Heterologous expression of novobiocin and clorobiocin biosynthetic gene clusters

Abstract

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. Lambda-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage phiC31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.

FIG. 1.

Structures of novobiocin and clorobiocin.

FIG. 2.

Cosmid constructs containing the novobiocin biosynthetic gene cluster and their integration into the S. coelicolor chromosome. (a) Cosmid constructs nov-BG1, nov-AE6, and nov-AE4. P, PstI restriction site; T3 and T7, T3 and T7 promoter of the SuperCos1 vector; tet, tetracycline resistance gene; neo, neomycin/kanamycin resistance gene; int and attP, integrase gene and attachment site of phage φC31; gyrB^R, gyrase B resistance gene. Fragment sizes resulting from digestion with PstI are indicated. The cosmid backbone is out of scale. (b) Schematic representation of site-specific integration of constructs nov-BG1, nov-AE6, and nov-AE4 (see reference for details of the integration mechanism). (c) Southern blot analysis of the S. coelicolor (S. c.) M512 parental strain; S. coelicolor M512 integration mutants harboring nov-BG1, nov-AE6, or nov-AE4; and the respective cosmid constructs. M, DIG-labeled DNA Molecular Weight Marker VII (Roche). Genomic and cosmid DNA were digested with PstI. The DIG-labeled cosmid nov-BG1 was used as a probe. The 14.0-kb band resulting from site-specific integration overlaps with the 13.8-kb band from the cosmid inserts.

FIG. 3.

Cosmid constructs containing the clorobiocin biosynthetic gene cluster and their integration into the S. coelicolor chromosome. (a) Cosmid constructs clo-BG1 and clo-AE2. B, BglII restriction site; T3 and T7, T3 and T7 promoter of the SuperCos1 vector; gyrB^R, gyrase B resistance gene; parY^R, topoisomerase IV resistance gene. Fragment sizes resulting from digestion with BglII are indicated. The cosmid backbone is out of scale. (b) Schematic representation of site-specific integration of constructs clo-BG1 and clo-AE2 (see reference for details of the integration mechanism). (c) Southern blot analysis of S. coelicolor (S. c.) M512 parental strain, of S. coelicolor M512 integration mutants harboring clo-BG1 or clo-AE2, and of the respective cosmid constructs. M, DIG-labeled DNA Molecular Weight Marker VII (Roche). Genomic and cosmid DNA were digested with BglII. The DIG-labeled cosmid clo-BG1 was used as a probe.

FIG. 4.

HPLC analyses of secondary metabolites. (a) S. coelicolor M512 parental strain (detection at 305 nm). (b) S. coelicolor harboring nov-BG1 (c) S. coelicolor M512 parental strain (detection at 340 nm). (d) S. coelicolor harboring clo-BG1.

FIG. 5.

Gene deletion using an apramycin resistance cassette containing flanking XbaI and SpeI recognition sites. aac(3)IV, apramycin resistance gene; P, promoter of the apramycin resistance gene.