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-Poly-L-lysine (-PL) is produced by Streptomyces albulus NBRC14147 as a secondary metabolite and can be detected only when the fermentation broth has an acidic pH during the stationary growth phase. Since strain NBRC14147 produces -PL-degrading enzymes, the original chain length of the -PL polymer product synthesized by -PL synthetase (Pls) is unclear. Here, we report on the identification of the gene encoding the -PL-degrading enzyme (PldII), which plays a central role in -PL degradation in this strain. A knockout mutant of the pldII gene was found to produce an -PL of unchanged polymer chain length, demonstrating that the length is not determined by -PL-degrading enzymes but rather by Pls itself and that the 25 to 35 L-lysine residues of -PL represent the original chain length of the polymer product synthesized by Pls in vivo. Transcriptional analysis of pls and a kinetic study of Pls further suggested that the Pls catalytic function is regulated by intracellular ATP, high levels of which are required for full enzymatic activity. Furthermore, it was found that acidic pH conditions during -PL fermentation, rather than the inhibition of the -PL-degrading enzyme, are necessary for the accumulation of intracellular ATP.

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...

Studies on the production of L-arginine by fermentation using mutants of Corynebacterium (Brevibacterium), Bacillus, and Serratia have been conducted since the 1960s. More recently, the breeding of L-arginine production strains by gene recombination techniques using Escherichia coli has been investigated. To produce L-arginine efficiently by fermentation, it is necessary to breed strains with a strong biosynthetic pathway to L-arginine. Because L-arginine is biosynthesized from the precursor L-glutamic acid through ornithine and citrulline, the use of strains with a high capability for producing L-glutamic acid is desirable. Corynebacterium (Brevibacterium), which is well known in the production of L-glutamic acid, was selected as a starting strain for the breeding of an L-arginine producer and has been used on a commercial scale. Regarding the fermentation conditions, as for other amino acids, L-arginine fermentation is controlled by regulating pH near the neutral point. Due to its high oxygen requirement, L-arginine production is seriously impaired without sufficient oxygen. Advanced purification methods are necessary to obtain highly pure L-arginine from the fermentation broth. After fermentation is complete, bacterial cells and proteins are removed by means of a membrane or centrifugation, and impurities are removed by means of an ion-exchange resin or activated carbon. Highly pure L-arginine crystals can be obtained through concentration at the end of the process.

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