Open AccessInactivation of P haeodactylum tricornutum urease gene using transcription activator‐like effector nuclease‐based targeted mutagenesis
Author(s) -
Weyman Philip D.,
Beeri Karen,
Lefebvre Stephane C.,
Rivera Josefa,
McCarthy James K.,
Heuberger Adam L.,
Peers Graham,
Allen Andrew E.,
Dupont Christopher L.
Publication year - 2015
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/pbi.12254
Subject(s) - phaeodactylum tricornutum , biology , urease , biochemistry , mutant , gene , transcription activator like effector nuclease , effector , southern blot , urea cycle , microbiology and biotechnology , enzyme , genome , diatom , arginine , botany , genome editing , amino acid
Summary Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome‐editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge‐based studies and bioengineering. Using a new technique, transcription activator‐like effector nucleases ( TALEN s), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum , was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA . The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build‐up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.