Molecular cloning, nucleotide sequencing, and characterization of purF gene encoding 5'-phosphoribosyl-1'-pyrophosphate (PRPP) amidotransferase of Brevibacterium ammoniagenes

Abstract

Brevibacterium ammoniagenes and related bacteria are important for the industrial production of the flavor-enhancing nucleotides, inosine-5``- monophosphate (IMP) and guanosine-5``-monophosphate (GMP). Guanine analogue, 6``-mercaptoguanine (MG), can strongly inhibit the biosynthesis of purine ribonucleotides and act as a "pseudofeedback inhibitor". MG-resistant mutants have been obtained by loss of the IMP-GMP pyrophosphorylase activity with a decreased capacity to form MG ribonucleotide. Br. ammoniagenes can convert MG to its nucleotide. Therefore, it followed that increased IMP production, by conferring MG-resistance, may be due to the release of the major regulatory enzyme of purine biosynthetic pathway, 5``-phosphoribosyl-1``- pyrophosphate (PRPP):glutamine amidotransferase (EC2.4.2.14) (hereafter PRPP amidotransferase),from feedback inhibition. Thus, we have attempted to clone the gene encoding PRPP amidotransferase of Br. ammoniagenes by cloning the DNA fragment conferring MG-resistance to the wild type strain Br. ammoniagenes ATCC6872. In the current study, 6``-mercaptoguanosine (MGS) was used as analogue other than MG because of its good solubility in water. PRPP amidotransferase has been studied in many organisms and genes or cDNA sequences encoding this enzyme have been cloned from many organisms. IMP-overproducing strain Br. ammoniagenes IPR-1 was constructed by conferring MGS-resistance. From the IPR-1 strain, DNA fragment conferring MGS-resistance to the wild-type strain ATCC6872 was cloned and its nucleotide sequence was determined. Several ORFs were identified in the sequence and one of the ORFs showed high degree of homology with genes or cDNA sequences encoding PRPP amidotransferases of other organisms. This DNA fragment also conferred the increased activity of PRPP amidotransferase to the host cell. Therefore the ORF was designated as purF. When this DNA fragment was expressed in the IMP-producing host cells, IPR-1, IMP production was incre...