title: Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis.
abstract: The demand on the biotechnological production of proteins for pharmaceutical, medical and industrial applications is steadily growing. For the production of challenging proteins, we aim to establish a novel expression platform in the well characterized eukaryotic microorganism Ustilago maydis. In filaments of this fungus, secretion of the endochitinase Cts1 depends on mRNA transport along microtubules, which is mediated by the key RNA-binding protein Rrm4. Here, we report two important findings: (i) Cts1 secretion occurs via a novel unconventional route and (ii) this secretory mechanism can be exploited for the export of active heterologous proteins. Initially, we used β-glucuronidase (Gus) as a reporter for unconventional secretion. This bacterial enzyme is inactivated by N-glycosylation during its passage through the conventional eukaryotic secretory pathway. By contrast, in our system Gus was exported in its active form by fusion to Cts1 confirming its secretion by an unconventional route. As a proof-of-principle for economically important biopharmaceuticals we expressed an active single-chain antibody. Importantly, the novel protein export pathway circumvents N-glycosylation which is advantageous in many applications, e.g., to avoid undesired immune reactions in humans. Thus, the unconventional Cts1 secretion machinery has a high potential for the production of biotechnologically relevant proteins.
authors.affiliation: Heinrich-Heine University Düsseldorf, Institute for Microbiology/Bldg. 26.12, 40225 Düsseldorf, Germany.
authors.forename: Janpeter
authors.initials: J
authors.lastname: Stock
authors.forename: Parveen
authors.initials: P
authors.lastname: Sarkari
authors.forename: Saskia
authors.initials: S
authors.lastname: Kreibich
authors.forename: Thomas
authors.initials: T
authors.lastname: Brefort
authors.forename: Michael
authors.initials: M
authors.lastname: Feldbrügge
authors.forename: Kerstin
authors.initials: K
authors.lastname: Schipper
mesh_terms: D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods*; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D005966:Glucuronidase; D006031:Glycosylation; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics; D014588:Ustilago / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D002688:Chitinases; D005966:Glucuronidase