Identification of uncharacterized enzymes using multiplexed measurement of biochemical activities

尽管进行了近两个世纪的研究，但我们对代谢生物化学的理解并不完整。糖酵解、TCA循环、磷酸戊糖途径、核苷酸合成和氨基酸生物合成的核心过程在生命的三个领域都高度保守。这种保守性使生物化学家能够建立一张新陈代谢图，其中包含了从大肠杆菌到发芽酵母再到牛心脏等各种生物的实验数据。使用这种广泛的模型系统构建代谢图导致了过于笼统的理解，因此关于特定生物体的信息可能是不完整的或不正确的。除了这些概括和错误分配之外，还有更多的遗漏之罪，其中编码预期步骤的酶的基因尚未被克隆。代谢中的这些未知步骤阻碍了对生化过程的准确预测，阻碍了将酵母工程用于生产食品、生物化学品和生物燃料的工业发酵目的的努力。拟议中的项目将扫描基因组，以识别催化生化活动的酶，这些酶已经被观察到，但从未与基因联系起来。该项目开创了一种集合策略，允许对整个酵母基因组进行有效的筛选，以获得感兴趣的酶活性。

Despite nearly two centuries of research, our understanding of metabolic biochemistry is incomplete. The core processes of glycolysis, the TCA cycle, the pentose phosphate pathway, nucleotide synthesis, and amino acid biosynthesis are highly conserved across the three domains of life. This conservation has allowed biochemists to build a metabolic chart incorporating experimental data derived from organisms ranging from E. coli to budding yeast to bovine heart. Construction of a metabolic map using this wide array of model systems has led to an over-generalized understanding so that information about a particular organism may be incomplete or incorrect. In addition to these generalizations and mis-assignments are far more numerous sins of omission in which the gene encoding the enzyme for an anticipated step has not been cloned. These uncharacterized steps in metabolism prevent accurate predictions of biochemical processes, hampering efforts to engineer yeast for purposes of industrial fermentations used to manufacture foodstuffs, biochemicals, and biofuels. The proposed project will scan the genome to identify enzymes catalyzing biochemical activities that have been observed but never have been linked to a gene. This project pioneers a pooling strategy that will allow the efficient screening of the entire yeast genome for enzyme activities of interest.