Investigating the biosynthesis of Sch-642305 in the fungus Phomopsis sp. CMU-LMA

Abstract

Sch-642305 is an unusual bicyclic 10-membered macrolide produced by the filamentous fungus Phomopsis sp. CMU-LMA for which no biosynthetic evidence exists. Here, we generate a draft genome sequence of the producing organism and discover the biosynthetic gene cluster responsible for formation of Sch-642305. Targeted gene disruptions together with reconstitution of the pathway in the heterologous host Aspergillus oryzae dissect key chemical steps and shed light on a series of oxidoreductions occuring in the pathway.

Scheme 1. A) Incorporation of labelled acetates and ¹⁸O₂ into SCH 1 and brefeldin A 5; (B) sch biosynthetic gene cluster and comparison to the bref BGC and PVSCH BGC; (C) hypothetical biosynthetic route to SCH 1 proposed by Snider and Zhou on the basis of the fungal metabolite mutolide 3 (a) and on the basis of benquoine 4 (b). Incorporation of labelled sodium [1-¹³C]-acetate and ¹⁸O₂ is displayed for 3.

Fig. 1. Secondary metabolites concomitantly isolated with SCH 1. Compounds in blue were previously isolated from Phomopsis sp. CMU-LMA but not observed in this study.

Fig. 2. Targeted gene disruption in Phomopsis sp. CMU-LMA. ELSD chromatograms of KO mutants for: (A) schPKS; (B) schR7; (C) schR3; (D) schR2; (E) schR5. ** = unrelated to SCH 1 biosynthesis. X-axis in minutes throughout.

Scheme 2. Proposed biosynthesis of SCH 1 and route to shunt metabolites (green). Compounds in black were isolated in this study, compounds in blue were previously isolated from Phomopsis sp. CMU-LMA. Compounds in grey were not observed but suggested as plausible intermediates on the pathway to 1.

Fig. 3. (A) Biotransformation of SCH 1 by A. oryzae NSAR1. ES⁺ traces of fed 1 to A. oryzae, a standard of 1 and WT control; (B) heterologous expression of the full sch BGC. Diode array detector (DAD) traces of pathway expression and WT control; (C) heterologous expression of the full sch BGC and comparison to fed 1 to A. oryzae. ES⁺ traces of fed 1 (top) and full sch BGC (bottom).

Fig. 4. DAD chromatograms of fungal extracts from the heterologous expression of early biosynthetic genes in A. oryzae NSAR1. Top, co-expression of schPKS, schR1, schR2, schR3, schR7; middle, co-expression of schPKS, schR1, schR2; bottom, NSAR1 WT.