School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China.
J Agric Food Chem. 2023 Jun 7;71(22):8551-8557. doi: 10.1021/acs.jafc.3c02340. Epub 2023 May 22.
Cell-free expression systems have emerged as a potent and promising platform for the biosynthesis of chemicals by reconstituting in vitro expressed enzymes. Here, we report cell-free biosynthesis of cinnamyl alcohol (cinOH) with enhanced productivity by using the Plackett-Burman experimental design for multifactor optimization. Initially, four enzymes were individually expressed in vitro and directly mixed to reconstitute a biosynthetic route for the synthesis of cinOH. Then, the Plackett-Burman experimental design was used to screen multiple reaction factors and found three crucial parameters (i.e., reaction temperature, reaction volume, and carboxylic acid reductase) for the cinOH production. With the optimum reaction conditions, approximately 300 μM of cinOH was synthesized after 10 h of cell-free biosynthesis. Extending the production time to 24 h also increased the production to a maximum yield of 807 μM, which is nearly 10 times higher than the initial yield without optimization. This study demonstrates that cell-free biosynthesis can be combined with other powerful optimization methodologies such as the Plackett-Burman experimental design for enhanced production of valuable chemicals.
无细胞表达系统已成为通过体外表达酶重新构建来合成化学物质的有力且有前途的平台。在这里，我们通过使用 Plackett-Burman 实验设计进行多因素优化，报告了增强生产力的肉桂醇（cinOH）的无细胞生物合成。最初，将四种酶分别进行体外表达，然后直接混合以重新构建合成 cinOH 的生物合成途径。然后，使用 Plackett-Burman 实验设计筛选多个反应因素，并找到了 cinOH 生产的三个关键参数（即反应温度、反应体积和羧酸还原酶）。在最佳反应条件下，无细胞生物合成 10 小时后可合成约 300 μM 的 cinOH。将生产时间延长至 24 小时也将产量提高到最大 807 μM，这比未经优化的初始产量高近 10 倍。这项研究表明，无细胞生物合成可以与其他强大的优化方法（如 Plackett-Burman 实验设计）结合使用，以提高有价值化学品的产量。
