July 2013  Vol. 23  No. 7 J. Microbiol. Biotechnol. (2013), 23(7), 923–931 http://dx.doi.org/10.4014/jmb.1304.04057 jmb Metabolomic Response of Chlamydomonas reinhardtii to the Inhibition of Target of Rapamycin (TOR) by Rapamycin Do Yup Lee 1 * and Oliver Fiehn 2 * 1 Department of Advanced Fermentation Fusion Science and Technology, Kookmin University, Seoul 136-702, Republic of Korea 2 Genome Center, University of California, Davis, CA 95616, USA Introduction Rapamycin, known as sirolimus, is an immunosuppressant drug used to prevent rejection in organ transplantation [26], and is produced by the bacterial species Streptomyces hygroscopicus [32]. Rapamycin binds the cytosolic protein FK-binding protein 12 (FKBP12), inhibits downstream signaling from the target of rapamycin (TOR) proteins [10], and eventually hinders cellular growth and proliferation, making this molecule an important potential target for various diseases. The evolutionarily conserved protein kinase (PK) TOR, is ubiquitously present in organisms, from yeast to plants and humans. Yeast genetics screened the identification of TOR as one mediator of the toxic effect of rapamycin in yeast [13], followed by the discovery of the mechanistic TOR (mTOR) as the physical target of rapamycin in mammalian cells [3]. TOR in yeasts and mammals is known to integrate diverse external and internal signals, and regulates cellular metabolism and growth by keeping a persistent level of nutrient supply, ribosome biogenesis, and mRNA translation [33]. Several reports have also shown the molecular and physiological functionalities of TOR in the plant kingdom, notably for Arabidopsis [12, 20, 22]. However the growth of Arabidopsis thaliana and other plants such as Oryza sativa, Nicotiana tabacum, or Brassica napus is not sensitive to rapamycin [22], probably due to a generic incapability of the plant FKBP12 to bind rapamycin, which may hinder the progress of the research on TOR signaling in photosynthetic organisms. Recently, it has been noted that TOR inhibition by rapamycin inhibits the growth of the photosynthetic unicellular alga Chlamydomonas reinhardtii [22] and following studies successfully identified the molecular evidence and cellular physiology in C. reinhardtii [5, 6, 24]. Considering these results, we carried out an analysis of rapamycin-induced effects on Chlamydomonas cells with a Received: April 23, 2013 Revised: May 1, 2013 Accepted: May 2, 2013 First published online June 3, 2013 *Corresponding authors O.F. Phone: +1-530-754-8258; Fax: +1-530-754-9658; E-mail: ofiehn@ucdavis.edu D.Y.L. Phone: +82-2-910-5733; Fax: +82-2-910-5733; E-mail: rome73@kookmin.ac.kr upplementary data for this paper are available on-line only at http://jmb.or.kr. pISSN 1017-7825, eISSN 1738-8872 Copyright © 2013 by The Korean Society for Microbiology and Biotechnology Rapamycin, known as an inhibitor of Target of Rapamycin (TOR), is an immunosuppressant drug used to prevent rejection in organ transplantation. Despite the close association of the TOR signaling cascade with various scopes of metabolism, it has not yet been thoroughly investigated at the metabolome level. In our current study, we applied mass spectrometric analysis for profiling primary metabolism in order to capture the responsive dynamics of the Chlamydomonas metabolome to the inhibition of TOR by rapamycin. Accordingly, we identified the impact of the rapamycin treatment at the level of metabolomic phenotypes that were clearly distinguished by multivariate statistical analysis. Pathway analysis pinpointed that inactivation of the TCA cycle was accompanied by the inhibition of cellular growth. Relative to the constant suppression of the TCA cycle, most amino acids were significantly increased in a time-dependent manner by longer exposure to rapamycin treatment, after an initial down-regulation at the early stage of exposure. Finally, we explored the isolation of the responsive metabolic factors into the rapamycin treatment and the culture duration, respectively. Keywords: Metabolomics, mass spectrometry, Chlamydomonas reinhardtii, target of rapamycin S S