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

Abstract

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.

Authors:
 [1];  [2];  [2];  [2];  [2];  [3];  [4];  [5];  [2]
  1. J. Craig Venter Inst., La Jolla, CA (United States). Dept. of Synthetic Biology and Bioenergy
  2. J. Craig Venter Inst., La Jolla, CA (United States). Dept. of Microbial and Environmental Genomics
  3. Colorado State Univ., Fort Collins, CO (United States). Proteomics and Metabolomics Facility
  4. Colorado State Univ., Fort Collins, CO (United States). Dept. of Biology
  5. J. Craig Venter Inst., La Jolla, CA (United States). Dept. of Microbial and Environmental Genomics; Univ. of California, San Diego, CA (United States). Scripps Inst. of Oceanography. Integrative Oceanography Division
Publication Date:
Research Org.:
J. Craig Venter Inst., La Jolla, CA (United States); Colorado State Univ., Fort Collins, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Science Foundation (NSF); Gordon and Betty Moore Foundation (United States)
OSTI Identifier:
1237106
Alternate Identifier(s):
OSTI ID: 1237107; OSTI ID: 1455144
Grant/Contract Number:  
SC0008593; SC0006719; SC0008595; MCB-1024913; MCB-1129303; GBMF3828
Resource Type:
Journal Article: Published Article
Journal Name:
Plant Biotechnology Journal
Additional Journal Information:
Journal Volume: 13; Journal Issue: 4; Journal ID: ISSN 1467-7644
Publisher:
Society for Experimental Biology; Association of Applied Biology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; diatoms; genome editing; urease; metabolomics; Phaeodactylum tricornutum; transcription activator-like effector nucleases

Citation Formats

Weyman, Philip D., Beeri, Karen, Lefebvre, Stephane C., Rivera, Josefa, McCarthy, James K., Heuberger, Adam L., Peers, Graham, Allen, Andrew E., and Dupont, Christopher L. Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis. United States: N. p., 2014. Web. doi:10.1111/pbi.12254.
Weyman, Philip D., Beeri, Karen, Lefebvre, Stephane C., Rivera, Josefa, McCarthy, James K., Heuberger, Adam L., Peers, Graham, Allen, Andrew E., & Dupont, Christopher L. Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis. United States. doi:10.1111/pbi.12254.
Weyman, Philip D., Beeri, Karen, Lefebvre, Stephane C., Rivera, Josefa, McCarthy, James K., Heuberger, Adam L., Peers, Graham, Allen, Andrew E., and Dupont, Christopher L. Fri . "Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis". United States. doi:10.1111/pbi.12254.
@article{osti_1237106,
title = {Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis},
author = {Weyman, Philip D. and Beeri, Karen and Lefebvre, Stephane C. and Rivera, Josefa and McCarthy, James K. and Heuberger, Adam L. and Peers, Graham and Allen, Andrew E. and Dupont, Christopher L.},
abstractNote = {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.},
doi = {10.1111/pbi.12254},
journal = {Plant Biotechnology Journal},
number = 4,
volume = 13,
place = {United States},
year = {Fri Oct 10 00:00:00 EDT 2014},
month = {Fri Oct 10 00:00:00 EDT 2014}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1111/pbi.12254

Citation Metrics:
Cited by: 26 works
Citation information provided by
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