Applying Unconventional Secretion in Ustilago maydis for the Export of Functional Nanobodies

Abstract

Exploiting secretory pathways for production of heterologous proteins is highly advantageous with respect to efficient downstream processing. In eukaryotic systems the vast majority of heterologous proteins for biotechnological application is exported via the canonical endoplasmic reticulum–Golgi pathway. In the endomembrane system target proteins are often glycosylated and may thus be modified with foreign glycan patterns. This can be destructive for their activity or cause immune reactions against therapeutic proteins. Hence, using unconventional secretion for protein expression is an attractive alternative. In the fungal model Ustilago maydis, chitinase Cts1 is secreted via an unconventional pathway connected to cell separation which can be used to co-export heterologous proteins. Here, we apply this mechanism for the production of nanobodies. First, we achieved expression and unconventional secretion of a functional nanobody directed against green fluorescent protein (Gfp). Second, we found that Cts1 binds to chitin and that this feature can be applied to generate a Gfp-trap. Thus, we demonstrated the dual use of Cts1 serving both as export vehicle and as purification tag. Finally, we established and optimized the production of a nanobody against botulinum toxin A and hence describe the first pharmaceutically relevant target exported by Cts1-mediated unconventional secretion.

mRNA transport in fungal top models

“…Central remaining questions are how protein translation and transport are synchronized for efficient delivery of the translation products, and how specific local delivery is achieved. Finally, basic research on mRNA transport led to the discovery of a novel mechanism of unconventional secretion, which is currently being exploited for export of heterologous proteins for industrial application . In essence, studying eukaryotic microorganisms as role models deciphers fundamental processes of intracellular trafficking.…”

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1

“…Here, we use Cts1 as a carrier for valuable heterologous proteins. Exploiting the unconventional secretion route brings the advantage that N- glycosylation is circumvented and thus, sensitive proteins like bacterial enzymes can be exported in an active state ( Sarkari et al, 2014 ; Terfrüchte et al, 2017 ; Stoffels et al, 2019 ). The screen will be adapted to select for mutants with enhanced marker secretion to eliminate existing bottlenecks and thus enhance yields ( Terfrüchte et al, 2018 ).…”

Establishing Polycistronic Expression in the Model Microorganism Ustilago maydis

“…Besides its role as a model system, U. maydis is currently being developed as a production chassis for a wide range of biotechnological relevant compounds. This includes itaconic acid as a chemical platform molecule for biofuels, ustilagic acid and mannosylerythritol lipids (MEL) as biosurfactants, and various antibody formats as valuable proteins ( Teichmann et al, 2010 ; Feldbrügge et al, 2013 ; Sarkari et al, 2014 ; Terfrüchte et al, 2017 ; Becker et al, 2020 ). Furthermore, strains were generated to utilise cellobiose, xylan and polygalacturonic acid as a carbon source in the yeast phase, so that plant cell wall components including pectin can be used as starting point for sustainable production ( Geiser et al, 2016 ; Müller et al, 2018 ; Stoffels et al, 2020 ).…”