Regulation of Geldanamycin Biosynthesis by Cluster-Situated Transcription Factors and the Master Regulator PhoP

Abstract

Geldanamycin and the closely related herbimycins A, B, and C are benzoquinone-type ansamycins with antitumoral activity. They are produced by Streptomyces hygroscopicus var. geldanus, Streptomyces lydicus and Streptomyces autolyticus among other Streptomyces strains. Geldanamycins interact with the Hsp-90 chaperone, a protein that has a key role in tumorigenesis of human cells. Geldanamycin is a polyketide antibiotic and the polyketide synthase contain seven modules organized in three geldanamycin synthases genes named gdmAI, gdmAII, and gdmAIII. The loading domain of GdmI activates AHBA, and also related hydroxybenzoic acid derivatives, forming geldanamycin analogues. Three regulatory genes, gdmRI, gdmRII, and gdmRIII were found associated with the geldanamycin gene cluster in S. hygroscopicus strains. GdmRI and GdmRII are LAL-type (large ATP binding regulators of the LuxR family) transcriptional regulators, while GdmRIII belongs to the TetR-family. All three are positive regulators of geldanamycin biosynthesis and are strictly required for expression of the geldanamycin polyketide synthases. In S. autolyticus the gdmRIII regulates geldanamycin biosynthesis and also expression of genes in the elaiophylin gene cluster, an unrelated macrodiolide antibiotic. The biosynthesis of geldanamycin is very sensitive to the inorganic phosphate concentration in the medium. This regulation is exerted through the two components system PhoR-PhoP. The phoRP genes of S. hygroscopicus are linked to phoU encoding a transcriptional modulator. The phoP gene was deleted in S. hygroscopicus var geldanus and the mutant was unable to grow in SPG medium unless supplemented with 5 mM phosphate. Also, the S. hygroscopicus pstS gene involved in the high affinity phosphate transport was cloned, and PhoP binding sequences (PHO boxes), were found upstream of phoU, phoRP, and pstS; the phoRP-phoU sequences were confirmed by EMSA and nuclease footprinting protection assays. The PhoP binding sequence consists of 11 nucleotide direct repeat units that are similar to those found in S. coelicolor Streptomyces avermitilis and other Streptomyces species. The available genetic information provides interesting tools for modification of the biosynthetic and regulatory mechanisms in order to increase geldanamycin production and to obtain new geldanamycin analogues with better antitumor properties.

Keywords: Streptomyces hygroscopicus; antitumorals; biosynthesis; geldanamycins; gene cluster; phosphate control.

Figure 1

Geldanamycin structure and biosynthesis pathway. (A) Structures of geldanamycin and herbimycin. (B) Geldanamycin biosynthetic pathway. The enzymes involved in every step are indicated.

Figure 2

Cluster of geldanamycin genes in S. hygroscopicus NRRL3602. Cluster of geldanamycin genes in S. hygroscopicus NRRL3602 [18]. The name of each gene is indicated above it. Similar gene clusters have been found in other S. hygroscopicus strains and in S. autolyticus with the minor differences described in the text. Genes for the geldanamycin precursor AHBA [19], named gdnKEAOP, and located separately in the genome, are shown below. The names of the geldanamycin genes given by different authors and function of the encoded enzymes are shown in Table 2.

Figure 3

Effect of the regulatory genes on geldanamycin production. (A) Production of geldanamycin by S. hygroscopicus var. geldanus NRRL3602 transformed with the empty plasmid pRA (black circles), and the pRA plasmid [38] carrying insertions with the genes gdmRI (white triangles), gdmRII (white squares), and gdmRI+gdmRII (white circles). (B) HPLC analysis of geldanamycin production by S. hygroscopicus var. geldanus NRRL3602 (upper panel) and S. hygroscopicus ΔgdmRII (lower panel) in which gdmRII was deleted. The arrow indicates the geldanamycin peak.

Figure 4

Phosphate control of geldanamycin production. (A) Organization of the pho genes and the surrounding region in a NotI DNA fragment of S. hygroscopicus var. geldanus NRRL 3602. Restriction sites for XhoI (X) and SalI (S) are indicated. (B) Production of geldanamycin in SPG medium (white circles) and SPG medium supplemented with 5 mM (gray circles) and 10 mM inorganic phosphate (black circles).

Figure 5

Localization of PHO boxes in the intergenic region phoRP-PhoU of S. hygroscopicus var. geldanus NRRL 3602. (A) Sequence of the intergenic region phoRP-phoU showing in red the nucleotides belonging to the phoP and phoU genes. The regions I, II, and III used in EMSA analysis are indicated. (B) Footprinting analysis of DNA containing regions II and III. The upper line (in red) corresponds to the parental strain DNA without the PhoP^DBD-GST protein. The lower line (in blue) corresponds to the same DNA sequence protected by addition of PhoP^DBD-GST protein. PhoP^DBD-GST protein (2 μM) was used in the assay as described by Sola-Landa et al. [66].