Identification by Heterologous Expression and Gene Disruption of VisA as l -Lysine 2-Aminotransferase Essential for Virginiamycin S Biosynthesis in Streptomyces virginiae

Abstract

The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics. Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety. The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35°C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into ⌬ 1 -piperidine 2-carboxylic acid. A visA deletion mutant of S. virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant. No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation. However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M 1 at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis. These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine.

Identification and Characterization of the Pyridomycin Biosynthetic Gene Cluster of Streptomyces pyridomyceticus NRRL B-2517

“…The streptogramin 3-HPA is derived from lysine (14,36,37), but the labeling experiment indicated that both pyridyl moieties of pyridomycin originate from L-aspartic acid, glycerol, and/or pyruvate, but lysine is not incorporated (11). Pyridyl ring formation from aspartate is known from the primary metabolism NAD biosynthetic pathway (39) (path a in Fig.…”

“…Biosynthesis of the Pyridyl Moieties-PyrB is an L-lysine 2-aminotransferase similar to VisA (61% identity and 70% similarity) involved in 3-HPA formation for virginiamycin biosyn-thesis in Streptomyces virginiae (14,36) and to NikC (56% identity and 69% similarity) that catalyzes the initial reaction for converting lysine to the pyridyl residue in nikkomycin D in Streptomyces tendae (37). However, L-aspartic acid instead of L-lysine (along with glycerol or pyruvic acid) was incorporated into the two pyridyl residues in pyridomycin.…”

Divergent Roles in Arabidopsis thaliana Development and Defense of Two Homologous Genes, ABERRANT GROWTH AND DEATH2 and AGD2-LIKE DEFENSE RESPONSE PROTEIN1, Encoding Novel Aminotransferases

“…Precedent for an aminotransferase having such a function comes from Streptomyces virginae, which uses Lys 2-aminotransferase to synthesize a cyclohexadepsipeptide antibiotic (Namwat et al, 2002).…”

Streptogramins, Oxazolidinones, and Other Inhibitors of Bacterial Protein Synthesis

“…VisA has been purified and shown to be a pyridoxal-phosphatedependent Lys 2-aminotransferase capable of generating 1-piperidine 2-carboxylate. 33 Inactivation of the visA gene in S. virginiae completely blocked biosynthesis of virginiamycin S 1 , and addition of 3-hydroxypicolinic acid but not pipecolic acid to the culture medium rescued this null phenotype, suggesting that VisA is required for formation of the former ( Figure 7). 33 Paradoxically, the intracellular levels of 1-piperidine 2-carboxylic acid in the visA null mutant were comparable to the wild-type organism.…”

“…Sequencing of DNA downstream from known regulatory genes of virginiamycin S biosynthesis in S. virginiae identified four genes, visA-D, predicted to be involved in amino acid biosynthesis. 33 Both visA and visC are predicted to encode enzymes that generate 1-piperidine 2-carboxylic acid. VisA has been purified and shown to be a pyridoxal-phosphatedependent Lys 2-aminotransferase capable of generating 1-piperidine 2-carboxylate.…”