Abstract
The yeasts belonging to the Wickerhamiella and Starmerella genera (W/S clade) share a distinctive evolutionary history marked by loss and subsequent reinstatement of alcoholic fermentation mediated by horizontal gene transfer events. Species in this clade also share unusual features of metabolism, namely the preference for fructose over glucose as carbon source, a rare trait known as fructophily. Here we show that fructose may be the preferred sugar in W/S-clade species because, unlike glucose, it can be converted directly to mannitol in a reaction with impact on redox balance. According to our results, mannitol is excreted to the growth medium in appreciable amounts along with other fermentation products such as glycerol and ethanol but unlike the latter metabolites mannitol production increases with temperature. We used comparative genomics to find genes involved in mannitol metabolism and established the mannitol biosynthesis pathway in W/S-clade species Starmerella bombicola using molecular genetics tools. Surprisingly, mannitol production seems to be so important that St. bombicola (and other W/S-clade species) deploys a novel pathway to mediate the conversion of glucose to fructose, thereby allowing cells to produce mannitol even when glucose is the sole carbon source. Using targeted mutations and 13C-labeled glucose followed by NMR analysis of end-products, we showed that the novel mannitol biosynthesis pathway involves fructose-6-phosphate as an intermediate, implying a key role for a yet unknown fructose-6-P phosphatase. We hypothesize that mannitol production contributed to mitigate the negative effects on redox balance of the ancient loss of alcoholic fermentation in the W/S clade. Presently, mannitol also seems to play a role in stress protection.
Highlights
Fungi are excellent models in which to explore the evolution of metabolism, as shown by multiple studies focused on secondary metabolite production by filamentous fungi (Spatafora and Bushley, 2015; Wisecaver and Rokas, 2015; Theobald et al, 2018; Keller, 2019)
To find out whether mannitol production is a widespread trait in the W/S clade, we quantified mannitol in the culture supernatants of representatives of five W/Sclade species, namely C. magnoliae, St. bacillaris, St. bombicola, W. domercqiae and W. versatilis, cultivated in rich medium containing either 10% glucose or 10% fructose as carbon and energy source
We found that all species but one produced variable amounts of mannitol in addition to ethanol and glycerol, and that more mannitol was produced from fructose than from glucose (Figure 1A)
Summary
Fungi are excellent models in which to explore the evolution of metabolism, as shown by multiple studies focused on secondary metabolite production by filamentous fungi (Spatafora and Bushley, 2015; Wisecaver and Rokas, 2015; Theobald et al, 2018; Keller, 2019). Comparative genomics of a large and ever-increasing number of yeast genomes is beginning to shed light on the evolution of metabolic traits in this sub-phylum, far revealing a general trend of loss of metabolic capabilities that implies a common ancestor metabolically more complex than most extant species (Shen et al, 2018) These losses most often resulted in narrowing spectra of nutrient assimilation, but we reported recently that even a central metabolic feature like alcoholic fermentation, which usually plays a crucial role in the metabolism of yeasts, either as the main pathway for energy production or as a means to circumvent oxygen shortage, was lost in a lineage within the Saccharomycotina currently comprising approximately 100 species belonging to the genera Wickerhamiella and Starmerella (referred to as the W/S clade) (Gonçalves et al, 2018). This remains to be empirically demonstrated, the unusually high number of horizontally acquired genes in the W/S clade suggests that other aspects of metabolism were impacted during evolution of the lineage (Gonçalves et al, 2018; Shen et al, 2018; Kominek et al, 2019)
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