O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in E. coli. OBP (P1, P2, or P3) was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in E. coli. Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research.

Mercury pollution is a kind of heavy metal pollution with great harm and strong toxicity which exists worldwide. Some microorganisms can convert highly toxic methylmercury into inorganic mercury compounds with significantly reduced toxicity. This is an effective means of methylmercury pollution remediation. As a safe microorganism with great potential in the remediation of heavy metal pollution, Rhodotorula mucilaginosa has not been studied in the remediation of methylmercury. Here, a R. mucilaginosa strain Rm4 with high methylmercury resistance was obtained by wild-strain screening. Its minimal inhibitory concentration and minimum lethal concentration reached 3 and 6 mg/L, respectively. At the same time, Rm4 can also degrade methylmercury. Unlike the traditional microbial methylmercury degradation pathways, R. mucilaginosa ’s genome does not encode the organomercury lyase gene MerB. However, transcriptomic analysis revealed that the glutathione reductase of R. mucilaginosa responds to the methylmercury degradation process. Structural domain analysis and molecular docking experiments suggest that the glutathione reductase of R. mucilaginosa has the potential to directly or indirectly participate in methylmercury degradation metabolism. Metabolic indicator tests of engineered strains overexpressing the glutathione reductase encoding gene also support this notion. IMPORTANCE: The remediation of methylmercury pollution is crucial for environmental health. The ability of Rhodotorula mucilaginosa to resist and degrade methylmercury offers a new avenue for bioremediation efforts. Understanding the metabolic pathways involved, particularly the role of glutathione reductase, enhances our knowledge of how Rhodotorula mucilaginosa responds to methylmercury and opens up new possibilities for future research in bioremediation strategies.

TurboID, a proximity labeling method based on mutant biotin ligase, is an efficient new technique for recognizing protein–protein interactions and has been successfully applied to living cells. Sialic acid is typically the terminal monosaccharide attached to many glycoproteins and plays many important roles in many biological processes. However, the lack of enrichment methods for the terminal sialic acid glycosylation in vivo hinders the identification and analysis of this glycosylation. Here, we introduced TurboID to identify the terminal sialic acid glycosylation in living cells. SpCBM, the carbohydrate-binding domain of sialidase from Streptococcus pneumoniae, was fused with TurboID and overexpressed in HeLa cells. After streptavidin-based purification and detection by mass spectrometry (MS), we identified 31 terminal sialic acid N-glycosylated sites and 1359 putative terminal sialic acid glycosylated proteins, many of which were located in the cytoplasm and nucleus.