A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis.

The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-ß-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-ß-lysine by ORF 5, followed by transfer to the T domain of ORF 18. In contrast, L-ß-lysine molecules adenylated by ORF 19 are used to elongate an L-ß-lysine peptide chain on ORF 18, a reaction unexpectedly catalyzed by ORF 19 itself. Finally, the C domain of ORF 18 catalyzes the condensation of L-ß-lysine oligopeptides covalently bound to ORF 18 with a freely diffusible intermediate to release the ST products. These results highlight an unusual activity for an A domain and unique mechanisms of crosstalk within NRPS machinery.

Mechanism of epsilon-poly-L-lysine production and accumulation revealed by identification and analysis of an epsilon-poly-L-lysine-degrading enzyme.

Epsilon-poly-L-lysine (epsilon-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 epsilon-PL-degrading enzymes, the original chain length of the epsilon-PL polymer product synthesized by epsilon-PL synthetase (Pls) is unclear. Here, we report on the identification of the gene encoding the epsilon-PL-degrading enzyme (PldII), which plays a central role in epsilon-PL degradation in this strain. A knockout mutant of the pldII gene was found to produce an epsilon-PL of unchanged polymer chain length, demonstrating that the length is not determined by epsilon-PL-degrading enzymes but rather by Pls itself and that the 25 to 35 L-lysine residues of epsilon-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 epsilon-PL fermentation, rather than the inhibition of the epsilon-PL-degrading enzyme, are necessary for the accumulation of intracellular ATP.

Mutational analysis of the three tandem domains of ε-poly-L-lysine synthetase catalyzing the L-lysine polymerization reaction.

ε-Poly-l-lysine (ε-PL) synthetase (Pls) is a nonribosomal peptide synthetase (NRPS)-like enzyme with three tandem domains to catalyze the l-lysine polymerization reaction. Mutational analysis of the three tandem domains demonstrated that the interconnected action of all three domains is essential for the enzyme activity.

Development of a recombinant ε-poly-L-lysine synthetase expression system to perform mutational analysis.

ε-Poly-L-lysine (ε-PL) synthetase (Pls), which is a membrane protein with adenylation and thiolation domains characteristic of the nonribosomal peptide synthetases, catalyzes polymerization of L-lysine molecules (25-mer to 35-mer). Here, we report on the development of a recombinant Pls expression system that allowed us to perform a site-directed mutational analysis.

Production of arginine by fermentation.

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.