The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo-and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single-and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows highefficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno-and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.
The steady-state kinetic parameters of pyridoxal 5’-phosphate-dependent
recombinant methionine γ -lyase from three pathogenic bacteria,
Clostridium tetani, Clostridium sporogenes,
and Porphyromonas gingivalis, were determined in β-
and γ-elimination reactions. The enzyme from C. sporogenes is
characterized by the highest catalytic efficiency in the γ-elimination reaction
of L-methionine. It was demonstrated that the enzyme from
these three sources exists as a tetramer. The N-terminal poly-histidine
fragment of three recombinant enzymes influences their catalytic activity and
facilitates the aggregation of monomers to yield dimeric forms under denaturing
conditions. The cytotoxicity of methionine γ-lyase from C.
sporogenes and C. tetani in comparison with
Citrobacter freundii was evaluated using K562, PC-3, LnCap,
MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4–1.3 U/ml),
PC-3 (IC50=0.1–0.4 U/ml), and MCF7 (IC50=0.04–3.2 U/ml)
turned out to be the most sensitive cell lines.
O-acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5 0 -phosphate-dependent enzyme involved in microbial methionine biosynthesis. In this study, we report gene cloning, protein purification, and some biochemical characteristics of OAHS from Clostridioides difficile. The enzyme is a tetramer with molecular weight of 185 kDa. It possesses a high activity in the reaction of L-homocysteine synthesis, comparable to reported activities of OAHSes from other sources. OAHS activity is inhibited by metabolic end product L-methionine. L-Propargylglycine was found to be a suicide inhibitor of the enzyme. Substrate analogue N γ -acetyl-L-2,4-diaminobutyric acid is a competitive inhibitor of OAHS with K i = 0.04 mM. Analysis of C. difficile genome allows to suggest that the bacterium uses the way of direct sulfhydrylation for the synthesis of Lmethionine. The data obtained may provide the basis for further study of the role of OAHS in the pathogenic bacterium and the development of potential inhibitors.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.