Department of Chemical and Biomolecular Engineering, Rice University, USA.
Department of Chemical and Biomolecular Engineering, Rice University, USA; Department of Bioengineering, Rice University, USA.
Metab Eng. 2014 May;23:100-15. doi: 10.1016/j.ymben.2014.02.011. Epub 2014 Feb 22.
The modularity and versatility of an engineered functional reversal of the β-oxidation cycle make it a promising platform for the synthesis of longer-chain (C≥4) products. While the pathway has recently been exploited for the production of n-alcohols and carboxylic acids, fully capitalizing on its potential for the synthesis of a diverse set of product families requires a system-level assessment of its biosynthetic capabilities. To this end, we utilized a genome scale model of Escherichia coli, in combination with Flux Balance Analysis and Flux Variability Analysis, to determine the key characteristics and constraints of this pathway for the production of a variety of product families under fermentative conditions. This analysis revealed that the production of n-alcohols, alkanes, and fatty acids of lengths C3-C18 could be coupled to cell growth in a strain lacking native fermentative pathways, a characteristic enabling product synthesis at maximum rates, titers, and yields. While energetic and redox constraints limit the production of target compounds from alternative platforms such as the fatty acid biosynthesis and α-ketoacid pathways, the metabolic efficiency of a β-oxidation reversal allows the production of a wide range of products of varying length and functionality. The versatility of this platform was investigated through the simulation of various termination pathways for product synthesis along with the use of different priming molecules, demonstrating its potential for the efficient synthesis of a wide variety of functionalized compounds. Overall, specific metabolic manipulations suggested by this systems-level analysis include deletion of native fermentation pathways, the choice of priming molecules and specific routes for their synthesis, proper choice of termination enzymes, control of flux partitioning at the pyruvate node and the pentose phosphate pathway, and the use of an NADH-dependent trans-enoyl-CoA reductase instead of a ferredoxin-dependent enzyme.
该工程化的β-氧化循环功能反转具有模块化和多功能性，使其成为合成更长链（C≥4）产物的有前途的平台。虽然该途径最近已被用于生产正醇和羧酸，但要充分利用其合成各种产物家族的潜力，需要对其生物合成能力进行系统水平的评估。为此，我们利用大肠杆菌的基因组规模模型，结合通量平衡分析和通量可变性分析，确定了该途径在发酵条件下生产各种产物家族的关键特性和限制。这项分析表明，在缺乏天然发酵途径的菌株中，n-醇、烷烃和 C3-C18 长度的脂肪酸的生产可以与细胞生长偶联，这一特性使产物能够以最大速率、浓度和产率进行合成。虽然能量和氧化还原限制了来自替代平台（如脂肪酸生物合成和α-酮酸途径）的目标化合物的生产，但β-氧化逆转的代谢效率允许生产具有不同长度和功能的各种产物。通过模拟不同的产物合成终止途径和使用不同的引发分子，研究了该平台的多功能性，证明了其合成各种功能化化合物的潜力。总的来说，这种系统水平分析建议进行特定的代谢操作，包括删除天然发酵途径、选择引发分子及其合成的特定途径、选择合适的终止酶、控制丙酮酸节点和戊糖磷酸途径的通量分配，以及使用 NADH 依赖性反式烯酰辅酶 A 还原酶代替依赖铁氧还蛋白的酶。
