Monasone Naphthoquinone Biosynthesis and Resistance in Monascus Fungi

Abstract

Despite the important biological activities of natural product naphthoquinones, the biosynthetic pathways of and resistance mechanisms against such compounds remain poorly understood in fungi. Here, we report that the genes responsible for the biosynthesis of Monascus naphthoquinones (monasones) reside within the gene cluster for Monascus azaphilone pigments (MonAzPs). We elucidate the biosynthetic pathway of monasones by a combination of comparative genome analysis, gene knockouts, heterologous coexpression, and in vivo and in vitro enzymatic reactions to show that this pathway branches from the first polyketide intermediate of MonAzPs. Furthermore, we propose that the monasone subset of biosynthetic genes also encodes a two-tiered resistance strategy in which an inducible monasone-specific exporter expels monasones from the mycelia, while residual intracellular monasones may be rendered nontoxic through a multistep reduction cascade.IMPORTANCE The genes for Monascus naphthoquinone (monasone) biosynthesis are embedded in and form a composite supercluster with the Monascus azaphilone pigment biosynthetic gene cluster. Early biosynthetic intermediates are shared by the two pathways. Some enzymes encoded by the supercluster play double duty in contributing to both pathways, while others are specific for one or the other pathway. The monasone subcluster is independently regulated and inducible by elicitation with competing microorganisms. This study illustrates genomic and biosynthetic parsimony in fungi and proposes a potential path for the evolution of the mosaic-like azaphilone-naphthoquinone supercluster. The monasone subcluster also encodes a two-tiered self-resistance mechanism that models resistance determinants that may transfer to target microorganisms or emerge in cancer cells in case of naphthoquinone-type cytotoxic agents.

Keywords: Monascus spp.; naphthoquinone; nested biosynthetic pathway; resistance mechanism; supercluster.

FIG 1

A branching pathway yields MonAzPs and monasone congeners in M. ruber M7. The reactive polyketide intermediate 1 gives rise to MonAzPs on one branch (represented by rubropunctatin 7a and monascorubrin 7b) and to naphthoquinone congeners on the other branch (represented by trihydroxynaphthalene 2, monasone A [compound 3], monasone B [compound 4], tetralindione 8, and trihydroxytetralone MA-1 [compound 9]). The structures of the boxed compounds were elucidated by LC-MS/MS and NMR analysis (data for compounds 2, 3, 4, 8 and 9 are in Table S8 and Fig. S10 [19]).

FIG 2

A conserved MrPigB regulon in MABGC-like gene clusters. (a) qRT-PCR analysis of the MABGC genes of the M. ruber M7 wild-type (WT), ΔmrpigB, and ΔmrpigC knockout strains, measured from monoculture or during cocultivation with Penicillium expansum ATCC 7861. Gene expression levels from monoculture are taken as the basis of comparison, with the means and standard deviations calculated from measurements from three biological replicates for each strain/cultivation condition shown. (b) MABGC-like gene clusters in filamentous fungal genomes. Arrows with identical colors indicate orthologous genes; white arrows show nonorthologous genes. (c) Proteins encoded by MABGC-related SM gene clusters of various fungi. Proteins highlighted in red are encoded by all MABGC-like gene clusters. Genes encoding orthologues of the MFS transporter MrPigP (highlighted in pink) are present in the majority of the MABGC-like gene clusters.

FIG 3

Biosynthesis of monasones during in vivo and vitro enzymatic reactions with MrPigA and MrPigN. (a) Metabolite profiles (reversed-phase HPLC traces recorded at 280 nm with a photodiode array detector) of fermentation extracts of A. oryzae M-2-3 expressing the indicated MABGC genes. (b) In vitro enzymatic assays with the indicated purified MABGC enzymes (reversed-phase HPLC traces recorded at 280 nm with a photodiode array detector). (c) Quantification of trihydroxynaphthalene 2 in enzymatic reactions with the indicated enzymes after 10 min or 30 min. Yields of compound 2 are shown in micrograms per milliliter as the means ± standard deviations (SDs) from three independent experiments of three replicates each, n = 9. Statistical analysis with Student's t test revealed that there was a significant difference between group MrPigA and group MrPigA+MrPigN at P < 0.05.

FIG 4

MrPigH-mediated reductive transformation of monasones. (a) Proposed metabolic grid for the enzymatic reduction of monasone A (compound 3) by MrPigH and MrPigC or a similar ketoreductase under aerobic or anaerobic conditions. The structures of the boxed compounds were elucidated by LC-HRMS/MS and NMR analysis (Table S8 and Fig. S10 [19]). (b) Time course analysis of the reduction of monasone A (compound 3) into compounds 10 and 11 by recombinant MrPigH under aerobic conditions. (c) Time course analysis of the reduction of monasone A (compound 3) into compounds 14 and 17 by MrPigH under anaerobic conditions. (d) Reduction of monasone B (compound 4) by recombinant MrPigH into compound 12 under aerobic conditions and to compound 16 under anaerobic conditions. Reconstituted enzymatic reactions were performed in HEPES buffer (pH 7.0) containing 1.5 mM substrate and 1.5 mM NADPH at 30°C, and metabolites were detected by reversed-phase HPLC at 280 nm with a photodiode array detector.

FIG 5

MrPigP is an inducible naphthoquinone transporter. (a) Growth of wild-type (WT) M. ruber M7, the ΔmrpigP knockout mutant, and the mrpigC-complemented knockout strain (CΔmrpigP) on PDA plates containing 32 μg/ml or 64 μg/ml monasone A (compound 3) at 30°C for 5 days. (b) MICs of the WT, ΔmrpigP, and CΔmrpigP strains evaluated after cultivation in PDB at 30°C for 5 days. (c) Flux assay measuring the intracellular concentration of monasone A (compound 3) in the WT, ΔmrpigP, and CΔmrpigP strains after immersion in 32 μg/ml monasone A for 6 h. Statistical analysis using Student's t test revealed that there was a significant difference (P < 0.05) between group ΔmrpigP and either group WT or group CΔmrpigP. Groups WT and CΔmrpigP were not significantly different. (d) Relative transcription levels of the mrpigP gene after growth of the wild-type strain on different monasone A (compound 3) concentrations. The β-actin gene was used as an internal standard to normalize expression levels. Extracellular (e) and intracellular (f) MonAzP concentrations in the WT and ΔmrpigP strains. The strains were cultivated in PDB medium at 30°C for 10 days (mid-production phase). MonAzPs measured: Monc, monascin; Ank, ankaflavin; Rubt, rubropunctatin; Monb, monascorubrin; Rubm, rubropunctamine; Monm, monascorubramine. Statistical analysis using Student's t test revealed no significant differences between groups WT and ΔmrpigP for any of these compounds. Data from all quantitative experiments are shown as the means ± SDs from three independent experiments of three replicates each, n = 9.

FIG 6

Model for monasone biosynthesis, export, and reductive detoxification in M. ruber M7. Thick black arrows show steps in monasone biosynthesis and recycling; thin black arrows indicate steps in MonAzP biosynthesis; blue arrows represent monasone export; dotted and solid red arrows show aerobic and anaerobic monasones detoxification steps, respectively; black block arrows indicate genes for monasone and/or MonAzP biosynthesis and resistance; gray block arrows show genes involved only in MonAzP biosynthesis.