Nystatin biosynthesis and transport: nysH and nysG genes encoding a putative ABC transporter system in Streptomyces noursei ATCC 11455 are required for efficient conversion of 10-deoxynystatin to nystatin

Abstract

The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxynystatin, a nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both nystatin and 10-deoxynystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of nystatin and 10-deoxynystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.

FIG. 1.

ABC transporter system encoded by the nysG and nysH genes in S. noursei. (A) Predicted structural features of the NysH and NysG proteins. SP, signal peptide; TM, transmembrane helix; WA, Walker A motif; WB, Walker B motif; LH, Linton-Higgins motif. (B) Genotypes of the nysH and nysG mutants compared to the wild-type (WT) strain.

FIG. 2.

DAD-HPLC isoplots of culture extracts from S. noursei ATCC 11455 (WT) and transporter mutants. Peaks for nystatin and a 910-Da nystatin analogue are indicated.

FIG. 3.

¹H NMR (400 MHz) spectra of DMSO-d₆ solutions (5 mM) at 25°C of nystatin showing the H-10 proton at 3.2 ppm (A) and of 10-deoxynystatin showing the H-10 proton at 1.4 to 1.6 ppm (B). An impurity in the 10-deoxynystatin sample is indicated by an “X” in panel B.

FIG. 4.

Results of washing experiments confirming the efflux of both nystatin and 10-deoxynystatin from the S. noursei wild-type strain and transporter mutants. Numbers above the bars reflect the actual amounts (g/liter) of nystatin and 10-deoxynystatin in the fermentation broth prior to washing (averages of two experiments).

FIG. 5.

Effect of sodium o-vanadate on biosynthesis of nystatin and 10-deoxynystatin by S. noursei wild-type strain and ΔnysH (AHH2) and ΔnysG (AHG13) mutants. Average data from three parallel experiments are presented.