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Systematic Applications of Metabolomics in Metabolic Engineering
Metabolites 2012, 2(4), 1123-1138; doi:10.3390/metabo2041123
Article

Changes in Primary and Secondary Metabolite Levels in Response to Gene Targeting-Mediated Site-Directed Mutagenesis of the Anthranilate Synthase Gene in Rice

1
, 2
, 2
, 2,3
, 2,4
 and 1,5,*
1 Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan 2 Riken Plant Science Center, Yokohama, Kanagawa 230-0045, Japan 3 Organization of Advanced Sciences and Technology, Kobe University, Kobe, Hyogo 657-8501, Japan 4 Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan 5 Kihara Institute of Biological Research, Yokohama City University, Yokohama, Kanagawa 244–0813, Japan
* Author to whom correspondence should be addressed.
Received: 16 October 2012 / Revised: 4 December 2012 / Accepted: 9 December 2012 / Published: 18 December 2012
(This article belongs to the Special Issue Metabolomics in Metabolic Engineering)
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Abstract

Gene targeting (GT) via homologous recombination allows precise modification of a target gene of interest. In a previous study, we successfully used GT to produce rice plants accumulating high levels of free tryptophan (Trp) in mature seeds and young leaves via targeted modification of a gene encoding anthranilate synthase—a key enzyme of Trp biosynthesis. Here, we performed metabolome analysis in the leaves and mature seeds of GT plants. Of 72 metabolites detected in both organs, a total of 13, including Trp, involved in amino acid metabolism, accumulated to levels >1.5-fold higher than controls in both leaves and mature seeds of GT plants. Surprisingly, the contents of certain metabolites valuable for both humans and livestock, such as γ-aminobutyric acid and vitamin B, were significantly increased in mature seeds of GT plants. Moreover, untargeted analysis using LC-MS revealed that secondary metabolites, including an indole alkaloid, 2-[2-hydroxy-3-β-d-glucopyranosyloxy-1-(1H-indol-3-yl)propyl] tryptophan, also accumulate to higher levels in GT plants. Some of these metabolite changes in plants produced via GT are similar to those observed in plants over expressing mutated genes, thus demonstrating that in vivo protein engineering via GT can be an effective approach to metabolic engineering in crops.
Keywords: gene targeting; indole alkaloid; metabolic engineering; rice; tryptophan gene targeting; indole alkaloid; metabolic engineering; rice; tryptophan
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Saika, H.; Oikawa, A.; Nakabayashi, R.; Matsuda, F.; Saito, K.; Toki, S. Changes in Primary and Secondary Metabolite Levels in Response to Gene Targeting-Mediated Site-Directed Mutagenesis of the Anthranilate Synthase Gene in Rice. Metabolites 2012, 2, 1123-1138.

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