14 research outputs found
Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii
To explore the potential of Ashbya gossypii as a
host for the expression of recombinant proteins and to
assess whether protein secretion would be more similar to
the closely related Saccharomyces cerevisiae or to other
filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase
I (CBHI) from the fungus Trichoderma reesei were
successfully expressed in A. gossypii from plasmids
containing the two micron sequences from S. cerevisiae,
under the S. cerevisiae PGK1 promoter. The native signal
sequences of EGI and CBHI were able to direct the
secretion of EGI and CBHI into the culture medium in A.
gossypii. Although CBHI activity was not detected using 4-
methylumbelliferyl-ÎČ-D-lactoside as substrate, the protein
was detected by Western blot using monoclonal antibodies.
EGI activity was detectable, the specific activity being
comparable to that produced by a similar EGI producing S.
cerevisiae construct. More EGI was secreted than CBHI, or
more active protein was produced. Partial characterization
of CBHI and EGI expressed in A. gossypii revealed
overglycosylation when compared with the native T. reesei
proteins, but the glycosylation was less extensive than on
cellulases expressed in S. cerevisiae.Fundação para a CiĂȘncia e a Tecnologia (FCT
Fungal chitinases: diversity, mechanistic properties and biotechnological potential
Chitin derivatives, chitosan and substituted chito-oligosaccharides have a wide spectrum of applications ranging from medicine to cosmetics and dietary supplements. With advancing knowledge about the substrate-binding properties of chitinases, enzyme-based production of these biotechnologically relevant sugars from biological resources is becoming increasingly interesting. Fungi have high numbers of glycoside hydrolase family 18 chitinases with different substrate-binding site architectures. As presented in this review, the large diversity of fungal chitinases is an interesting starting point for protein engineering. In this review, recent data about the architecture of the substrate-binding clefts of fungal chitinases, in connection with their hydrolytic and transglycolytic abilities, and the development of chitinase inhibitors are summarized. Furthermore, the biological functions of chitinases, chitin and chitosan utilization by fungi, and the effects of these aspects on biotechnological applications, including protein overexpression and autolysis during industrial processes, are discussed in this review
SPLIFF: A Single-Cell Method to Map Protein-Protein Interactions in Time and Space
Protein interactions occur at certain times and at specific cellular places. The past years have seen a massive accumulation of binary protein-protein interaction data. The rapid increase of this context-free information necessitates robust methods to monitor protein interactions with temporal and spatial resolution in single cells. We have developed a simple split-ubiquitin-based method (SPLIFF) that uses the ratio of two fluorescent reporters as a signal for protein-protein interactions. One protein of the pair of interest is attached to the linear fusion of mCherry, the C-terminal half of ubiquitin, and GFP (mCherry-Cub-GFP). The other potential binding partner is expressed as a C-terminal fusion to the N-terminal half of ubiquitin (Nub). Upon co-expression the interaction between the two proteins of interest induces the reassociation of Nub and Cub to the native-like ubiquitin. GFP is subsequently cleaved from the C-terminus of Cub and degraded whereas the red-fluorescent mCherry stays attached to the Cub-fusion protein. We first implemented this method in the model yeast Saccharomyces cerevisiae. One fusion protein is expressed in cells of the a-mating type and the complementary fusion protein in cells of the α-mating type. Upon mixing, both cell types fuse and the Nub- and Cub-fusion proteins are free to interact. The red and green fluorescence is monitored by two-channel fluorescence time-lapse microcopy. The moment of cell fusion defines the start of the analysis. The calculated ratio of green to red fluorescence allows mapping the spatiotemporal interaction profiles of the investigated proteins in single cells
A fluorescent reporter for mapping cellular proteinâprotein interactions in time and space
Functional characterization of extracellular chitinase encoded by the YlCTS1 gene in a dimorphic yeast Yarrowia lipolytica
Identification of genome-wide binding sites of heat shock factor 1, Hsf1, under basal conditions in the human pathogenic yeast, Candida albicans
Glucanases and Chitinases
In many yeast and fungi, ÎČ-(1,3)-glucan and chitin are essential components of the cell wall, an important structure that surrounds cells and which is responsible for their mechanical protection and necessary for maintaining the cellular shape. In addition, the cell wall is a dynamic structure that needs to be remodelled along with the different phases of the fungal life cycle or in response to extracellular stimuli. Since ÎČ-(1,3)-glucan and chitin perform a central structural role in the assembly of the cell wall, it has been postulated that ÎČ-(1,3)-glucanases and chitinases should perform an important function in cell wall softening and remodelling. This review focusses on fungal glucanases and chitinases and their role during fungal morphogenesis.This work was supported by grants from the Spanish Government to CR (BFU2017-84508-P) and CRV (BIO2015-70195-C2-1-R) and from Junta de Castilla y LeĂłn to CR (SA116G19). The IBFG is supported by Programa âEscalera de Excelenciaâ from Junta de Castilla y LeĂłn (CLU-2017-03) and University of Salamanca. All Spanish funding is co-sponsored by the European Union FEDER programme.Peer reviewe