Matsui H, Kawasaki H, Shimaoka M, Kurahashi O
Fermentation & Biotechnology Laboratories, Ajinomoto Co., Inc., Kawasaki-shi, Kanagawa, Japan.
Biosci Biotechnol Biochem. 2001 Mar;65(3):570-8. doi: 10.1271/bbb.65.570.
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.
为了从野生型微生物中获得肌苷高产菌株，已知添加腺嘌呤需求、去除嘌呤核苷水解活性、消除反馈抑制以及抑制嘌呤核苷酸生物合成途径中的关键酶是必不可少的。因此，在大肠杆菌菌株W3110中对purA（腺嘌呤需求）、deoD（去除嘌呤核苷磷酸化酶活性）、purR（解除嘌呤核苷酸生物合成途径的调控抑制）进行了破坏，并且使磷酸核糖焦磷酸（PRPP）酰胺转移酶对5'-单磷酸腺苷（AMP）和5'-单磷酸鸟苷（GMP）的反馈抑制不敏感，然后评估了肌苷的产量。在使用携带编码脱敏PRPP酰胺转移酶的PRPP酰胺转移酶基因（purF）的质粒的情况下，使用purF破坏突变体作为宿主菌株。发现这四种基因型的创新带来了少量肌苷积累。此外，大肠杆菌的腺嘌呤营养缺陷型突变体显示出不适当的腺嘌呤利用，因为其生长不能有效地响应添加的腺嘌呤浓度。由于大肠杆菌中腺苷脱氨酶的存在是众所周知的，并且认为其参与腺嘌呤利用，构建并测试了破坏腺苷脱氨酶基因（add）的突变体。该突变体缺乏purF、purA、deoD、purR和add基因，并作为质粒携带脱敏的purF，每升积累约1克肌苷。尽管我们研究了purR破坏和purF基因改良的效果，但出乎意料的是，该突变体的肌苷产量并未增加。
