Fig 1.
The polyether tetronates tetromadurin (1), tetronasin (2), and tetronomycin (3).
Table 1.
Primers used in this study.
Table 2.
Plasmids used in this study.
Table 3.
Bacteria strains used in this study.
Fig 2.
Predicted functions of genes in the mad gene cluster.
ATa = malonyl-CoA selective AT domain (acetate unit). ATp = (2S)-methylmalonyl-CoA selective AT domain (propionate unit). ATm = predicted (2R)-methoxymalonyl-ACP selective AT domain.
Fig 3.
The tetromadurin biosynthetic gene cluster (the mad gene cluster).
The total size of the mad gene cluster is 110 kbp. A total of 32 were predicted to comprise the mad gene cluster. Genes are colour coded according to their predicted function.
Fig 4.
The proposed biosynthesis pathway of tetromadurin (part 1).
(I) The seven PKS enzymes (MadAI-MadAVIII) and predicted linear tetromadurin intermediates of the mad gene cluster. ATa domains contain the amino acid motifs associated with malonyl-CoA (acetate unit) incorporation. ATp domains contain the amino acid motifs associated with (2S)-methylmalonyl-CoA (propionate unit) incorporation. The ATm domain is predicted to recognise (2R)-methoxymalonyl-ACP. ERD domains are predicted to create a D-configured α-methyl group. B1 KRs are predicted to create D-configured α-methyl and β-hydroxyl groups. The KS domain in MadAI is a KSQ domain. The red crosses indicate domains that are predicted to be inactive on the basis of their amino acid sequence. (II) The key biosynthetic precursor 1,3-bisphosphoglycerate, involved in both tetronate formation and (2R)-methoxymalonyl-ACP biosynthesis. (III) Formation of glyceryl-ACP by the FkbH-like enzyme Mad14 and the ACP Mad12. (IV) Oxidation of glyceryl-ACP by the dehydrogenase Mad11 to form 2-hydroxy-3-oxopropionyl-ACP. (V) Oxidation of 2-hydroxy-3-oxopropionyl-ACP by the dehydrogenase Mad13 to form hydroxymalonyl-ACP. (VI) Methylation of hydroxymalonyl-ACP by the O-methyltransferase Mad15 to form (2R)-methoxymalonyl-ACP. (VII) The (2R)-methoxylmalonyl-ACP is incorporated by the MadAVI_AT_13. (VIII) Synthesis of a second pool of glyceryl-ACP by FkbH-like Mad7 and ACP Mad8. (IX) Glyceryl-ACP is a substrate of the FabH-like protein Mad16 that catalyses tetronate formation and concomitant chain release from the PKS. Bonds/atoms have been coloured red to highlight the chemical change.
Fig 5.
The proposed biosynthetic pathway of tetromadurin (part 2).
(X) In the first step of tetrahydrofuran formation, MadC catalyses epoxidation of the C24-C25 and C28-C29 E double bonds. (XI) The epoxide hydrolase MadB catalyses formation of the two tetrahydrofuran rings through a cascade epoxide ring-opening; (XII) Acetylation of the C41 hydroxyl group is catalysed by Mad17. (XIII) Elimination of the acetyl group by Mad18 forms the C40-C41 exocyclic double bond. (XIV) Hydroxylation of C36 by Mad29. It is possible this hydroxylation event takes place earlier in the biosynthesis (XV) Mad10 catalyses the formation of an oxadecalin containing intermediate 10. (XVI) The equivalent oxadecalin intermediate in tetronasin biosynthesis acquired a water at C3 to form a hemiacetal [7]. Given the structural similarity between tetromadurin and tetronasin, it is likely the tetromadurin oxadecalin intermediate can be hydrated at C3 to form 11. (XVII) Mad31 catalyses formation of the tetrahydropyran ring and dismantles the oxadecalin ring to form 12. (XVIII) Mad30 then catalyses hydroxylation of C38, forming tetromadurin 1. Bonds/atoms have been coloured red to highlight the chemical change.
Fig 6.
Hypothetical ACP-bound linear tetromadurin backbone.
13: the hypothetical linear tetromadurin intermediate predicted to be produced by the MadAI-MadAVII arrangement. 14: the hypothetical linear tetromadurin intermediate predicted from the structure of tetromadurin itself.
Fig 7.
HPLC-MS analysis of A. verrucosospora Δmad10 and A. verrucosospora Δmad31.
a, The structure and molecular weight of tetromadurin (1) produced by A. verrucosospora. b, Extracted m/z = 783.8 HPLC-MS spectra from A. verrucosospora wild type (WT), A. verrucosospora Δmad10, and A. verrucosospora Δmad10 pIB139-mad10. c, Extracted m/z = 783.8 HPLC-MS spectra from A. verrucosospora WT, A. verrucosospora Δmad31, and A. verrucosospora Δmad31 pIB139-mad31. All data are representative of three independent experiments.
Fig 8.
Novel tetromadurin analogue produced by A. verrucosospora Δmad10.
a, Total ion current (TIC) spectra of the crude organic extracts from A. verrucosospora wild type (WT) and A. verrucosospora Δmad10. The other peaks represent unrelated species. Tetromadurin (1) was produced by A. verrucosospora and eluted at 19.8 min. A novel tetromadurin-related metabolite (named T-17) was produced by A. verrucosospora Δmad10, eluting at 17.4 min. b, HPLC-PDA (photodiode array) spectra of the crude organic extracts from A. verrucosospora WT and A. verrucosospora Δmad10. c, Positive ion mode mass spectrum of tetromadurin (1) and T-17: *[M+Na]+, **[M+NH4]+, ***[M+H+, ****[M+H−H2O]+, *****[M+H−2.H2O]+. For tetromadurin (1): m/z [C42H64O12+Na]+ = 783.81; m/z [C42H64O12+NH4]+ = 778.82; m/z [C42H64O12+H]+ = 761.84; m/z [C42H64O12+H−H2O]+ = 739.84. For T-17: m/z [C42H64O11+Na]+ = 767.69; m/z [C42H64O11+NH4]+ = 762.58; m/z [C42H64O11+H]+ = 745.63; m/z [C42H64O11+H−H2O]+ = 727.70; m/z [C42H64O11+H−H2O]+ = 727.70; m/z [C42H64O11+H−2.H2O]+ = 709.79. d, UV absorption spectra of tetromadurin (1) (λmax = 252 nm, 300 nm, MeOH) and T-17 (λmax = 236 nm, 300 nm, MeOH). The ♦ indicates an unrelated compound that is also detectable in the WT strain.
Fig 9.
A. verrucosospora Δmad10 no longer produces tetromadurin but produces a tetromadurin derivative, T-17. The mass spectrum of T-17 indicates that it is missing a primary hydroxyl group. The primary hydroxyl groups of tetromadurin are located at C36 and C38. Two possible structures of T-17 are therefore possible where either: a, the C38 hydroxyl group (15) or b, the C36 hydroxyl group (16) is absent. c, the structure of the tetronasin derivative 17 produced by Streptomyces longisporoflavus Δtsn11 [7].