Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
J Biotechnol. 2012 Jan;157(1):113-23. doi: 10.1016/j.jbiotec.2011.09.019. Epub 2011 Sep 22.
Each of the twelve enzymes for glycolytic fermentation, eleven from Escherichia coli and one from Saccharomyces cerevisiae, have been over-expressed in E. coli and purified with His-tags. Simple assays have been developed for each enzyme and they have been assembled for fermentation of glucose to ethanol. Phosphorus-31 NMR revealed that this in vitro reaction accumulates fructose 1,6-bisphosphate while recycling the cofactors NAD(+) and ATP. This reaction represents a defined ATP-regeneration system that can be tailored to suit in vitro biochemical reactions such as cell-free protein synthesis. The enzyme from S. cerevisiae, pyruvate decarboxylase 1 (Pdc1; EC 4.1.1.1), was identified as one of the major 'flux controlling' enzymes for the reaction and was replaced with an evolved version of Pdc1 that has over 20-fold greater activity under glycolysis reaction conditions. This substitution was only beneficial when the ratio of glycolytic enzymes was adjusted to suit greater Pdc1 activity.
十二种糖酵解发酵酶中的每一种，包括十一种来自大肠杆菌和一种来自酿酒酵母，都在大肠杆菌中过表达并带有 His 标签进行纯化。为每种酶开发了简单的测定方法，并将它们组装起来用于葡萄糖发酵生成乙醇。磷-31 NMR 显示，该体外反应积累果糖 1,6-二磷酸，同时循环利用辅因子 NAD(+)和 ATP。该反应代表了一个定义明确的 ATP 再生系统，可以根据体外生化反应进行定制，例如无细胞蛋白合成。来自酿酒酵母的酶，丙酮酸脱羧酶 1（Pdc1；EC 4.1.1.1），被鉴定为该反应的主要“通量控制”酶之一，并被一种经过进化的 Pdc1 取代，其在糖酵解反应条件下的活性超过 20 倍。只有当调整糖酵解酶的比例以适应更大的 Pdc1 活性时，这种替代才是有益的。
