Insights into the biosynthesis of the benzoquinone ansamycins geldanamycin and herbimycin, obtained by gene sequencing and disruption

Abstract

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.

FIG. 1.

Proposed pathway for herbimycin biosynthesis, starting from the ansamycin starter unit AHBA, followed by PKS processing through seven chain extension units (modules [mod.] 1 to 7) to give the hypothetical intermediate progeldanamycin, followed by formation of herbimycin A by PKS tailoring enzymes.

FIG. 2.

Comparison of a 90-kb section of the herbimycin biosynthetic gene cluster with the genes responsible for geldanamycin production. PKS genes (A1, A2, and A3) and the PKS domains for both the hbm and gdm cluster are given in blue, with the modular organization indicated above each PKS gene by broad arrows. PKS tailoring genes are in green, and differences in gene organization or homology are indicated in red. “No homology” or “end of homology” indicates that these DNA regions are present but have <20% similarity; “missing in hbm” indicates that this region of DNA is absent in the herbimycin-producing gene cluster, whereas the comparable region contains the gdmF and gdmM genes in the geldanamycin producer.

FIG. 3.

Structures of geldanamycin and monophenolic analogs. Distinctions from the geldanamycin structure are highlighted, and missing functions are indicated by open circles.

FIG. 4.

ahba-B biosynthetic gene cluster in S. hygroscopicus AM 3602.