Efficient synthesis of 2’-deoxyguanosine by employing an engineered purine nucleoside phosphorylase from Brevibacterium acetylicum in one-pot cascade

Abstract

Abstract 2’-deoxyguanosine is a key medical intermediate which could be applied for the synthesis of anti-cancer drug and biomarker in type 2 diabetes. In present study, an enzymatic cascade for the efficient synthesis of 2’-deoxyguanosine was proposed by employing thymidine phosphorylase from Escherichia coli (EcTP) and purine nucleoside phosphorylase from Brevibacterium acetylicum (BaPNP) in a one-pot whole cell catalysis. Semi-rational design of BaPNP was performed to enhance its activity, resulting a best triple variant BaPNP-Mu3 (E57A/T189S/L243I), with an overall 5.6-fold higher yield of 2'-deoxyguanosine as compared with BaPNP-Mu0. Molecular dynamics simulation revealed that the engineering of BaPNP-Mu3 led to a larger and more flexible substrate entrance channel, which might contribute to its catalytic performance. Furthermore, by coordinating the expression of BaPNP-M3 and EcTP, a robust whole cell catalyst W05 was constructed, which could produce 14.8 mM 2’-deoxyguanosine with a high time-space yield (1.32 g/L/h) and therefore was very competitive for industrial applications.