Improved expression of single-chain antibodies in Ustilago maydis

Sarkari, Parveen; Reindl, Michèle; Stock, Janpeter; Müller, Olaf; Kahmann, Regine; Feldbrügge, Michael; Schipper, Kerstin

doi:10.1016/j.jbiotec.2014.06.028

Cited by 40 publications

(66 citation statements)

References 51 publications

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“…Passage through the ER-Golgi apparatus pathway could lead to proteolytic processing of Cts2, similar to the zymogenic properties of yeast chitin synthase 3 (58) or activation of killer toxins by the protease Kex2 (59). Consistent with this, the S. cerevisiae chitinase ScCts1p, which mediates cytokinesis, is secreted in a signal peptide-dependent manner and requires glycosylation in the ER, whereas ScCts2p, like Cts1, lacks an N-terminal secretion signal (9,19,60). Hence, in general, chitinases seem to employ different modes of secretion, which might be required for their differential regulation or might be caused by their ability to bind sugar residues, which would interfere with glycosylation.…”

Section: Discussionmentioning

confidence: 79%

Chitinases Are Essential for Cell Separation in Ustilago maydis

et al. 2015

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Chitin is an essential component of the fungal cell wall, providing rigidity and stability. Its degradation is mediated by chitinases and supposedly ensures the dynamic plasticity of the cell wall during growth and morphogenesis. Hence, chitinases should be particularly important for fungi with dramatic morphological changes, such as Ustilago maydis. This smut fungus switches from yeast to filamentous growth for plant infection, proliferates as a mycelium in planta, and forms teliospores for spreading. Here, we investigate the contribution of its four chitinolytic enzymes to the different morphological changes during the complete life cycle in a comprehensive study of deletion strains combined with biochemical and cell biological approaches. Interestingly, two chitinases act redundantly in cell separation during yeast growth. They mediate the degradation of remnant chitin in the fragmentation zone between mother and daughter cell. In contrast, even the complete lack of chitinolytic activity does not affect formation of the infectious filament, infection, biotrophic growth, or teliospore germination. Thus, unexpectedly we can exclude a major role for chitinolytic enzymes in morphogenesis or pathogenicity of U. maydis. Nevertheless, redundant activity of even two chitinases is essential for cell separation during saprophytic growth, possibly to improve nutrient access or spreading of yeast cells by wind or rain.

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Section: Discussionmentioning

confidence: 79%

Chitinases Are Essential for Cell Separation in Ustilago maydis

et al. 2015

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“…1A). These two constitutive promoters were previously described to mediate high (P otef ) and very high (P oma ) gene expression during axenic growth (47). A Golden Gate cloning strategy was applied in which flanking regions of about 1 kb in length were combined with a resistance cassette module containing the respective artificial promoter (40).…”

Section: Resultsmentioning

confidence: 99%

“…This would provide a means to fine-tune the expression of genes for hydrolytic enzymes in a way that allows for a good balance between high activity, efficient secretion, and cell growth. In this respect, the enhancement of the stability and amount of the secreted enzymes by reducing the proteolytic potential (47) and manipulating the unfolded protein response will also be a focal point (68,69). Strategies to expand substrate repertoire and complexity.…”

Section: Discussionmentioning

confidence: 99%

“…This strategy might also be a good means to provide flexibility by adapting the activated strains and their ratios depending on the substrate to be degraded. In U. maydis, it is furthermore possible to enable the simultaneous activation of multiple CAZymes in a single strain using resistance marker recycling (47,75). That way, specialized strains for the degradation of different lignocellulose-building blocks could be developed.…”

Section: Discussionmentioning

confidence: 99%

“…Both parts were combined with a PCR fragment containing P oma in a three-fragment ligation. The 1.3-kb PCR product containing P oma was amplified from pUMa2113 (pRabX1PomaGus-SHH-Cts 1 ubi1 3= untranslated region [UTR] [47]) using primers oRL1855 and oRL1856 and digested with XbaI and SfiI to be compatible for ligation.…”

Section: Methodsmentioning

confidence: 99%

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Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

Geiser

Reindl

Blank

et al. 2016

Appl Environ Microbiol

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The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and ␤-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCEThis study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future. O ne central aim of a sustainable bioeconomy is the switch from fossil-to bio-based production of platform chemicals and other valuable substances. To date, the most promising feedstock for biorefineries is lignocellulosic nonfood plant biomass (1). Lignocellulose is a complex composite mainly consisting of cellulose, hemicelluloses, pectin, and lignin (2). The selective conversion of lignocellulosic biomass comprises different steps: pret...

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A Reporter System to Study Unconventional Secretion of Proteins Avoiding N-Glycosylation in Ustilago maydis

Stock¹,

Terfrüchte²,

Schipper³

2016

Methods in Molecular Biology

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Unconventional secretion of proteins in eukaryotes is characterized by the circumvention of the Endoplasmic Reticulum (ER). As a consequence proteins exported by unconventional pathways lack N-glycosylation, a post-transcriptional modification that is initiated in the ER during classical secretion. We are exploiting the well-established enzyme β-glucuronidase (GUS) to assay unconventional protein secretion (UPS). This bacterial protein is perfectly suited for this purpose because it carries a eukaryotic N-glycosylation motif. Modification of this residue by attachment of sugar moieties during the passage of the ER apparently causes a very strong reduction in GUS activity. Hence, this enzyme can only be secreted in an active state, if the export mechanism does not involve ER passage. Here, we describe a reporter system applied in the corn smut fungus Ustilago maydis that is based on this observation and can be used to test if candidate proteins are secreted to the culture supernatant via alternative pathways avoiding N-glycosylation. Importantly, this system is the basis for the establishment of genetic screens providing mechanistic insights into unknown UPS pathways in the future.

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Improved expression of single-chain antibodies in Ustilago maydis

Cited by 40 publications

References 51 publications

Chitinases Are Essential for Cell Separation in Ustilago maydis

Chitinases Are Essential for Cell Separation in Ustilago maydis

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

A Reporter System to Study Unconventional Secretion of Proteins Avoiding N-Glycosylation in Ustilago maydis

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