Abstract
Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. The kinase Don3 is essential for functional assembly of the fragmentation zone and hence, for release of Cts1-fusion proteins. Here, we are first to develop regulatory systems for unconventional protein secretion using Don3 as a gatekeeper to control when export occurs. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional don3 regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.
Highlights
Recombinant proteins are ubiquitous biological products with versatile industrial, academic, and medical applications [1,2]
The nature of a protein largely influences the choice of a particular expression system, and not every protein is adequately expressed in the standard platform of choice [7]
We build on our mechanistic knowledge on Cts1 export to establish the first regulatory systems to control the unconventional secretion of heterologous proteins in
Summary
Recombinant proteins are ubiquitous biological products with versatile industrial, academic, and medical applications [1,2]. Secretory systems are advantageous because the protein product is exported into the medium allowing for economic and straightforward downstream processing workflows [8]. Due to their extraordinary secretion capacities and inexpensive cultivation, fungal expression hosts are promising candidates for novel platforms and already the preferred hosts for the production of proteases and other hydrolytic enzymes [9,10]. It is important to further develop tailor-made strategies to provide a broad repertoire of potent fungal host organisms and enable the economic production of all relevant requested proteins in their functional form
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