2'-脱氧鸟苷是一种关键的药物中间体，可用于合成抗癌药物和2型糖尿病生物标志物。本研究提出了一种酶级联催化体系，利用大肠杆菌嘧啶核苷酸磷酸化酶（EcTP）和乙酸布氏杆菌嘌呤核苷酸磷酸化酶（BaPNP）在一个反应体系中进行整细胞催化，高效合成2'-脱氧鸟苷。BaPNP经半理性设计改良，得到了最佳三重突变体BaPNP-Mu3（E57A/T189S/L243I），其2'-脱氧鸟苷产量比野生型BaPNP（BaPNP-Mu0）提高5.6倍。分子动力学模拟表明，对BaPNP-Mu3进行工程改造导致其底物进入通道更大、更灵活，可能有助于其催化效率。此外，通过协调BaPNP-Mu3和EcTP的表达，构建了一个稳健的整细胞催化剂W05，能够高产2'-脱氧鸟苷（14.8 mM，转化率74.0%），并具有高的时间-空间产率（1.32 g/L/h），因此在工业应用上具有很大竞争力。© 2023. 作者授予Springer Nature B.V.独家许可。

2'-deoxyguanosine is a key medicinal intermediate that could be used to synthesize anti-cancer drug and biomarker in type 2 diabetes. In this study, an enzymatic cascade using thymidine phosphorylase from Escherichia coli (EcTP) and purine nucleoside phosphorylase from Brevibacterium acetylicum (BaPNP) in a one-pot whole cell catalysis was proposed for the efficient synthesis of 2'-deoxyguanosine. BaPNP was semi-rationally designed to improve its activity, yielding the best triple variant BaPNP-Mu3 (E57A/T189S/L243I), with a 5.6-fold higher production of 2'-deoxyguanosine than that of wild-type BaPNP (BaPNP-Mu0). Molecular dynamics simulation revealed that the engineering of BaPNP-Mu3 resulted in a larger and more flexible substrate entrance channel, which might contribute to its catalytic efficiency. Furthermore, by coordinating the expression of BaPNP-Mu3 and EcTP, a robust whole cell catalyst W05 was created, capable of producing 14.8 mM 2'-deoxyguanosine (74.0% conversion rate) with a high time-space yield (1.32 g/L/h) and therefore being very competitive for industrial applications.© 2023. The Author(s), under exclusive licence to Springer Nature B.V.