A NOVEL PROCESS OF INOSINE 5'-MONOPHOSPHATE PRODUCTION USING OVEREXPRESSED GUANOSINE INOSINE KINASE/

A novel process for producing inosine 5'-monophosphate (5'-IMP) has be en demonstrated. The process consists of two sequential bioreactions; the first is a fermentation of inosine by a mutant of Corynebacterium ammoniagenes, and the second is a unique phosphorylating reaction of i nosine by guanosine/inosine kinase (GIKase). GIKase was produced by an Escherichia coli recombinant strain, MC1000(pIK75), which overexpress ed the enzyme up to 50% of the total cellular protein. The overproduci ng plasmid, pIK75, which was randomly screened out from deletion plasm ids with various lengths of intermediate sequence between the E. coli trpL Shine-Dalgarno sequence, derived from the vector plasmid, and the start codon of the GIKase structural gene. In pIK75, the start ATG wa s placed 16 bp downstream of the trpL Shine-Dalgarno sequence under th e control of the E. coli trp promoter. Fermentation of inosine and its phosphorylation were sequentially performed in a 5-1 jar fermenter. A t the end of inosine fermentation by C. ammoniagenes KY13761, culture broth of MC1000(pIK75) was mixed with that of KY13761 to start the pho sphorylating reaction. Inosine in the reaction mixture was stoichiomet rically phosphorylated, and 91 mM 5'-IMP accumulated in a 12-h reactio n. This new biological process has advantages over traditional methods for producing 5'-IMP.