Engineered Synthesis of 7-Oxo- and 15-Deoxy-15-Oxo-Amphotericins: Insights into Structure-Activity Relationships in Polyene Antibiotics

Abstract

Site-directed mutagenesis and gene replacement were used to inactivate two ketoreductase (KR) domains within the amphotericin polyketide synthase in Streptomyces nodosus. The KR12 domain was inactivated in the DeltaamphNM strain, which produces 16-descarboxyl-16-methyl-amphotericins. The resulting mutant produced low levels of the expected 15-deoxy-15-oxo analogs that retained antifungal activity. These compounds can be useful for further chemical modification. Inactivation of the KR16 domain in the wild-type strain led to production of 7-oxo-amphotericin A and 7-oxo-amphotericin B in good yield. 7-oxo-amphotericin B was isolated, purified, and characterized as the N-acetyl methyl ester derivative. 7-oxo-amphotericin B had good antifungal activity and was less hemolytic than amphotericin B. These results indicate that modification at the C-7 position can improve the therapeutic index of amphotericin B.

New Nystatin-Related Antifungal Polyene Macrolides with Altered Polyol Region Generated via Biosynthetic Engineering of Streptomyces noursei

“…Numerous attempts to generate less-toxic chemical analogues of AmB have been reported (2). Recently, these have been complemented by a series of genetically engineered amphotericin analogues, some of which showed considerably reduced in vitro toxicity (11,12,20,22). We have previously constructed an S. noursei mutant, GG5073SP, producing heptaenic nystatin analogue S44HP (4), which displayed considerably improved antifungal activity but also increased hemolytic activity compared to that of nystatin (7).…”

A labile point in mutant amphotericin polyketide synthases

“…There are no clear precedents where inactivation of a KR domain in one module prevents ketoreduction by the next. KR16 and KR12 mutants formed 3,5 diketoacyl and 3,5,7-triketoacyl intermediates, respectively, that are extended to form full length macrolactones (Power et al, 2008). The behaviour of the KR10 amphotericin PKSs is therefore surprising.…”

“…These products undergo no further enzymatic processing, and are released to accumulate as pyrones. This finding was unexpected because inactivation of KR12 or KR16 did not block subsequent steps in polyketide chain assembly (Power et al, 2008). The co-incidental loss of two AmphC modules from strain KR10-1 raised the possibility that early termination might result from further unplanned deletions downstream of ACP11.…”

The Combinatorial Biosynthesis of “Unnatural” Products with Polyketides

“…Power et al [37] inactivated the KR domain of amphotericin PKS, replaced the hydroxyl at C-7 of amphotericin B with a ketone group, and produced the 15-deoxy-15-oxo-amphotericin B (14 shown in Fig. 5) and 7-oxo-amphotericin B (15 shown in Fig.…”