FunOrder: A robust and semi-automated method for the identification of essential biosynthetic genes through computational molecular co-evolution.

Secondary metabolites (SMs) are a vast group of compounds with different structures and properties that have been utilized as drugs, food additives, dyes, and as monomers for novel plastics. In many cases, the biosynthesis of SMs is catalysed by enzymes whose corresponding genes are co-localized in the genome in biosynthetic gene clusters (BGCs). Notably, BGCs may contain so-called gap genes, that are not involved in the biosynthesis of the SM. Current genome mining tools can identify BGCs, but they have problems with distinguishing essential genes from gap genes. This can and must be done by expensive, laborious, and time-consuming comparative genomic approaches or transcriptome analyses. In this study, we developed a method that allows semi-automated identification of essential genes in a BGC based on co-evolution analysis. To this end, the protein sequences of a BGC are blasted against a suitable proteome database. For each protein, a phylogenetic tree is created. The trees are compared by treeKO to detect co-evolution. The results of this comparison are visualized in different output formats, which are compared visually. Our results suggest that co-evolution is commonly occurring within BGCs, albeit not all, and that especially those genes that encode for enzymes of the biosynthetic pathway are co-evolutionary linked and can be identified with FunOrder. In light of the growing number of genomic data available, this will contribute to the studies of BGCs in native hosts and facilitate heterologous expression in other organisms with the aim of the discovery of novel SMs. Author summary: The discovery and description of novel fungal secondary metabolites promises novel antibiotics, pharmaceuticals, and other useful compounds. A way to identify novel secondary metabolites is to express the corresponding genes in a suitable expression host. Consequently, a detailed knowledge or an accurate prediction of these genes is necessary. In fungi, the genes are co-localized in so-called biosynthetic gene clusters. Notably, the clusters may also contain genes that are not necessary for the biosynthesis of the secondary metabolites, so-called gap genes. We developed a method to detect co-evolved genes within the clusters and demonstrated that essential genes are co-evolving and can thus be differentiated from the gap genes. This adds an additional layer of information, which can support researchers with their decisions on which genes to study and express for the discovery of novel secondary metabolites. [ABSTRACT FROM AUTHOR]