A novel process of inosine 5′-monophosphate production using overexpressed guanosine/inosine kinase

Abstract

A novel process for producing inosine 5'-monophosphate (5'-IMP) has been 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 inosine by guanosine/inosine kinase (GIKase). GIKase was produced by an Escherichia coli recombinant strain, MC1000(pIK75), which overexpressed the enzyme up to 50% of the total cellular protein. The overproducing plasmid, pIK75, which was randomly screened out from deletion plasmids 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 was placed 16 bp downstream of the trpL Shine-Dalgarno sequence under the control of the E. coli trp promoter. Fermentation of inosine and its phosphorylation were sequentially performed in a 5-1 jar fermenter. At the end of inosine fermentation by C. ammoniagenes KY13761, culture broth of MC1000(pIK75) was mixed with that of KY13761 to start the phosphorylating reaction. Inosine in the reaction mixture was stoichiometrically phosphorylated, and 91 mM 5'-IMP accumulated in a 12-h reaction. This new biological process has advantages over traditional methods for producing 5'-IMP.

An Efficient Method for Quantitative Determination of Cellular ATP Synthetic Activity

“…The bacterial ATP recycling system was reported and used for supplying ATP for industrial production of several compounds. [1][2][3][4][5] In these processes, bacterial cells were chemically treated to get permeability for substrates and products including ATP. Although permeable cells lost the ability of growing, enzymatic activity for ATP synthesis was maintained and used for ATP supply to ATP-dependent enzymes.…”

Phosphate Starvation-Inducible Gene ushA Encodes a 5′ Nucleotidase Required for Growth of Corynebacterium glutamicum on Media with Nucleotides as the Phosphorus Source

“…Corynebacterium ammoniagenes is another member of the Corynebacteriaceae and is used for biotechnological production of flavor enhancers, such as 5Ј-IMP (20). In such a process, inosine is synthesized de novo by intracellular dephosphorylation of 5Ј-IMP.…”

“…In such a process, inosine is synthesized de novo by intracellular dephosphorylation of 5Ј-IMP. Inosine secreted into the culture medium can be converted to 5Ј-IMP chemically or enzymatically, and efficient producing strains need to be devoid of extracellular 5Ј nucleotidase activity (20 …”

Effects ofeddandpgiDisruptions on Inosine Accumulation inEscherichia coli

“…1) Other methods combine the fermentation and phosphorylation of purine nucleosides. Phosphorylation can be achieved through chemical 2) and enzymatic 3,4) methods. Studies on the microbial production of purine nucleosides have been done exclusively in mutants of Bacillus subtilis [5][6][7] and Corynebacterium ammoniagenes.…”