Physical linkage of metabolic genes in fungi is an adaptation against the accumulation of toxic intermediate compounds

Abstract

Genomic analyses have proliferated without being tied to tangible phenotypes. For example, although coordination of both gene expression and genetic linkage have been offered as genetic mechanisms for the frequently observed clustering of genes participating in fungal metabolic pathways, elucidation of the phenotype(s) favored by selection, resulting in cluster formation and maintenance, has not been forthcoming. We noted that the cause of certain well-studied human metabolic disorders is the accumulation of toxic intermediate compounds (ICs), which occurs when the product of an enzyme is not used as a substrate by a downstream neighbor in the metabolic network. This raises the hypothesis that the phenotype favored by selection to drive gene clustering is the mitigation of IC toxicity. To test this, we examined 100 diverse fungal genomes for the simplest type of cluster, gene pairs that are both metabolic neighbors and chromosomal neighbors immediately adjacent to each other, which we refer to as "double neighbor gene pairs" (DNGPs). Examination of the toxicity of their corresponding ICs shows that, compared with chromosomally nonadjacent metabolic neighbors, DNGPs are enriched for ICs that have acutely toxic LD50 doses or reactive functional groups. Furthermore, DNGPs are significantly more likely to be divergently oriented on the chromosome; remarkably, ∼40% of these DNGPs have ICs known to be toxic. We submit that the structure of synteny in metabolic pathways of fungi is a signature of selection for protection against the accumulation of toxic metabolic intermediates.

Keywords: gene cluster; gene orientation; inborn error of metabolism; secondary metabolism; specialized metabolism.

Fig. 1.

Avoidance of toxic IC accumulation is a phenotype favored by selection to drive gene clustering in fungi. Several human genes that, when disrupted, lead to inborn errors of metabolism have fungal orthologs that are clustered. For example, galactosemia arises when any one of three enzymatic steps in the galactose pathway is disrupted (A). Type 1 galactosemia, the most severe form, leads to accumulation of galactose-1-phosphate, a toxic inhibitor of glycolysis. In many fungi, this pathway is clustered. Similarly, tyrosinemia arises when any one of three different genes in the tyrosine catabolic pathway is disrupted, with symptoms that vary in severity according to the properties of the accumulating IC (B). Tyrosinemia type I, the most damaging form that frequently leads to hepatic cancer, stems from loss of the final step in the pathway, leading to accumulation of fumarylacetoacetate, a mutagenizing and alkylating IC. In many fungi, the genes participating in this pathway are also clustered. Our hypothesis (C) is that natural selection against the accumulation of toxic ICs results in the clustering of the genes that handle them. We tested this hypothesis on the simplest type of gene cluster, gene pairs that are neighbors in the metabolic network, i.e., share an IC, and that are also immediately adjacent neighbors on the chromosome, which we refer to as DNGPs. Topologically, the two genes composing a DNGP can have four possible orientations relative to each other on the chromosome (D): one divergent orientation, one convergent orientation, and two different colinear orientations depending on gene order.

Fig. 2.

DNGPs handle more toxic ICs than background. Two independent measures confirm the toxicity of ICs handled by DNGPs. (A) The set of divergently oriented DNGPs is more likely to have LD₅₀ doses of higher toxicity than background across multiple IC LD₅₀ dose cutoffs. The x axis shows the LD₅₀ dose (mg/kg) and the y axis the size of divergently, colinearly, and convergently oriented DNGPs relative to the expected background. (B) The divergent and colinear sets of DNGPs are enriched relative to background for ICs with reactive functional groups, but the set of convergently oriented DNGPs is depleted for reactive ICs. Furthermore, divergent DNGPs are enriched for ICs with highly reactive functional groups; in contrast, colinearly oriented DNGPs are more enriched when all reactive functional groups are included. Enrichment is calculated as the fraction of ICs with reactive functional groups in each set of DNGPs divided by the fraction of all metabolic gene pairs with ICs containing reactive functional groups. (C) Almost 40% of the ICs handled by divergent and colinear DNGPs are known to be toxic, even with limited annotation of IC toxicity and excluding likely cases of cryptic toxicity. Thus, reduced accumulation of toxic ICs is likely to be an important phenotypic target of selection leading to DNGP formation. (Difference from background: **P = 1.5 × 10⁻⁶; *P = 8.2 × 10⁻³).

Fig. 3.

The IC betaine aldehyde, produced by a DNGP, contains a highly reactive functional group. In many organisms, osmotic shock is mitigated by the formation of the osmoprotectant betaine. (A) This pathway, which is encoded by a DNGP, produces a reactive toxic IC, betaine aldehyde, during the conversion of choline to betaine. Betaine can reach high concentrations during osmotic stress, an indication of substantial flux that must be regulated to match environmental conditions (48). Betaine aldehyde toxicity is indicated by its use as an alternative to antibiotics for selection of transformants in plant species that lack the pathway (22). (B) The DNGP is divergently oriented in 23 different fungal species; 3 of these species have two DNGP copies. The Phytophthora oomycetes, nonfungal water molds that occupy an ecological niche similar to fungi, also have this DNGP, albeit in a colinear orientation, which suggests that their genomes have evolved in parallel to adapt to a common selection pressure imposed by intermittent high flux through this pathway.