Biosynthetic pathway of cephabacins in Lysobacter lactamgenus: molecular and biochemical characterization of the upstream region of the gene clusters for engineering of novel antibiotics.

The cephabacins, one of the beta-lactam antibiotics, are produced by Lysobacter lactamgenus. The previous studies the cephabacin biosynthesis were limited to a gene cluster that encodes the gene products responsible for the biosynthesis of the cephem nucleus. The long-term goal of this research is to elucidate the metabolic diversity and biosynthetic pathway of cephabacins and to design and/or discover new pharmacologically active compounds by engineering the cephabacin biosynthetic pathway in L. lactamgenus. In this study, we have cloned and sequenced a 24-kb fragment of a DNA locus upstream of the previously reported but incomplete putative ORF9 of L. lactamgenus. This contains three putative ORFs (the complete ORF9, ORF10, and ORF11) transcribed in the same direction and one putative ORF (ORF12) in the opposite direction. The isolated DNA locus extends the previously cloned part of the DNA locus containing the genes responsible for biosynthesis of the cephem nucleus up to 45 kb. The 42-kb fragment of the 45-kb gene cluster is located between a potential TATA box just upstream of the ORF11 and a termination loop just downstream of the previously reported bla gene. The complete ORF9 contains three nonribosomal peptide synthetase (NRPS) modules and one polyketide synthase (PKS) module and the ORF11 contains one NRPS module. The complete ORF9 also contains a putative thioesterase domain at the C-terminal end. We predicted the amino acid specificity of the four NRPSs by generating specificity binding pockets and expressed one of the NRPSs to confirm the amino acid specificity. The adenylation domain of the NRPS1, which is the last module of the NRPSs, showed significant amino acid specificity for L-arginine. These findings are in perfect agreement with the composition that was expected for the structure of cephabacins which contain an acetate residue, an L-arginine, and one to three L-alanines at the C-3' position of the cephem nucleus of cephabacins. The ORF10, encoding a putative ABC transporter which might be involved in conferring resistance against cephabacins, was identified between the complete ORF9 and the ORF11. Therefore, the complete ORF9, ORF10, ORF11 reported here and the other genes previously reported constitute an operon for the biosynthesis of cephabacins in L. lactamgenus. Based on our results, the biosynthetic pathways of acetate and elongated peptide moieties and a mechanism by which cephabacins are assembled by connecting the peptide moiety synthesized by the gene products of the complete ORF9 and the ORF11 to the C-3' position of the cephem nucleus synthesized by the gene products of pcbAB, pcbC, cefE, cefF, and cefD have been elucidated.