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Applied and Environmental Microbiology, August 2009, p. 5001-5008, Vol. 75, No. 15
0099-2240/09/$08.00+0     doi:10.1128/AEM.00715-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of the Central Metabolic Pathways in Thermoanaerobacter sp. Strain X514 via Isotopomer-Assisted Metabolite Analysis ^,

Xueyang Feng,¹ Housna Mouttaki,² Lu Lin,^2,3 Rick Huang,¹ Bing Wu,¹ Christopher L. Hemme,² Zhili He,² Baichen Zhang,⁴ Leslie M. Hicks,⁴ Jian Xu,³ Jizhong Zhou,² and Yinjie J. Tang^1*

Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, Missouri 63130,¹ Institute for Environmental Genomics and Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019,² Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, People's Republic of China,³ Donald Danforth Plant Science Center, 975 N. Warson Rd., St. Louis, Missouri 63132⁴

Received 26 March 2009/ Accepted 5 June 2009

Thermoanaerobacter sp. strain X514 has great potential in biotechnology due to its capacity to ferment a range of C₅ and C₆ sugars to ethanol and other metabolites under thermophilic conditions. This study investigated the central metabolism of strain X514 via ¹³C-labeled tracer experiments using either glucose or pyruvate as both carbon and energy sources. X514 grew on minimal medium and thus contains complete biosynthesis pathways for all macromolecule building blocks. Based on genome annotation and isotopic analysis of amino acids, three observations can be obtained about the central metabolic pathways in X514. First, the oxidative pentose phosphate pathway in X514 is not functional, and the tricarboxylic acid cycle is incomplete under fermentative growth conditions. Second, X514 contains (Re)-type citrate synthase activity, although no gene homologous to the recently characterized (Re)-type citrate synthase of Clostridium kluyveri was found. Third, the isoleucine in X514 is derived from acetyl coenzyme A and pyruvate via the citramalate pathway rather than being synthesized from threonine via threonine ammonia-lyase. The functionality of the citramalate synthase gene (cimA [Teth514_1204]) has been confirmed by enzymatic activity assays, while the presence of intracellular citramalate has been detected by mass spectrometry. This study demonstrates the merits of combining ¹³C-assisted metabolite analysis, enzyme assays, and metabolite detection not only to examine genome sequence annotations but also to discover novel enzyme activities.

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* Corresponding author. Mailing address: Department of Energy, Environmental and Chemical Engineering, Washington University, One Brookings Drive, Box 1180, St. Louis, MO 63130. Phone: (314) 935-3441. Fax: (314) 935-7211. E-mail: yinjie.tang{at}seas.wustl.edu

Published ahead of print on 12 June 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, August 2009, p. 5001-5008, Vol. 75, No. 15
0099-2240/09/$08.00+0     doi:10.1128/AEM.00715-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.