Kües, Ursula

[["dc.bibliographiccitation.issue","1"],["dc.bibliographiccitation.journal","AMB Express"],["dc.bibliographiccitation.volume","9"],["dc.contributor.author","Yin, Qiang"],["dc.contributor.author","Zhou, Gang"],["dc.contributor.author","Peng, Can"],["dc.contributor.author","Zhang, Yinliang"],["dc.contributor.author","Kües, Ursula"],["dc.contributor.author","Liu, Juanjuan"],["dc.contributor.author","Xiao, Yazhong"],["dc.contributor.author","Fang, Zemin"],["dc.date.accessioned","2021-06-01T10:48:06Z"],["dc.date.available","2021-06-01T10:48:06Z"],["dc.date.issued","2019"],["dc.description.abstract","Abstract Engineering of fungal laccases with optimum catalytic activity at alkaline pH has been a long-lasting challenge. In this study, a mutant library containing 3000 clones was obtained by error-prone PCR to adapt the optimum pH of a fungal laccase Lcc9 from the basidiomycete Coprinopsis cinerea . After three rounds of functional screening, a mutant with three amino acid changes (E116K, N229D, I393T) named PIE5 was selected. PIE5 showed an optimum pH of 8.5 and 8.0 against guaiacol and 2,6-DMP when expressed in Pichia pastoris , representing the first fungal laccase that possesses an optimum pH at an alkaline condition. Site directed mutagenesis disclosed that N229D contributed the most to the optimum pH increment. A single N229D mutation caused an increase in optimum pH by 1.5 units. When used in indigo dye decolorization, PIE5 efficiently decolorized 87.1 ± 1.1% and 90.9 ± 0.3% indigo dye at the optimum conditions of pH 7.0–7.5 and 60 °C, and with either methyl 3,5-dimethoxy-4-hydroxybenzoate or 2,2′-azino-bis(3-ethylbenzothazoline-6-sulfonate) as the mediator. In comparison, the commercially available fungal laccase TvLac from Trametes villosa decolorized 84.3 ± 1.8% of indigo dye under its optimum conditions (opt. pH 5.0 and 60 °C). The properties of an alkaline-dependent activity and the high indigo dye decolorization ability (1.3-fold better than the parental Lcc9) make the new fungal laccase PIE5 an alternative for specific industrial applications."],["dc.identifier.doi","10.1186/s13568-019-0878-2"],["dc.identifier.purl","https://resolver.sub.uni-goettingen.de/purl?gs-1/16397"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/85827"],["dc.language.iso","en"],["dc.notes.intern","DOI-Import GROB-425"],["dc.notes.intern","Merged from goescholar"],["dc.relation.eissn","2191-0855"],["dc.rights","CC BY 4.0"],["dc.rights.uri","https://creativecommons.org/licenses/by/4.0"],["dc.title","The first fungal laccase with an alkaline pH optimum obtained by directed evolution and its application in indigo dye decolorization"],["dc.type","journal_article"],["dc.type.internalPublication","yes"],["dc.type.version","published_version"],["dspace.entity.type","Publication"]]

[["dc.bibliographiccitation.issue","1"],["dc.bibliographiccitation.journal","Fungal Biology and Biotechnology"],["dc.bibliographiccitation.volume","7"],["dc.contributor.author","Dörnte, Bastian"],["dc.contributor.author","Peng, Can"],["dc.contributor.author","Fang, Zemin"],["dc.contributor.author","Kamran, Aysha"],["dc.contributor.author","Yulvizar, Cut"],["dc.contributor.author","Kües, Ursula"],["dc.date.accessioned","2021-06-01T10:48:07Z"],["dc.date.available","2021-06-01T10:48:07Z"],["dc.date.issued","2020"],["dc.description.abstract","Abstract Background Two reference strains have been sequenced from the mushroom Coprinopsis cinerea , monokaryon Okayama 7/#130 (OK130) and the self-compatible homokaryon AmutBmut. An adenine-auxotrophy in OK130 ( ade8-1 ) and a para -aminobenzoic acid (PABA)-auxotrophy in AmutBmut ( pab1-1 ) offer selection markers for transformations. Of these two strains, homokaryon AmutBmut had been transformed before to PABA-prototrophy and with the bacterial hygromycin resistance marker hph , respectively. Results Gene ade8 encodes a bifunctional enzyme with an N-terminal glycinamide ribonucleotide synthase (GARS) and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain required for steps 2 and 5 in the de novo biosynthesis of purines, respectively. In OK130, a missense mutation in ade8-1 rendered residue N231 for ribose recognition by the A loop of the GARS domain into D231. The new ade8 + vector p Cc Ade8 complements the auxotrophy of OK130 in transformations. Transformation rates with p Cc Ade8 in single-vector and co-transformations with ade8 + -selection were similarly high, unlike for trp1 + plasmids which exhibit suicidal feedback-effects in single-vector transformations with complementation of tryptophan synthase defects. As various other plasmids, unselected p Cc Ade8 helped in co-transformations of trp1 strains with a trp1 + -selection vector to overcome suicidal effects by transferred trp1 + . Co-transformation rates of p Cc Ade8 in OK130 under adenine selection with nuclear integration of unselected DNA were as high as 80% of clones. Co-transformation rates of expressed genes reached 26–42% for various laccase genes and up to 67% with lcc9 silencing vectors. The bacterial gene hph can also be used as another, albeit less efficient, selection marker for OK130 transformants, but with similarly high co-transformation rates. We further show that the pab1-1 defect in AmutBmut is due to a missense mutation which changed the conserved PIKGT motif for chorismate binding in the C-terminal PabB domain to PIEGT in the mutated 4-amino-4-deoxychorismate synthase. Conclusions ade8-1 and pab1-1 auxotrophic defects in C. cinerea reference strains OK130 and AmutBmut for complementation in transformation are described. p Cc Ade8 is a new transformation vector useful for selection in single and co-transformations of the sequenced monokaryon OK130 which was transformed for the first time. The bacterial gene hph can also be used as an additional selection marker in OK130, making in combination with ade8 + successive rounds of transformation possible."],["dc.description.sponsorship","Open-Access-Publikationsfonds 2021"],["dc.identifier.doi","10.1186/s40694-020-00105-0"],["dc.identifier.purl","https://resolver.sub.uni-goettingen.de/purl?gs-1/17606"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/85835"],["dc.language.iso","en"],["dc.notes.intern","DOI-Import GROB-425"],["dc.notes.intern","Merged from goescholar"],["dc.relation.eissn","2054-3085"],["dc.rights","CC BY 4.0"],["dc.rights.uri","https://creativecommons.org/licenses/by/4.0"],["dc.title","Selection markers for transformation of the sequenced reference monokaryon Okayama 7/#130 and homokaryon AmutBmut of Coprinopsis cinerea"],["dc.type","journal_article"],["dc.type.internalPublication","yes"],["dc.type.subtype","original_ja"],["dc.type.version","published_version"],["dspace.entity.type","Publication"]]