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  <doc>
    <id>62364</id>
    <completedYear/>
    <publishedYear>2024</publishedYear>
    <thesisYearAccepted/>
    <language>eng</language>
    <pageFirst>1</pageFirst>
    <pageLast>231</pageLast>
    <pageNumber/>
    <edition/>
    <issue/>
    <volume/>
    <type>doctoralthesis</type>
    <publisherName/>
    <publisherPlace>Berlin</publisherPlace>
    <creatingCorporation>Freie Universität Berlin, Universitätsbibliothek</creatingCorporation>
    <contributingCorporation/>
    <belongsToBibliography>0</belongsToBibliography>
    <completedDate>--</completedDate>
    <publishedDate>--</publishedDate>
    <thesisDateAccepted>2024-11-27</thesisDateAccepted>
    <title language="eng">Genetic tools for targeted genome editing and random mutagenesis of the rock-inhabiting black fungus Knufia petricola</title>
    <abstract language="eng">Rock-inhabiting black fungi are ubiquitously found on sun-exposed natural and human-made surfaces. These stresstolerant biofilm-formers are known to force the deterioration of the colonized surfaces. Black fungi belong to different classes of Ascomycota (Arthoniomycetes, Eurotiomycetes, and Dothideomycetes) but evolved the same morpho-physiological adaptations such as strong melanized multilayered cell walls and slow yeast-like or meristematic growth to resist extreme environmental conditions. The rock-inhabitant Knufia petricola was chosen as representative of the polyphyletic group of black fungi for studying mineral weathering, biofilm formation, and interaction with phototrophic microbes. For understanding the molecular basis of these traits, the genetic information and its regulation must be explored. Thus, the genome was sequenced, first -omics datasets obtained and protocols for the transformation of protoplasts and CRISPR/Cas9-based genome editing were implemented to enable genetic studies.&#13;
In scope of this work, the transformation protocol was optimized to improve the regeneration of K. petricola protoplasts. The successful implementation of multiplexed CRISPR/Cas9 and three additional selection marker systems (geneticin/nptII, glufosinate/bar, chlorimuron ethyl/sur) extended the existing strategies for targeted genome editing. While in the first promoter studies only constitutive promoters (PoliC, PgpdA) were identified as appropriate for expression of fluorescent reporter genes in K. petricola, the functionality of an inducible promoter (synthetic TET-on promoter system) could later be demonstrated. The identification and validation of defined genomic insertion sites enabled the neutral or color-based selection (igr1-5 or pks1, phs1, ade2) as well as the detection of correctly integrated expression constructs. Using the color-based transformant screening (black – pink/white/rose) accelerated the selection of transformants, especially for localization and protein-protein interaction studies (e.g., by studying bimolecular fluorescence complementation). Additionally, the fusion of two or three genes via the viral P2A motif was shown to allow the expression of multiple genes from a single expression cassette/integration site. Finally, the established tools enabled the implementation of a forward genetics tool (customized Ac/Ds transposon system) for regulatable in-vivo mutagenesis of K. petricola. The isolation and characterization of revertants/Ds reinsertion mutants demonstrated the functionality of the system which marks an important milestone for assigning functions to yet unknown species- and/or trait-specific genes. In sum, the genetic engineering toolbox is now fully equipped which paves the way for exploring the biology of K. petricola and other black fungi.</abstract>
    <enrichment key="opus.source">publish</enrichment>
    <author>Eileen A. Erdmann</author>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Microcolonial fungi</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Genetic engineering</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>CRISPR/Cas9-mediated genome editing</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>DHN melanin</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Transposon mutagenesis</value>
    </subject>
    <collection role="ddc" number="628">Sanitär- und Kommunaltechnik; Umwelttechnik</collection>
    <collection role="institutes" number="">4 Material und Umwelt</collection>
    <collection role="themenfelder" number="">Umwelt</collection>
    <collection role="themenfelder" number="">Umwelt-Material-Interaktionen</collection>
    <collection role="fulltextaccess" number="">Datei im Netzwerk der BAM verfügbar ("Closed Access")</collection>
    <collection role="literaturgattung" number="">Graue Literatur</collection>
    <collection role="institutes" number="">4.0 Abteilungsleitung und andere</collection>
    <collection role="themenfelder" number="">Biokorrosion</collection>
    <thesisGrantor>Freie Universität Berlin</thesisGrantor>
  </doc>
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