International Center for Biotechnology, Osaka University, Yamadaoka 2-1, Suita, Osaka, 565-0871, Japan.
Department of Life Science and Biotechnology, Kansai University, Yamate-cho 3-3-35, Suita, Osaka, 564-8680, Japan.
Chembiochem. 2022 Aug 17;23(16):e202200210. doi: 10.1002/cbic.202200210. Epub 2022 Jun 14.
Adenosine triphosphate (ATP) is an essential cofactor for energy-dependent enzymatic reactions that occur during in vitro biochemical conversion. Recently, an enzyme cascade based on non-oxidative glycolysis, which uses starch and orthophosphate as energy and phosphate sources, respectively, for the regeneration of ATP from adenosine diphosphate, has been developed (Wei et al., ChemCatChem 2018, 10, 5597-5601). However, the 12 enzymes required for this system hampered its practical usability and further testing potential. Here, we addressed this issue by constructing co-expression vectors for the simultaneous gene expression of the 12 enzymes in a single expression strain. All enzymes were sourced from (hyper)thermophiles, which enabled a one-step purification via a heat-treatment process. We showed that the combination of the two enabled the ATP regeneration system to function in a single recombinant Escherichia coli strain. Additionally, this work provides a strategy to rationally design and control proteins expression levels in the co-expression vectors.
三磷酸腺苷（ATP）是一种必需的辅酶，用于体外生化转化过程中发生的能量依赖的酶促反应。最近，一种基于非氧化糖酵解的酶级联反应被开发出来，该反应分别使用淀粉和正磷酸盐作为能量和磷酸源，从二磷酸腺苷中再生 ATP（Wei 等人，ChemCatChem 2018, 10, 5597-5601）。然而，该系统所需的 12 种酶阻碍了其实际可用性和进一步测试的潜力。在这里，我们通过在单个表达菌株中同时表达 12 种酶的共表达载体来解决这个问题。所有的酶都来源于（超）嗜热菌，这使得它们可以通过热处理过程进行一步纯化。我们表明，这两种方法的结合使 ATP 再生系统能够在单个重组大肠杆菌菌株中发挥作用。此外，这项工作为在共表达载体中合理设计和控制蛋白质表达水平提供了一种策略。
