Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891

Abstract

The novobiocin biosynthetic gene cluster from Streptomyces spheroides NCIB 11891 was cloned by using homologous deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratase gene fragments as probes. Double-stranded sequencing of 25.6 kb revealed the presence of 23 putative open reading frames (ORFs), including the gene for novobiocin resistance, gyrB(r), and at least 11 further ORFs to which a possible role in novobiocin biosynthesis could be assigned. An insertional inactivation experiment with a dNDP-glucose 4, 6-dehydratase fragment resulted in abolishment of novobiocin production, since biosynthesis of the deoxysugar moiety of novobiocin was blocked. Heterologous expression of a key enzyme of novobiocin biosynthesis, i.e., novobiocic acid synthetase, in Streptomyces lividans TK24 further confirmed the involvement of the analyzed genes in the biosynthesis of the antibiotic.

FIG. 1

Hypothetical biosynthetic pathway of novobiocin. Roman numerals refer to the putative assignments of genes identified in the biosynthetic reactions (see Table 1). SAM, S-adenosylmethionine.

FIG. 2

Organization of the novobiocin biosynthetic gene cluster. (A) Map of the core sequence, obtained by double-strand sequencing of S. spheroides genomic DNA (cosmid 9-6G). The roman numerals after the proposed gene functions refer to the reactions marked in Fig. 1. (B) Schematic representation of constructs used for heterologous expression of novobiocic acid synthetase. ABC, ATP-binding cassette.

FIG. 3

Comparison of the conserved core motifs of the deduced amino acid (aa) sequence of NovH with other peptide synthetase modules and with NovL. Alignment was performed by using the DNASIS program. GRSA, gramicidin S-synthetase I from Bacillus cereus (32); PCZA361.18, peptide synthetase from Amycolatopsis orientalis (64); SpsnbDE, pristinamycin I synthetase 3 from Streptomyces pristinaespiralis (11); TycA, tyrocidine synthetase 1 from Bacillus brevis (44); SvsnbDE, virginiamycin S synthetase from Streptomyces virginiae (12). The consensus line contains capital letters for amino acids with 100% conservation and lowercase letters for conservative amino acid substitutions. Motifs A1 to A8 are core motifs of the adenylation domain, and motif T represents the 4′-phosphopantetheine cofactor attachment site (40).

FIG. 4

Insertional gene inactivation of deoxysugar biosynthesis. (A) Schematic presentation of the integration event. The 520-bp FspI-PstI-DNA fragment used as the probe is indicated as a black arrow. Relevant restriction sites are identified as follows: A, AccI; E, EcoRI; F, FspI; P, PvuII. tsr, Thiostrepton resistance. Hatched arrows indicate genes. (B) Southern analysis of S. spheroides NCIB 11891 wild type (WT) and a disrupted mutant. Lanes 1 and 2, genomic DNA of the wild type restricted with PvuII in lane 1 (expected band at 1.07 kb) and with AccI in lane 2 (expected band at 1.47 kb); lanes 3 and 4, genomic DNA of an integration mutant restricted with PvuII in lane 3 (expected bands at 1.07 and 0.91 kb) and AccI in lane 4 (expected bands at 1.47 and 3.98 kb).

FIG. 5

HPLC chromatogram of secondary metabolites of S. spheroides NCIB 11891 (wild type, dotted line) and of an integration mutant (solid line). Peak 1, novobiocic acid; peak 2, novobiocin. See text for further explanations.