Accurate prediction of secondary metabolite gene clusters in filamentous fungi

Abstract

Biosynthetic pathways of secondary metabolites from fungi are currently subject to an intense effort to elucidate the genetic basis for these compounds due to their large potential within pharmaceutics and synthetic biochemistry. The preferred method is methodical gene deletions to identify supporting enzymes for key synthases one cluster at a time. In this study, we design and apply a DNA expression array for Aspergillus nidulans in combination with legacy data to form a comprehensive gene expression compendium. We apply a guilt-by-association-based analysis to predict the extent of the biosynthetic clusters for the 58 synthases active in our set of experimental conditions. A comparison with legacy data shows the method to be accurate in 13 of 16 known clusters and nearly accurate for the remaining 3 clusters. Furthermore, we apply a data clustering approach, which identifies cross-chemistry between physically separate gene clusters (superclusters), and validate this both with legacy data and experimentally by prediction and verification of a supercluster consisting of the synthase AN1242 and the prenyltransferase AN11080, as well as identification of the product compound nidulanin A. We have used A. nidulans for our method development and validation due to the wealth of available biochemical data, but the method can be applied to any fungus with a sequenced and assembled genome, thus supporting further secondary metabolite pathway elucidation in the fungal kingdom.

Fig. 1.

Venn diagram of SMs found on three different solid media. The number of different metabolites is sorted according to which media the metabolites have been identified on. The number of metabolites unable to be confidently identified are noted in parentheses. Details can be found in Dataset S1, and the chemical structures are illustrated in Fig. S1.

Fig. 2.

Identification of the sterigmatocystin biosynthetic cluster. (A) Gene expression profiles across 44 experiments for the 24 genes (marked in black in B) predicted to be in the sterigmatocystin biosynthetic cluster (liquid and solid cultures are marked for reference). The expression profile of AN7811(stcO) is marked in blue. (B) Illustration of the values of the gene CS for the 24 genes and the two immediate neighbors. Genes included in the predicted cluster are marked in black. AN7811(stcO) did not have a CS above the used cutoff of 2.13 denoting clustering but was added due to the similarity of the expression profile, as shown in blue. The predicted extent of the cluster corresponds with the cluster as originally described by Brown et al. (17), when correcting for the fact that the gene models have changed since then. Full data for all predicted clusters may be found in Dataset S2.

Fig. 3.

Cross-chromosomal clustering. Matrix diagram of the correlation between 67 predicted and known biosynthetic genes. Each square in the matrix shows the compounded squared Spearman correlation coefficient for comparison of the expression profile of the genes color-coded from 0 (white) to 1 (green). Genes are sorted horizontally according to their location on the chromosomes (marked in orange) and vertically according to their scores (Left, marked with a dendrogram). (Right) Genes located in the same clusters are highlighted with a gray box, which is connected with a gray bracket in one case. Genes with known cross-chemistry are marked with a black bracket. An example of cross-chemistry found in this study is marked with a red bracket. Seven putative superclusters are marked. Further details of the clusters may be found in Fig. S4.

Fig. 4.

Proposed absolute structure of nidulanin A. Details on the structural elucidation are available in SI Text.

Fig. P1.

Comparison of the gene cluster known to be required for biosynthesis of emericellamide to the predictions of the described method. (A) Gene expression plots of the five genes, AN2545–AN2549, known to be required for emericellamide biosynthesis. (B) Chromosomal map of the emericellamide gene cluster and surrounding genes. The clustering score (CS) evaluating coregulation is shown for the genes in the columns and in the numbers above the columns. Note how the expression pattern of AN2546, which does not contribute to emericellamide biosynthesis (3), deviates with a statistically insignificant clustering score (CS < 2.13). Genes surrounding the cluster exhibit dissimilar expression patterns (expression values not shown). NRPS, nonribosomal peptide synthetase; PKS, polyketide synthase.