Investigation of various genotype characteristics for inosine accumulation in Escherichia coli W3110.
For the derivation of an inosine-overproducing strain from the wild type microorganism, it is known that the addition of an adenine requirement, removal of purine nucleoside hydrolyzing activity, removal of the feedback inhibition, and repression of key enzymes in the purine nucleotides biosynthetic pathway are essential. Thus, the disruption of purA (adenine requirement), deoD (removal of purine nucleosides phosphorylase activity), purR (derepression of the regulation of purine nucleotides biosynthetic pathway), and the insensitivity of the feedback inhibition of phosphoribosylpyrophosphate (PRPP) amidotransferase by adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) were done in the Escherichia coli strain W3110, and then the inosine productivity was estimated. In the case of using a plasmid harboring the PRPP amidotransferase gene (purF) that encoded a desensitized PRPP amidotransferase, purF disrupted mutants were used as the host strains. It was found that the innovation of the four genotypes brought about a small amount of inosine accumulation. Furthermore, an adenine auxotrophic mutant of E. coli showed inappropriate adenine use because its growth could not respond efficiently to the concentration of adenine added. As the presence of adenosine deaminase is well known in E. coli and it is thought to be involved in adenine use, a mutant disrupted adenosine deaminase gene (add) was constructed and tested. The mutant, which is deficient in purF, purA, deoD, purR, and add genes, and harboring the desensitized purF as a plasmid, accumulated about 1 g of inosine per liter. Although we investigated the effects of purR disruption and purF gene improvement, unexpectedly an increase in the inosine productivity could not be found with this mutant.
Matsui
H
H
Fermentation & Biotechnology Laboratories, Ajinomoto Co., Inc., Kawasaki-shi, Kanagawa, Japan. hiroshi_matsui@ajinomoto.com
Kawasaki
H
H
Shimaoka
M
M
Kurahashi
O
O
D000243:Adenosine Deaminase / Q000235:genetics / Q000502:physiology*; D000265:Adenylosuccinate Synthase / Q000235:genetics / Q000502:physiology*; D000582:Amidophosphoribosyltransferase / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005838:Genotype; D007288:Inosine / Q000096:biosynthesis*; D016297:Mutagenesis, Site-Directed; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics / Q000502:physiology*; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C065825:PurR protein, Bacteria; C489317:PurR protein, E coli; D012097:Repressor Proteins; D007288:Inosine; D011683:Purine-Nucleoside Phosphorylase; D000582:Amidophosphoribosyltransferase; D000243:Adenosine Deaminase; D000265:Adenylosuccinate Synthase