Fig 1.
Phthoxazolin A production in the S. avermitilis progeny.
(A) Chemical structure of phthoxazolin A. (B) HPLC chromatograms of MeOH extracts from S. avermitilis KA-320 (top), S. avermitilis SUKA22 (middle), and S. avermitilis K139 (bottom). mAU, milliabsorbance units at 275 nm. Phthoxazolin A was detected at a retention time of 33.9 min, and is indicated by an inverted triangle.
Fig 2.
Genetic organization of the phthoxazolin A biosynthetic gene cluster.
Arrows indicate the direction of transcription and relative gene size. ORFs predicted to participate in phthoxazolin A biosynthesis are shaded. The proposed functions of individual ORFs are indicated here and summarized in Table 1.
Table 1.
Deduced functions of ORFs in the phthoxazolin A biosynthetic gene cluster.
Fig 3.
Phthoxazolin A production in the avaR3/ptxA double mutant.
(A) Schematic representation of the strategy for the ptxA gene disruption. ΔavaR3, avaR3 mutant; ΔavaR3 ΔptxA, avaR3/ptxA double mutant; ΔavaR3 ΔptxA/ptxA, ptxA-complemented avaR3/ptxA double mutant. (B) PCR analysis to confirm gene-disruption of the ptxA gene and its complementation. With the primer pair ptzA-tFw/ptzA-tRe, a fragment (4,193 bp) containing an intact ptxA gene or a fragment (3,067 bp) containing the mut-loxP-hph-mut-loxP was amplified with PCR. An intact ptxA gene (485 bp) was detected by using the primer pair ptzA-Fw/ptzA-Re. An internal region of aac(3)IV gene (974 bp) was amplified using the primer pair apr-Fw/apr-Re. (C) HPLC chromatograms of MeOH extracts from the avaR3/ptxA double mutant. mAU, milliabsorbance units at 275 nm. Phthoxazolin A is indicated by an inverted triangle.
Fig 4.
Proposed model for phthoxazolin A biosynthesis.
A, adenylation; ACP, acyl carrier protein; AT, acyltransferase; C, condensation; Cyp, cytochrome P450; DH, dehydratase; F, formylation; KS, ketosynthase; KS0, KS lacking His in the HTGTG motif; KR, ketoreductase; MT, methyltransferase; PCP, peptidyl carrier protein. The presumed inactive ACP domain of module 9 is shaded in black.