van der Werf Mariët J, Overkamp Karin M, Muilwijk Bas, Koek Maud M, van der Werff-van der Vat Bianca J C, Jellema Renger H, Coulier Leon, Hankemeier Thomas
TNO Quality of Life, PO Box 360, 3700 AJ Zeist, The Netherlands.
Mol Biosyst. 2008 Apr;4(4):315-27. doi: 10.1039/b717340g. Epub 2008 Feb 29.
Metabolomics is an emerging, powerful, functional genomics technology that involves the comparative non-targeted analysis of the complete set of metabolites in an organism. We have set-up a robust quantitative metabolomics platform that allows the analysis of 'snapshot' metabolomes. In this study, we have applied this platform for the comprehensive analysis of the metabolite composition of Pseudomonas putida S12 grown on four different carbon sources, i.e. fructose, glucose, gluconate and succinate. This paper focuses on the microbial aspects of analyzing comprehensive metabolomes, and demonstrates that metabolomes can be analyzed reliably. The technical (i.e. sample work-up and analytical) reproducibility was on average 10%, while the biological reproducibility was approximately 40%. Moreover, the energy charge values of the microbial samples generated were determined, and indicated that no biotic or abiotic changes had occurred during sample work-up and analysis. In general, the metabolites present and their concentrations were very similar after growth on the different carbon sources. However, specific metabolites showed large differences in concentration, especially the intermediates involved in the degradation of the carbon sources studied. Principal component discriminant analysis was applied to identify metabolites that are specific for, i.e. not necessarily the metabolites that show those largest differences in concentration, cells grown on either of these four carbon sources. For selected enzymatic reactions, i.e. the glucose-6-phosphate isomerase, triosephosphate isomerase and phosphoglyceromutase reactions, the apparent equilibrium constants (K(app)) were calculated. In several instances a carbon source-dependent deviation between the apparent equilibrium constant (K(app)) and the thermodynamic equilibrium constant (K(eq)) was observed, hinting towards a potential point of metabolic regulation or towards bottlenecks in biosynthesis routes. For glucose-6-phosphate isomerase and phosphoglyceromutase, the K(app) was larger than K(eq), and the results suggested that the specific enzymatic activities of these two enzymes were too low to reach the thermodynamic equilibrium in growing cells. In contrast, with triosephosphate isomerase the K(app) was smaller than K(eq), and the results suggested that this enzyme is kinetically controlled.
代谢组学是一项新兴的、强大的功能基因组学技术，它涉及对生物体中全套代谢物进行比较性非靶向分析。我们建立了一个强大的定量代谢组学平台，可用于分析“快照”代谢组。在本研究中，我们应用该平台对恶臭假单胞菌S12在四种不同碳源（即果糖、葡萄糖、葡萄糖酸盐和琥珀酸盐）上生长时的代谢物组成进行了全面分析。本文重点关注分析全面代谢组的微生物学方面，并证明代谢组可以可靠地进行分析。技术（即样品处理和分析）重现性平均为10%，而生物学重现性约为40%。此外，还测定了所产生的微生物样品的能荷值，结果表明在样品处理和分析过程中未发生生物或非生物变化。总体而言，在不同碳源上生长后，存在的代谢物及其浓度非常相似。然而，特定代谢物的浓度显示出很大差异，特别是在所研究碳源降解过程中涉及的中间体。应用主成分判别分析来鉴定对于在这四种碳源中的任何一种上生长的细胞具有特异性的代谢物，即不一定是那些浓度差异最大的代谢物。对于选定的酶促反应，即葡萄糖-6-磷酸异构酶、磷酸丙糖异构酶和磷酸甘油变位酶反应，计算了表观平衡常数（K(app)）。在几个实例中，观察到表观平衡常数（K(app)）与热力学平衡常数（K(eq)）之间存在碳源依赖性偏差，这暗示了潜在的代谢调节点或生物合成途径中的瓶颈。对于葡萄糖-6-磷酸异构酶和磷酸甘油变位酶，K(app)大于K(eq)，结果表明这两种酶的比酶活性太低，无法在生长细胞中达到热力学平衡。相反，对于磷酸丙糖异构酶，K(app)小于K(eq)
