Department of Chemical and Biological Engineering, University of Colorado, Boulder, 3415 Colorado Avenue, Boulder, CO, 80303, USA.
Department of Chemical and Biological Engineering, University of Colorado, Boulder, 3415 Colorado Avenue, Boulder, CO, 80303, USA.
Metab Eng. 2022 Jan;69:209-220. doi: 10.1016/j.ymben.2021.11.008. Epub 2021 Nov 23.
Cellular metabolism is a nonlinear reaction network in which dynamic shifts in enzyme concentration help regulate the flux of carbon to different products. Despite the apparent simplicity of these biochemical adjustments, their influence on metabolite biosynthesis tends to be context-dependent, difficult to predict, and challenging to exploit in metabolic engineering. This study combines a detailed kinetic model with a systematic set of in vitro and in vivo analyses to explore the use of enzyme concentration as a control parameter in fatty acid synthesis, an essential metabolic process with important applications in oleochemical production. Compositional analyses of a modeled and experimentally reconstituted fatty acid synthase (FAS) from Escherichia coli indicate that the concentration ratio of two native enzymes-a promiscuous thioesterase and a ketoacyl synthase-can tune the average length of fatty acids, an important design objective of engineered pathways. The influence of this ratio is sensitive to the concentrations of other FAS components, which can narrow or expand the range of accessible chain lengths. Inside the cell, simple changes in enzyme concentration can enhance product-specific titers by as much as 125-fold and elicit shifts in overall product profiles that rival those of thioesterase mutants. This work develops a kinetically guided approach for using ratiometric adjustments in enzyme concentration to control the product profiles of FAS systems and, broadly, provides a detailed framework for understanding how coordinated shifts in enzyme concentration can afford tight control over the outputs of nonlinear metabolic pathways.
细胞代谢是一个非线性反应网络，其中酶浓度的动态变化有助于调节碳向不同产物的通量。尽管这些生化调整显然很简单，但它们对代谢物生物合成的影响往往是依赖于上下文的、难以预测的，并且在代谢工程中难以利用。本研究将详细的动力学模型与系统的体外和体内分析相结合，探索了将酶浓度用作脂肪酸合成中的控制参数，脂肪酸合成是一种重要的代谢过程，在油脂化学品生产中有重要应用。对来自大肠杆菌的模型化和实验重建的脂肪酸合酶（FAS）进行组成分析表明，两种天然酶（一种混杂的硫酯酶和酮酰基合酶）的浓度比可以调节脂肪酸的平均长度，这是工程化途径的一个重要设计目标。这种比率的影响对其他 FAS 成分的浓度敏感，这可以缩小或扩大可访问链长的范围。在细胞内，酶浓度的简单变化可以将特定产物的比活提高多达 125 倍，并引起整体产物分布的变化，与硫酯酶突变体相当。这项工作开发了一种基于动力学的方法，用于使用酶浓度的比例调整来控制 FAS 系统的产物分布，并为理解酶浓度的协调变化如何能够对非线性代谢途径的输出进行严格控制提供了详细的框架。
