Application of genome-wide strategies for the mining of secondary metabolite biosynthesis pathways in filamentous fungi

2017-04-20Genome projects of filamentous fungi have generated an unprecedented amount of information, and in a moment in time often referred to as the “post-genomic era”, it is up to us to find ways to provide meaning to this immense quantity of genome sequence data. Fungi have long been known to be prolific producers of bioactive secondary metabolites, but bioinformatic analysis has showed that these organisms harbor the potential to produce far more secondary metabolites than are currently known. The work herein describes genome-wide strategies to approach two main challenges we now face: 1) finding ways to access the hidden natural products and 2) developing and/ or using genetic systems in fungal species to link secondary metabolites to their biosynthesis genes and elucidate their biosynthesis pathways. We have demonstrated the power of combining bioinformatics, molecular gene targeting, and natural product chemistry in secondary metabolite biosynthesis research. ❧ We initally used A. nidulans as the target species and took advantage of its well-established gene targeting system. First, genome-based deletion analysis in A. nidulans led us to find genes involved in the biosynthesis of xanthones located in at least three distinct loci in the genome. This was particularly interesting because it contradicted the general notion that fungal secondary metabolites are clustered. This highlighted the utility of genomics combined with efficient gene targeting to identify these dispersed genes. Next, we hypothesized that altering the expression of kinases would have an effect on secondary metabolite production and may activate silent gene clusters. We used a genome-wide kinase knockout library and screened its secondary metabolite profile and found that the mpkA deletion strain produced aspernidine A, a compound not previously isolated from A. nidulans. We performed additional gene deletions and determined the border of the biosynthesis gene cluster and proposed the biosynthesis pathway for aspernidine A. ❧ We then took advantage of the recent advances in genome sequencing of Penicillium species and chose Penicillium canescens as the target species. This species is a prolific producer of secondary metabolites but no previous work has been reported to link their metabolites to biosynthesis genes. This is due in large part to the lack of an efficient gene targeting system in this organism. We therefore developed a genetic system in P. canescens that would allow us to perform targeted gene manipulations rapidly and efficiently. We then applied this system and generated a genome-wide deletion library of polyketide synthase (PKS) genes and nonribosomal peptide synthetase (NRPS) genes. Using this library, we successfully linked four secondary metabolites, griseofulvin, xanthoepocin, 15-deoxyoxalicine B, and amauromine, to their core secondary metabolite biosynthesis genes. Interestingly, 15-deoxyoxalicine B is a structurally unique diterpenic meroterpenoid, a class of fungal metabolites whose biosynthesis mechanism remains largely unknown. We performed additional gene deletions and determined the gene cluster involved in its biosynthesis. We were able to isolate and characterize intermediates from the gene deletants, which allowed us to propose a biosynthesis pathway. We believe the gene targeting system and genome-wide PKS and NRPS deletion libraries will be important resources towards a systematic understanding of secondary metabolite biosynthesis in P. canescens