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.