Fushimi Pharmaceutical Co., Ltd.

Acetic acid bacteria convert environmental sugars and alcohols into acetic acid and various sugars through oxidative fermentation, resulting in the accumulation of these compounds at high concentrations in the culture medium. One such product is the rare sugar 5-keto-d-fructose (5-KF). In Gluconobacter species, 5-KF is transported into the cell and reduced to fructose in a single step by 5-ketofructose reductase, allowing entry into glycolysis. However, it remains unclear whether eukaryotic microorganisms can metabolize 5-KF or which genes are involved in this process. In this study, we investigated the ability of various yeasts to utilize 5-KF and identified genes involved in its metabolism. The model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were unable to grow on 5-KF, whereas the oleaginous yeast Lipomyces starkeyi efficiently metabolized this sugar. RNA-seq analysis of L. starkeyi grown on 5-KF revealed genes specifically upregulated in response to 5-KF. Based on gene annotation and expression profiles, a putative metabolic pathway was proposed. Gene knockout analyses showed that mutants deficient in specific steps of the pathway grew on downstream intermediates but failed to grow on upstream substrates, indicating loss of the corresponding enzymatic functions. These results suggest that L. starkeyi metabolizes 5-KF via a multistep pathway, 5-KF → l-sorbose → d-sorbitol → d-fructose. This study provides the first evidence of a 5-KF metabolic pathway in yeast, distinct from the single-step conversion to fructose observed in Gluconobacter species.

Immunoglobulin G (IgG) antibodies possess a conserved N-glycosylation site in the Fc domain. In FcγRIIIa affinity column chromatography, unglycosylated, hemiglycosylated, and fully glycosylated IgG retention times differ considerably. Using retention-time differences, 66 different trastuzumab antibodies with symmetric and asymmetric homogeneous glycans were prepared systematically, substantially expanding the scope of IgGs with homogeneous glycans. Using the prepared trastuzumab with homogeneous glycans, thermal stability and antibody-dependent cellular cytotoxicity were investigated. In some glycan series, a directly proportional relationship was observed between the thermal unfolding temperature (T_m) and the calorimetric unfolding heat (ΔH_cal). Antibody function could be deduced from the combination of a pair of glycans in an intact form. Controlling glycan structure through the combination of a pair of glycans permits the precise tuning of stability and effector functions of IgG. Overall, our technology can be used to investigate the effects of glycans on antibody functions.

Both pyruvylation and sialylation onto the terminus of oligosaccharides of N-glycoproteins seem to be structurally and functionally similar with a property of conferring negative charge. However, detailed molecular characteristics of pyruvylation and sialylation in vivo were elusive. Here, to investigate an effect of terminal pyruvylation to N-glycan on in vivo biodistribution and kinetics, we prepared human serum albumin (HSA) modified with pyruvylated N-glycan (PvG), conjugated with HiLyte Fluor 750 (FL750-PvGHSA). In vivo imaging by using FL750-PvGHSA revealed that terminally pyruvylated N-glycoalbumin was excreted like sialylated N-glycoalbumin, suggesting that pyruvylation mimics sialylation in in vivo biodistribution and kinetics of N-glycoproteins. Terminal pyruvylation onto N-glycans can be a potential tool for a novel glycoengineering strategy.