Functional analyses of oxygenases in jadomycin biosynthesis and identification of JadH as a bifunctional oxygenase/dehydrase

Abstract

A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase alpha (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon.

FIG. 1

a, partial gene organization map of jadomycin biosynthetic gene cluster. Genes responsible for UWM6 biosynthesis are indicated in gray, and genes studied in this paper are indicated in black. B, BamHI; K, KpnI. b, conserved motifs in JadF, JadH, and related proteins. Three conserved motifs are indicated in bold and marked on top. The highly conserved residues in motifs are marked with asterisk.

FIG. 2

a, diagram illustrating the construction of VS668 by disrupting jadH with an apramycin-resistance gene (Am^R). B, BamHI; N, NcoI, M, MluI; P, PstI. b, Southern blotting of S. venezuelae ISP5230 and VS668 genomic DNA digested with PstI. Lane M, digoxigeninlabeled λ DNA (HindIII- and EcoRI-digested); lane 1, S. venezuelae ISP5230 genomic DNA; lane 2, VS668 genomic DNA.

FIG. 3

a, diagram illustrating the construction of CH56 by in-frame deleting of jadA. H, HindIII; E, EcoRI; N, NdeI; B, BamHI; X, XhoI. b, PCR detection of jadA deletion. Lane M is DNA ladder marker, and lanes 1,2, and 3 are fragments obtained by PCR with pHK400A, CH56, and wild-type genomic DNA as template, respectively. c, Southern blotting of S. venezuelae ISP5230 and CH56 genomic DNA digested with XhoI. Lane M, digoxigenin-labeled λ DNA (HindIII- and EcoRI-digested); lane 1, S. venezuelae ISP5230 genomic DNA; lane 2, CH56 genomic DNA.

FIG. 4. SDS-PAGE of purified JadF and JadH (His-tagged)

Lane M, low molecular weight protein marker; Lanes 1 and 2 are two elution fractions of His₆-JadF; lanes 3 and 4 are two elution fractions of His₆-JadH.

FIG. 5

Proposed biosynthetic pathway of jadomycin and selected structures of angucycline polyketides, whose biosyntheses require similar ring B cleavages of angucyclinone precursors.