Next-generation sequencing approach for connecting secondary metabolites to biosynthetic gene clusters in fungi

Abstract

Genomics has revolutionized the research on fungal secondary metabolite (SM) biosynthesis. To elucidate the molecular and enzymatic mechanisms underlying the biosynthesis of a specific SM compound, the important first step is often to find the genes that responsible for its synthesis. The accessibility to fungal genome sequences allows the bypass of the cumbersome traditional library construction and screening approach. The advance in next-generation sequencing (NGS) technologies have further improved the speed and reduced the cost of microbial genome sequencing in the past few years, which has accelerated the research in this field. Here, we will present an example work flow for identifying the gene cluster encoding the biosynthesis of SMs of interest using an NGS approach. We will also review the different strategies that can be employed to pinpoint the targeted gene clusters rapidly by giving several examples stemming from our work.

Keywords: filamentous fungi; gene clusters; genome mining; next generation sequencing; secondary metabolites.

FIGURE 1

Next-generation sequencing (NGS) workflow for targeted secondary metabolite (SM) gene cluster discovery.

FIGURE 2

Comparative genomics and knowledge-based prediction strategies for targeted SM gene cluster discovery. New biosynthetic knowledge and insights into gene-to-molecular structure relationship would be useful for target SM gene cluster predictions and knowledge-based genome mining for discovery of novel SM compounds.

FIGURE 3

Comparative genomics approach for discovery of SM biosynthetic gene cluster. Comparison of the PKS genes in Penicillium aethiopicum and P. chrysogenum was utilized in searching for the griseofulvin and viridicatumtoxin gene cluster (A). In order to narrow down possible candidates for the tryptoquialanine gene cluster in P. aethiopicum, NRPS genes that are non-orthologous to P. chrysogenum NRPS genes but are orthologous to the NRPS genes of the tryptoquivaline producer A. clavatus were found (B). The echinocandin NRPS gene was found in Emericella rugulosa was found by searching for a hexamodule NRPS gene in E. rugulosa that is non-orthologous to A. nidulans NRPS genes (C).

FIGURE 4

Griseofulvin biosynthetic gene clusterin P. aethiopicum. Shown are the roles of the genes in the biogenesis of the compound. The non-reducing polyketide synthase (NR-PKS) gene gsfA is found via comparison of PKS genes in P. aethiopicum and P. chrysogenum. Two structural features, O-methylation and chlorination (in red text), were used as criteria to find the candidate gene clusters.

FIGURE 5

Viridicatumtoxin biosynthetic gene cluster in P. aethiopicum. As with the case for the griseofulvin, the NR-PKS gene vrtA is non-orthologous to PKS genes in P. chrysogenum. The presence of the prenyltransferase gene vrtC and the O-methyltransferase gene vrtF (in red text) led to the identification of the gene cluster.

FIGURE 6

Tryptoquialanine biosynthetic gene cluster in P. aethiopicum. The presence of acyltransferase gene tqaD and the anthranilic acid-activating A domain of TqaA (in red text) were clues to identification of the cluster.

FIGURE 7

Echinocandin B biosynthetic gene cluster in E. rugulosa. The gene cluster was found in two loci (ecd and hty). The ecd cluster contained a six module NRPS gene ecdA and an acyl-CoA ligase gene ecdI (in red text), indicating that the gene cluster is involved in the biosynthesis of a lipo-hexapeptide SM. The separate homotyrosine biosynthesis hty gene cluster at a different locus was located by searching for an isopropylmalate synthase (IPMS) homolog (in red text).