Biosynthesis of Deoxyamphotericins and Deoxyamphoteronolides by Engineered Strains of Streptomyces nodosus

Abstract

Amphotericin B is an antifungal antibiotic produced by Streptomyces nodosus. During biosynthesis of amphotericin, the macrolactone core undergoes three modifications: oxidation of a methyl branch to a carboxyl group, mycosaminylation, and hydroxylation. Gene disruption was undertaken to block two of these modifications. Initial experiments targeted the amphDIII gene, which encodes a GDP-D-mannose 4,6-dehydratase involved in biosynthesis of mycosamine. Analysis of products by mass spectrometry and NMR indicated that the amphDIII mutant produced 8-deoxyamphoteronolides A and B. This suggests that glycosylation with mycosamine normally precedes C-8 hydroxylation and that formation of the exocyclic carboxyl group can occur prior to both these modifications. Inactivation of the amphL cytochrome P450 gene led to production of novel polyenes with masses appropriate for 8-deoxyamphotericins A and B. These compounds retained antifungal activity and may be useful new antibiotics.

Phosphomannose isomerase and phosphomannomutase gene disruptions in Streptomyces nodosus: Impact on amphotericin biosynthesis and implications for glycosylation engineering

“…Disruption of both genes did not impair growth or production of amphotericin aglycones. A previous study reported inactivation of the amphDIII gene, which encodes a GDP-mannose 4, 6 dehydratase that catalyses the first step in the mycosamine-specific pathway (Byrne et al, 2003). The amphDIII mutant produced mostly 8-deoxyamphoteronolide A along with some mannosylamphoteronolide A.…”

“…Figure 6B). Previous work has shown that C-8 hydroxylation of the macrolactone ring occurs after mycosamine is added (Byrne et al, 2003). The free mannose content of the growth medium (12.19 µmoles/litre) would account for partial glycosylation of the total polyene (62.5 µmoles/litre).…”

“…Amphotericins A and B are potent antifungal agents whereas their aglycones have no activity (Byrne et al, 2003). Attempts were made to grow S. nodosus on defined liquid minimal media in which the mannose content could be controlled.…”

Engineered biosynthesis and characterisation of disaccharide-modified 8-deoxyamphoteronolides

“…nodosus amphL was described previously (Byrne et al, 2003). This is strain IMD2700 in culture collection WDCM227 maintained in University College Dublin.…”

“…This led us to investigate whether NypY might act efficiently on 8-deoxyamphotericins. An S. nodosus mutant is available in which the amphL gene is inactivated by insertion of a KC515 phage genome (Byrne et al, 2003). This strain produces 8-deoxyamphotericins A and B (Murphy et al, 2010) but contains a thiostrepton resistance gene within the embedded prophage DNA, and cannot be transformed with pIAGO-nypY which contains the same tsr selectable marker (De Poire et al, 2013).…”