Tackling destructive proteolysis of unconventionally secreted heterologous proteins in Ustilago maydis

Terfrüchte, Marius; Wewetzer, Sandra J.; Sarkari, Parveen; Stollewerk, Daniel; Franz‐Wachtel, Mirita; Maček, Boris; Schlepütz, Tino; Feldbrügge, Michael; Büchs, Jochen; Schipper, Kerstin

doi:10.1016/j.jbiotec.2018.07.035

Cited by 23 publications

(22 citation statements)

References 67 publications

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“…To identify the proteases responsible for product degradation, investigation of the proteins secreted into the medium can be performed. This may also be a substantial task since the number of secreted proteins in fungal cultivations may vary from 300 to 700 or more, depending on the growth conditions and the species (Jun et al 2013;Schmoll et al 2016;Terfrüchte et al 2018). In A. oryzae, more than 130 exoand endopeptidase genes have been identified (Kobayashi et al 2007).…”

Section: Improving Protein Quality and Stabilitymentioning

confidence: 99%

“…In Ustilago maydis, the use of proteomics identified 477 proteins in submerged cultivation of which 21 were annotated as proteases or peptidases. Surprisingly, engineered strains in which deletion of major protease genes has been introduced and not displaying detectable protease activity in casein-indicator plates still contained a significant protease activity (Terfrüchte et al 2018). As in many other fungi, the arsenal of proteases present in wild-type strains ensures that available protein sources can be degraded under different growth conditions even if loss of function mutations in major protease genes occur.…”

Section: Improving Protein Quality and Stabilitymentioning

confidence: 99%

See 1 more Smart Citation

Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories

Arnau

Yaver

Hjort

2020

Grand Challenges in Biology and Biotechnology

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Section: Improving Protein Quality and Stabilitymentioning

confidence: 99%

Section: Improving Protein Quality and Stabilitymentioning

confidence: 99%

Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories

Arnau

Yaver

Hjort

2020

Grand Challenges in Biology and Biotechnology

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“…Homologous recombination is used for precise and stable integration of DNA constructs at defined genomic loci (Loubradou et al, 2001). Resistance marker recycling allows the generation of marker free production strains as well as the generation of multiple gene deletions (Khrunyk et al, 2010;Terfrüchte et al, 2018). Importantly, online bioengineering procedures for fermentation of U. maydis are already implemented (Kreyenschulte et al, 2015;Müller et al, 2018).…”

Section: Production Of Plant and Fungal Sesquiterpenoids In U Maydismentioning

confidence: 99%

“…This is strongly supported by the discovery that valuable proteins like antibody formats can be exported in the culture medium by a novel unconventional secretion pathway (Sarkari et al, 2014). This pathway prevents undesired N-glycosylation of the product that is usually processed during conventional secretion (Stock et al, 2012;Sarkari et al, 2014;Terfrüchte et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Ustilago maydis Serves as a Novel Production Host for the Synthesis of Plant and Fungal Sesquiterpenoids

et al. 2020

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Sesquiterpenoids are important secondary metabolites with various pharma-and nutraceutical properties. In particular, higher basidiomycetes possess a versatile biosynthetic repertoire for these bioactive compounds. To date, only a few microbial production systems for fungal sesquiterpenoids have been established. Here, we introduce Ustilago maydis as a novel production host. This model fungus is a close relative of higher basidiomycetes. It offers the advantage of metabolic compatibility and potential tolerance for substances toxic to other microorganisms. We successfully implemented a heterologous pathway to produce the carotenoid lycopene that served as a straightforward read-out for precursor pathway engineering. Overexpressing genes encoding enzymes of the mevalonate pathway resulted in increased lycopene levels. Verifying the subcellular localization of the relevant enzymes revealed that initial metabolic reactions might take place in peroxisomes: despite the absence of a canonical peroxisomal targeting sequence, acetyl-CoA C-acetyltransferase Aat1 localized to peroxisomes. By expressing the plant (+)-valencene synthase CnVS and the basidiomycete sesquiterpenoid synthase Cop6, we succeeded in producing (+)valencene and α-cuprenene, respectively. Importantly, the fungal compound yielded about tenfold higher titers in comparison to the plant substance. This proof of principle demonstrates that U. maydis can serve as promising novel chassis for the production of terpenoids.

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“…This is strongly supported by the discovery that valuable proteins like antibody formats can be exported in the culture medium by a novel unconventional secretion pathway (Sarkari et al, 2014). This pathway prevents undesired glycosylation of the product that is usually processed during conventional secretion (Stock et al, 2012;Sarkari et al, 2014;Terfrüchte et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Ustilago maydisserves as a novel production host for the synthesis of plant and fungal sesquiterpenoids

Lee

Hilgers

Loeschke

et al. 2020

Preprint

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AbtractSesquiterpenoids are important secondary metabolites with various pharma-and nutraceutical properties. In particular, higher basidiomycetes possess a versatile biosynthetic repertoire for these bioactive compounds. To date, only a few microbial production systems for fungal sesquiterpenoids have been established. Here, we introduce Ustilago maydis as a novel production host. This model fungus is a close relative of higher basidiomycetes. It offers the advantage of metabolic compatibility and potential tolerance for substances toxic to other microorganisms. We successfully implemented a heterologous pathway to produce the carotenoid lycopene that served as a straightforward read-out for precursor pathway engineering. Overexpressing genes encoding enzymes of the mevalonate pathway resulted in increased lycopene levels. Verifying the subcellular localisation of the relevant enzymes revealed that initial metabolic reactions might take place in peroxisomes: despite the absence of a canonical peroxisomal targeting sequence, acetyl-CoA C-acetyltransferase Aat1 localised to peroxisomes. By expressing the plant (+)-valencene synthase CnVS and the basidiomycete sesquiterpenoid synthase Cop6, we succeeded in producing (+)-valencene and -cuprenene, respectively. Importantly, the fungal compound yielded about tenfold higher titres in comparison to the plant substance. This proof of principle demonstrates that U. maydis can serve as promising novel chassis for the production of terpenoids.

show abstract

Tackling destructive proteolysis of unconventionally secreted heterologous proteins in Ustilago maydis

Cited by 23 publications

References 67 publications

Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories

Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories

Ustilago maydis Serves as a Novel Production Host for the Synthesis of Plant and Fungal Sesquiterpenoids

Ustilago maydisserves as a novel production host for the synthesis of plant and fungal sesquiterpenoids

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