Open AccessMultiplex Genetic Engineering Exploiting Pyrimidine Salvage Pathway-Based Endogenous Counterselectable Markers
Author(s) -
Lukas Birštonas,
Alex Dallemulle,
Manuel S. LópezBerges,
Ilse D. Jacobsen,
Martin Offterdinger,
Beate Abt,
Maria Straßburger,
Ingo Bauer,
Oliver Schmidt,
Bettina Sarg,
Herbert Lindner,
Hubertus Haas,
Fabio Gsaller
Publication year - 2020
Publication title -
mbio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.562
H-Index - 121
eISSN - 2161-2129
pISSN - 2150-7511
DOI - 10.1128/mbio.00230-20
Subject(s) - selectable marker , biology , computational biology , gene , genetics , plasmid
This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Aspergillus fumigatus . Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.