Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis

Weyman, Philip D.; Beeri, Karen; Lefebvre, Stephane C.; Rivera, Josefa; McCarthy, James K.; Heuberger, Adam L.; Peers, Graham; Allen, Andrew E.; Dupont, Christopher L.

doi:10.1111/pbi.12254

Title: Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis

Journal Article · Fri Oct 10 00:00:00 EDT 2014 · Plant Biotechnology Journal

DOI:https://doi.org/10.1111/pbi.12254· OSTI ID:1237106

Weyman, Philip D. ^[1]; Beeri, Karen ^[2]; Lefebvre, Stephane C. ^[2]; Rivera, Josefa ^[2]; McCarthy, James K. ^[2]; Heuberger, Adam L. ^[3]; Peers, Graham ^[4]; Allen, Andrew E. ^[5]; Dupont, Christopher L. ^[2]

Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, La Jolla CA USA
Department of Microbial and Environmental Genomics, J. Craig Venter Institute, La Jolla CA USA
Proteomics and Metabolomics Facility, Colorado State University, Fort Collins CO USA
Department of Biology, Colorado State University, Fort Collins CO USA
Department of Microbial and Environmental Genomics, J. Craig Venter Institute, La Jolla CA USA, Scripps Institution of Oceanography, Integrative Oceanography Division, La Jolla CA USA

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 (TALENs), 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.

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Research Organization:: J. Craig Venter Inst., La Jolla, CA (United States); Colorado State Univ., Fort Collins, CO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF); Gordon and Betty Moore Foundation (United States)

Grant/Contract Number:: SC0008593; SC0006719; SC0008595; MCB-1024913; MCB-1129303; GBMF3828

OSTI ID:: 1237106

Alternate ID(s):: OSTI ID: 1237107; OSTI ID: 1455144

Journal Information:: Plant Biotechnology Journal, Journal Name: Plant Biotechnology Journal Vol. 13 Journal Issue: 4; ISSN 1467-7644

Publisher:: Society for Experimental Biology; Association of Applied BiologyCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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Cited by: 90 works

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59 BASIC BIOLOGICAL SCIENCES
diatoms
genome editing
urease
metabolomics
Phaeodactylum tricornutum
transcription activator-like effector nucleases

Title: Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis

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