Biosynthetic Pathway of Cephabacins in Lysobacter lactamgenus : Molecular and Biochemical Characterization of the Upstream Region of the Gene Clusters for Engineering of Novel Antibiotics 신규 항생물질 창출을 위한 유전자 뭉치 상단부의 분자 생화학적 특성 조사 = Lysobacter lactamgenus에서의 cephabacins 생합성 경로

다국어 초록 (Multilingual Abstract)

The cephabacins, one of the β-lactum antibiotics, are produced by Lysobacter lactamgenus. The previous studies the cephabacin biosyathesis 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 rescarch 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 tis study, we have clooed and sequenced a 24-Kb fragment of a DNA locus apstream of the previously report 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 lsolated DNA locus extends the previously clonod part of the DNA locus containing the genes responsible for biosynthesis of the cephem nucleus up to 45kb. 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 bia gene. The complete ORF9 contains three nonribosomal peptide synthetase (NRPS) modulcs and one polyketide synthese (PKS) module and the ORF11 contains one NRPS module. The complete ORF9 also contains a puttive thioesterase domain at the C-terminal end. We predicted the amino acid specificity of the foru NRPSs by generating specificity binding pockets and expressed one of the NRPSs to conflrm 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 agroement with the composition that was expected for the structure of cephbacins which coptain as 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 identifled between the complete ORF9 and the ORF11. Therefore, the complete ORF9, ORF10. ORF11 reported bere and the other genes previously reported constitute an operon for the biosynthesis of caphabacins in L. lactamgenus. Basod on our results, the biosynthetic pathways of acetate and elonganted peptide moietion and a mechanism by which cephabacins are assembled by conecting the peptide moiety synthesized by the gene products of the complete ORF9 and the ORF11 to the C-3' position of the cephem nucleus syatheslzed by the gene products of pcb AB, pcbC, cefE, cefF, and cefD have been elucidated.