The Corn Smut Fungus Ustilago maydis as an Alternative Expression System for Biopharmaceuticals

Sarkari, Parveen; Feldbrügge, Michael; Schipper, Kerstin

doi:10.1007/978-3-319-27951-0_7

Cited by 7 publications

(7 citation statements)

References 112 publications

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“…As an alternative application, the screen will be employed to optimize our recently established protein expression platform ( Feldbrügge et al, 2013 ; Sarkari et al, 2016 ). Here, we use Cts1 as a carrier for valuable heterologous proteins.…”

Section: Discussionmentioning

confidence: 99%

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1

et al. 2020

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Subcellular targeting of proteins is essential to orchestrate cytokinesis in eukaryotic cells. During cell division of Ustilago maydis , for example, chitinases must be specifically targeted to the fragmentation zone at the site of cell division to degrade remnant chitin and thus separate mother and daughter cells. Chitinase Cts1 is exported to this location via an unconventional secretion pathway putatively operating in a lock-type manner. The underlying mechanism is largely unexplored. Here, we applied a forward genetic screen based on UV mutagenesis to identify components essential for Cts1 export. The screen revealed a novel factor termed Jps1 lacking known protein domains. Deletion of the corresponding gene confirmed its essential role for Cts1 secretion. Localization studies demonstrated that Jps1 colocalizes with Cts1 in the fragmentation zone of dividing yeast cells. While loss of Jps1 leads to exclusion of Cts1 from the fragmentation zone and strongly reduced unconventional secretion, deletion of the chitinase does not disturb Jps1 localization. Yeast-two hybrid experiments indicate that the two proteins might interact. In essence, we identified a novel component of unconventional secretion that functions in the fragmentation zone to enable export of Cts1. We hypothesize that Jps1 acts as an anchoring factor for Cts1.

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Section: Discussionmentioning

confidence: 99%

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1

et al. 2020

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“…The fact that ER/Golgi-mediated co- and post-translational modifications are avoided by unconventionally secreted proteins offers new possibilities for applications in biotechnology and medicine [ 20 , 21 ]. On this basis, we have established a novel system for protein production in the corn smut fungus Ustilago maydis .…”

Section: Introductionmentioning

confidence: 99%

“…Two such strains are available: the first carries a deletion of the preproprotease convertase Kexin 2 (Kex2) that activates secreted proteases, and in the second the genes for five major proteases were deleted sequentially. In these adapted strains other targets like single-chain variable fragments could also be secreted in an active state [ 21 , 25 ].…”

Section: Introductionmentioning

confidence: 99%

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

Terfrüchte

Reindl

Jankowski

et al. 2017

IJMS

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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.

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“…Filamentous fungi harbor several homologs to glycosylationspecific genes studied from other organisms, possibly offering the opportunity to test posttranslationally modified proteins (Rosi-Marshall et al, 2007). Pathogens are often genetically tractable and promoters active at specific stages in the infection cycle are available, facilitating engineering of pathogens as Trojan horse delivery vehicles (Table 1); nowadays, some plant pathogens are even used as expression systems in biotechnology because of their exceptional protein folding and secretion capabilities (Royer et al, 1995;Sarkari et al, 2016). Therefore, we predict that the TH strategy of exploiting a pathogen to study secreted host proteins should be generalizable to a variety of host-pathogen interactions.…”

Section: Variety Of Monocots and Dicotsmentioning

confidence: 99%

Pathogen Trojan Horse Delivers Bioactive Host Protein to Alter Maize Anther Cell Behavior in Situ

et al. 2018

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Small proteins are crucial signals during development, host defense, and physiology. The highly spatiotemporal restricted functions of signaling proteins remain challenging to study in planta. The several month span required to assess transgene expression, particularly in flowers, combined with the uncertainties from transgene position effects and ubiquitous or overexpression, makes monitoring of spatiotemporally restricted signaling proteins lengthy and difficult. This situation could be rectified with a transient assay in which protein deployment is tightly controlled spatially and temporally in planta to assess protein functions, timing, and cellular targets as well as to facilitate rapid mutagenesis to define functional protein domains. In maize (), secreted ZmMAC1 (MULTIPLE ARCHESPORIAL CELLS1) was proposed to trigger somatic niche formation during anther development by participating in a ligand-receptor module. Inspired by Homer's Trojan horse myth, we engineered a protein delivery system that exploits the secretory capabilities of the maize smut fungus , to allow protein delivery to individual cells in certain cell layers at precise time points. Pathogen-supplied ZmMAC1 cell-autonomously corrected both somatic cell division and differentiation defects in mutant Zm anthers. These results suggest that exploiting host-pathogen interactions may become a generally useful method for targeting host proteins to cell and tissue types to clarify cellular autonomy and to analyze steps in cell responses.

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The Corn Smut Fungus Ustilago maydis as an Alternative Expression System for Biopharmaceuticals

Cited by 7 publications

References 112 publications

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1

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

Pathogen Trojan Horse Delivers Bioactive Host Protein to Alter Maize Anther Cell Behavior in Situ

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