfectant reduced the regurgitation of dextran, causing the accumulation of dextran in the ER. Therefore, these results demonstrated the first example to describe the intracellular trafficking and the regurgitation of TI-2 antigen dextran, suggesting the novel pathway of TI-2 antigen presentation to immune cells.