Chemical diversity of polyene macrolides produced by Streptomyces noursei ATCC 11455 and recombinant strain ERD44 with genetically altered polyketide synthase NysC

Abstract

The gram-positive bacterium Streptomyces noursei ATCC 11455 produces a complex mixture of polyene macrolides generally termed nystatins. Although the structures for nystatins A(1) and A(3) have been reported, the identities of other components of the nystatin complex remain obscure. Analyses of the culture extract from the S. noursei wild type revealed the presence of several nystatin-related compounds for which chemical structures could be suggested on the basis of their molecular weights, their UV spectra, and knowledge of the nystatin biosynthetic pathway. Nuclear magnetic resonance (NMR) studies with one of these polyene macrolides identified it as a nystatin analogue containing a mycarose moiety at C-35. A similar investigation was performed with the culture extract of the ERD44 mutant, which has a genetically altered polyketide synthase (PKS) NysC and which was previously shown to produce a heptaene nystatin analogue. The latter compound, tentatively named S44HP, and its derivative, which contains two deoxysugar moieties, were purified; and their structures were confirmed by NMR analysis. Nystatin analogues with an expanded macrolactone ring were also observed in the extract of the ERD44 mutant, suggesting that the altered PKS can "stutter" during the polyketide chain assembly. These data provide new insights into the biosynthesis of polyene macrolide antibiotics and the functionalities of PKSs and post-PKS modification enzymes.

FIG. 1.

Model for biosynthesis of nystatin A₁ in S. noursei ATCC 11455. PKS domains: KS, ketosynthase; AT, acyltransferase (acetate specific); mAT, acyltransferase (propionate specific); KR, ketoreductase; DH, dehydratase; ER, enoyl reductase; TE, thioesterase; ACP, acyl carrier protein. Post-PKS modifying enzymes: NysN, P450 monooxygenase (oxidation of methyl group at C-16); NysDI, mycosaminyl transferase; NysL, P450 monooxygenase (hydroxylation at C-10).

FIG. 2.

HPLC-DAD-MS analyses of polyene macrolides produced by S. noursei strains ATCC 11455 (A) and ERD44 (B). MWs (M+H)⁺ for monoisotopic peaks are indicated, with the errors (in parts per million) in the MWs of the proposed compounds given in parentheses.

FIG. 3.

Region of the spectrum of S44HP determined by DQF-COSY spectroscopy in DMSO-d₆ at 25°C. The empty square shows the absence of 27-H/28-H and 29-H/30-H correlations in the ethylene/methylene region. The asterisk indicates a peak from an impurity.

FIG. 4.

Structures of the various forms of nystatin A and its analogues. (A) Nystatin A₁ (10, 29); (B) NYST1070 (this work); (C) nystatin A₃ (49); (D) S44HP (this work); (E) NYST1068 (this work).

FIG. 4.

Structures of the various forms of nystatin A and its analogues. (A) Nystatin A₁ (10, 29); (B) NYST1070 (this work); (C) nystatin A₃ (49); (D) S44HP (this work); (E) NYST1068 (this work).

FIG. 5.

¹H chemical shift variations between NYST1068 and S44HP for the aglycone and mycosamine nonexchangeable protons. ax and eq, pseudo-axial and pseudo-equatorial orientations relative to the average plane of the macrocycle, respectively.

FIG. 6.

Elucidation of the structure of the l-mycarose moiety. (A) Single-headed arrows show correlations going from ¹³C to ¹H, as determined by ¹³C-HMBC spectroscopy. Dotted lines indicate some ¹H-¹H coupling constants. (B) Double-headed arrows refer to interproton ROEs.