Three Gram-negative, aerobic, rod-shaped bacterial strains were isolated, from the pollen of Japanese flowers, as producers of xylitol; these strains were subjected to a polyphasic taxonomic study. Phylogenetic analyses of the 16S rRNA gene sequences demonstrated that these three isolates formed a new cluster within a group of acetic acid bacteria in the α-Proteobacteria. The characteristics of the three isolates were as follows: (i) their predominant quinone was Q-10; (ii) their cellular fatty acid profile contained major amounts of 2-hydroxy acids and an unsaturated straight-chain acid (C18 : 1 ω7c); and (iii) their DNA G+C contents were in the range 51·9–52·3 mol%, which is around the lower limit of the reported range for the genera of acetic acid bacteria. The negligible or very weak productivity of acetic acid from ethanol and the osmophilic growth properties distinguished these strains from other acetic acid bacteria. The unique phylogenetic and phenotypic characteristics suggest that the three isolates should be classified within a novel genus and species with the proposed name Saccharibacter floricola gen. nov., sp. nov. The type strain is strain S-877T (=AJ 13480T=JCM 12116T=DSM 15669T).

A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. The Morganella morganii gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to the M. morganii PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced into Escherichia coli, and the transformants were screened for the production of 5-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of 5-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreased K m value for inosine was responsible for the increased productivity.Nucleotides are often used as food additives and as pharmaceutical intermediates. Among them, 5Ј-IMP and 5Ј-GMP are important, because they have a characteristic taste and are used as flavor potentiators in various foods. Purine nucleosides such as inosine (7, 9) and guanosine (8) can be produced efficiently by fermentation, and phosphorylation of nucleosides is a very efficient process for the large-scale production of 5Ј nucleotides.At present, there are two main phosphorylation methods. One is a chemical phosphorylation process that uses phosphoryl chloride (POCl 3 ) (22), and the other is an enzymatic phosphorylation process that uses inosine kinase of Escherichia coli (11,12). The chemical phosphorylation process is relatively complex, because it needs two reactors, for the fermentation and chemical reactions. The enzymatic phosphorylation process is simpler, because the enzymatic reaction can be carried out in the same reactor as the fermentation reaction. The inosine kinase reaction, however, requires ATP, and the ATP needs to be regenerated by resting cells of Corynebacterium ammoniagenes, which are used for the fermentative production of inosine. Therefore, applications of the enzymatic phosphorylation process are limited. Alternatively, an enzyme that catalyzes the synthesis of nucleotides by transfer of phosphate groups from low-energy phosphate esters to nucleosides was described by Brawerman and Chargaff (3) and Mitsugi and coworkers (10).Prompted by these findings, we have investigated a novel nucleoside phosphorylation reaction using the food additive pyrophosphate (PP i ), as shown in the following equation (9, 10): nucleoside ϩ PP i 3 nucleoside 5Ј-monophosphate ϩ P i acid phosphatase/phosphotransferase (EC 3.1.3.2). We purified and characterized a C5Ј-position selective pyrophosphatenucleoside phosphotransferase from a crude extract of Morganella morganii NCIMB10466 (2). The purified enzyme exhibited not only phosphotransferase activity but also phosphatase activity. On the basis of a kinetic study, it appeared to be a phosphatase with regioselective phosp...

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