One-pot Process Of 2’-deoxyguanylate And 2’-deoxyinosinate Catalyzed By A Multi-enzymatic System

Deoxynucleotide is a common kind of low molecular compouds in living organisms. It can be extensively used in intermediates of anticancer and antiviral drugs, and also plays an important role in food, industry and science area. However, biosysthesis of 2’-deoxyguanylate (dGMP) and 2’-deoxyinosinate (dIMP) was seldom mentioned in literatures. Herein, we developed a novel, one-pot multi-enzymatic cascade reaction to produce dGMP and dIMP. The reaction pathway involved purine nucleoside phosphorylase (PNPase) and acetate kinase (ACKase) from Escherichia coli,N-deoxyribosytransferase Ⅱ (NDT-Ⅱ) from Lactobacillus delbrueckii and deoxyguanosine kinase (dGKase) from Bacillus subtilis.Four kinds of single gene recombination strain were constructed, which was BL21/PNP, BL21/NDT-Ⅱ, BL21/dGK and BL21/ACK, respectively. In addition, using the enzyme characteristics of isocaudarner BamH Ⅰ and Bgl Ⅱ, gene yaaG and ack were cloned into the same plasmid pET-28a and therefore, a tandem recombination strain BL21/dGK-ACK was contructed, which could coexpress dGKase and ACKase simultaneously. During the reaction, the initial substrate guanosine was cleaved into guanine and ribose-1-phosphate by PNPase. Then, deoxyguanosine (dGR) was subsequently produced from a reaction between guanine and thymidine catalysed by NDT-Ⅱ. Finally, the intermediate dGR was phosphorylated to dGMP by dGKase and a cytidine triphosphate (CTP) regeneration system that utilised acetyl phosphate via ACKase. In addition, deoxyinosine (dIR) was produced from hypoxanthine (Ⅰ) and thymidine catalyzed by NDT-Ⅱ, and then the desired product dIMP was obtained from dIR catalyzed by dGKase, coupled UTP-regeneration. A very small amount of CTP or UTP was added because phosphate regeneration was efficient to transfer a phosphate group from acetyl phosphate to dGR or dIR. After 12 h of incubation, yield of dGMP was respectively 73.1% and 74.7% catalyzed by single gene recombination strain or by tandem recombination strain based on the addition of 5 mM guanosine and 5 mM thymidine. Under the same condition, the yield of dIMP was 81% after 5 hours when tandem recombination strain was used.