Production Of Uridine Monophosphate By Uridine-cytidine Kinase Coupled With Polyphosphate Kinase

Uridine monophosphate(UMP)has been wildly used as food additives,drugs and drug precursors in different fields.Uridine/cytidine kinase(UCK)is an important catalyst in the metabolic compensation pathway of nucleotides in organisms,which catalyzes the phosphorylation of uridine and cytidine into UMP and cytidine monophosphate.Polyphosphate kinase(PPK)can catalyze the reversible transfer reaction of y-phosphate from ATP to inorganic polyphosphate(PolyP).In this study,we used UCK and PPK for UMP synthesis,and systematically optimized the reaction conditions.We further produced UMP by combining uridine fermentation and phosphorylation.Moreover,we proposed the method for the in situ purification and immobilization of enzymes.Escherichia coli BL21(DE3)was used as the host bacteria,and pET-28a plasmid was used as the expression vector.UCK was derived from E.coli K12,Bacillus subtilis 168,Bacillus sp.45385,Deinococcus radiodurans and Thermus thermophilus HB8,respectively.PPK was derived from E.coli K12,Rhodobacter sphaeroides and Pseudomonas denitrificans,respectively.The selectivity of UCK to phosphate donors was investigated.Results showed that all the UCK could use GTP and CTP as the phosphate donors.However,UCK from D.radiodurans(UK4)and T.thermophilus HB8(UK5)could use ATP as the phosphate donor.We analyzed UK5 by bioinformatics,and investigated the effect of different PPK on the UK5-catalyzed UMP production.Finally,PPK from P.denitrificans was selected as the ATP regeneration enzyme.Through single-factor experiment,the optimal reaction conditions were confirmed and the titer and yield of UMP reached 190 mM and 95%,respectively.In order to further decrease the material cost,uridine was produced by fermentation using engineed E.coli UR6 strain,and UMP was prepared using uridine fermentation broth as the substrate.Results showed that when untreated uridine fermentation broth was used,166 mM UMP was produced during 3 h,with a yield of 83%.Further increase the reaction cycle resulted in the degradation of UMP to uridine.However,when trated uridine fermentation broth was used,186 mM UMP was produced during 3 h,with a yield of 93%,and the UMP was stable and not easy to degrade.In order to increase the utilization efficiency of enzymes,metal chelating resin D403 was used to adsorb nickel ions to form the immobilized carrier,and the immobilized enzymes were obtained by the covalent binding of immobilized carrier and histidine-tag enzymes.With the use of immobilized enzymes,5-batch catalyzation were realized,and the average titer and yield of UMP reached 90 mM and 95%,respectively.