Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada.
Biotechnol Bioeng. 2019 Dec;116(12):3269-3281. doi: 10.1002/bit.27146. Epub 2019 Sep 3.
Cell-free systems are growing in importance for the biosynthesis of complex molecules. These systems combine the precision of traditional chemistry with the versatility of biology in creating superior overall processes. Recently, a new synthetic pathway for the biosynthesis of isoprenoids using the substrate isopentenol, dubbed the isopentenol utilization pathway (IUP), was demonstrated to be a promising alternative to the native 2C-methyl-d-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathways. This simplified pathway, which contains a minimum of four enzymes to produce basic monoterpenes and only depends on ATP and isopentenol as substrates, allows for a highly flexible approach to the commercial synthesis of isoprenoid products. In this work, we use metabolic reconstitution to characterize this new pathway in vitro and demonstrate its use for the cell-free synthesis of mono-, sesquit-, and diterpenoids. Kinetic modeling and sensitivity analysis were also used to identify the most significant parameters for taxadiene productivity, and metabolic control analysis was employed to elucidate protein-level interactions within this pathway, which demonstrated that the IUP enzymatic system is primarily controlled by the concentration and kinetics of choline kinase (CK) and not regulated by any pathway intermediates. This is a significant advantage over the natural MEP or MVA pathways as it greatly simplifies future metabolic engineering efforts, both in vitro and in vivo, aiming at improving the kinetics of CK. Finally, we used the insights gathered to demonstrate an in vitro IUP system that can produce 220 mg/L of the diterpene taxadiene, in 9 hr, almost 3-fold faster than any system reported thus far.
无细胞体系在复杂分子的生物合成中变得越来越重要。这些系统将传统化学的精确性与生物学的多功能性结合起来，创造出更优越的整体工艺。最近，一种使用异戊烯醇作为底物的异戊二烯生物合成新途径，即异戊烯醇利用途径（IUP），被证明是一种有前途的替代天然 2C-甲基-D-赤藓糖醇-4-磷酸（MEP）和甲羟戊酸（MVA）途径的方法。这个简化的途径包含最少四种酶来生产基本的单萜类化合物，并且只依赖于 ATP 和异戊烯醇作为底物，为异戊二烯产品的商业合成提供了一种高度灵活的方法。在这项工作中，我们使用代谢重建来体外表征这条新途径，并证明其可用于无细胞合成单萜、倍半萜和二萜。我们还使用动力学建模和敏感性分析来确定影响紫杉醇产量的最重要参数，并且进行代谢控制分析来阐明该途径中的蛋白水平相互作用，结果表明 IUP 酶系统主要受胆碱激酶（CK）的浓度和动力学控制，而不受任何途径中间产物的调节。与天然 MEP 或 MVA 途径相比，这是一个显著的优势，因为它大大简化了未来的代谢工程努力，无论是在体外还是在体内，目的都是提高 CK 的动力学。最后，我们利用获得的见解展示了一个可以在 9 小时内生产 220mg/L 二萜紫杉醇的体外 IUP 系统，比迄今为止报道的任何系统都快近 3 倍。
