RAB Xpress

 

Unconventional secretion and its biotechnological applications in corn smut

Our group is mainly interested in a novel mechanism of unconventional secretion in the corn smut fungus Ustilago maydis. In different projects we aim on uncovering the molecular details of this mechanism and on applying it for protein production.

Uncovering the mechanism of unconventional secretion

Protein secretion in eukaryotic cells mostly occurs via the endomembrane system including endoplasmic reticulum (ER) and Golgi apparatus. Proteins directed to this route carry an N-terminal secretion signal that targets them to the ER. Within the endomembrane system proteins are post-translationally modified e.g. by N-glycosylation. Besides this well-established mechanism unconventional secretion mechanisms exist. These include vesicular and non-vesicular routes which are independent of the canonical signal peptides for classical secretion. In U. maydis we discovered an unconventional secretory mechanism for chitinase Cts1. In a basic research project we study this mechanism in detail in order to understand why it is employed by the cell. To this end, we identify components of the secretion pathway followed by their functional characterization.

Fig. 1. Gus reporter system to assay unconventional secretion on a fluorogenic substrate.The bacterial enzyme Gus cannot be secreted via the canonical secretion system (Sp-Gus) in an active state because it is sensitive to N-glycosylation (indicated by asterisk). In contrast Gus-Cts1 fusions show Gus activity in the culture supernatant demonstrating circumvention of ER and thus, its unconventional secretion. Cytoplasmic Gus deals as a control for cell lysis. (Feldbrügge et al., 2013)

 

Establishment and application of a novel protein expression platform

Establishing and applying novel protein expression systems is crucial in producing the full repertoire of economically relevant proteins in the area of white biotechnology. Although a variety of prokaryotic and eukaryotic expression systems is already successfully applied in the industrial biotechnology, there is a great need for novel expression platforms that allow heterologous expression of proteins that are currently difficult to produce. To fill existing gaps, we use the basidiomycete fungus Ustilago maydis as novel cellular platform for protein production. This eukaryotic microorganism is very well suited for biotechnological applications as it is amenable to genetic manipulation and mutants can be generated rapidly. Furthermore, biochemical and proteomic approaches are well established and easily applicable.
In an applied project, we use an unconventional secretion mechanism based on chitinase Cts1 in U. maydis to export heterologous proteins of interest, i.e. therapeutic antibody formats (Fig. 1). The advantage is that proteins secreted via this route are not N-glycosylated. This can be advantageous for expression of bacterial or therapeutic proteins. Currently, we are optimizing the system on different levels. For example, knowledge gained on the mechanism of secretion (see above) is applied for improving the secretion of heterologous target proteins. In parallel, we are scaling-up the system by establishing optimal cultivation and fermentation conditions.

Fig. 2. Cts1-mediated unconventional secretion of an anti-botulinum toxin nanobody. A Precipitated cell-free culture supernatants after different times of incubation. The full length fusion protein is depicted with a filled arrowhead. B Supernatant samples harvested at indicated time points were subjected to ELISA against BoNTA (biological triplicates). (Terfrüchte et al., 2016)

 

Biomass valorization – from biomass side-streams to valuable products

The conversion of biomass side-streams or waste to valuable substances is one main challenge in bioeconomy. As a plant pathogen, U. maydis contains a distinct set of hydrolytic enzymes that can be exploited for biomass degradation. These can be complemented by potent heterologous enzymes using conventional secretion for eukaryotic and unconventional secretion for bacterial enzymes. In addition, U. maydis is a natural producer of organic acids and glycolipids. Both are considered valuable substances with multiple biotechnological applications. Hence it is conceivable to use U. maydis for one-pot biomass conversion into valuable products. Together with cooperation partners (group of Dr. Nick Wieckx, RWTH Aachen) we provided first evidence, that U. maydis is able to convert simple biomass components into organic acid. Currently, this strategy is extended to more complex biomass sources.

Fig. 3. Production of organic acids from cellobiose.Cultivation of MB215 wild type (square) and the MB215 Pomabgl1 overexpression mutant (upside down triangle) in minimal cellobiose medium. A Growth curve. B Concentration of glucose (dashed lines) and cellobiose (solid lines) during cultivation of the strains in minimal cellobiose medium. C Production of malate (triangle) and itaconate (diamond) from cellobiose by MB215 Pomabgl1. (Geiser et al., 2016)
Verantwortlich für den Inhalt: E-Mail sendenProf. Dr. Michael Feldbrügge