A New Pathway for Mannitol Metabolism in Yeasts Suggests a Link to the Evolution of Alcoholic Fermentation

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.

The Wickerhamiella/Starmerella clade—A treasure trove for the study of the evolution of yeast metabolism

“…Because fructophilic yeasts of the W/S clade lost alcoholic fermentation early in their evolution, it seems plausible that mannitol production from fructose may have helped mitigate redox imbalances resulting from this loss. In accordance to this, most W/S‐clade species tested so far produce considerable amounts of mannitol (Gonçalves et al, ). However, unlike fructophilic lactic acid bacteria, fructophilic yeasts also make mannitol from glucose because they possess a pathway that mediates the conversion of glucose to fructose, with fructose‐6 phosphate as an intermediate (Gonçalves et al, ).…”

“…In accordance to this, most W/S‐clade species tested so far produce considerable amounts of mannitol (Gonçalves et al, ). However, unlike fructophilic lactic acid bacteria, fructophilic yeasts also make mannitol from glucose because they possess a pathway that mediates the conversion of glucose to fructose, with fructose‐6 phosphate as an intermediate (Gonçalves et al, ). This pathway enables the operation of the mannitol dehydrogenase reaction even when glucose is the only carbon source available.…”

Nutritional Compositions of Optimally Processed Umqombothi (a South African Indigenous Beer)

“…A build-up of mannitol was observed in the OPB (Figure 1). At high temperatures and high concentrations of fructose or sucrose, mannitol may be produced by yeasts, bacteria, or fungi [64,65]. Mannitol is an antioxidant and can be used as a nonmetabolizable sweetener for diabetics [66].…”

Genetic, Physiological, and Industrial Aspects of the Fructophilic Non-Saccharomyces Yeast Species, Starmerella bacillaris

“…This low ethanol yield, in addition to its low acetic acid production, of S. bacillaris compared to S. cerevisiae, reveals low activity of the acetaldehyde pathway in S. bacillaris, leading to a redistribution in the fluxes of the central carbon metabolism network [58]. Differently from other members of the W/S clade, which convert fructose directly into mannitol [31], S. bacillaris overproduces glycerol to maintain the NADH/NAD+ redox balance in the cells [31,58]. Thus, high glycerol levels are frequently reported for fermentations involving this yeast species [17,[58][59][60][61].…”

“…The specific features present in Starmerella versus Wickerhamiella subclades probably result from the remodeling of important fluxes in central carbon metabolism as well as the reinstatement of other metabolic pathways [28]. Thus, the genera Starmerella harbors an unusually large number of genes of alien origin, which were shown to reconstruct the fermentative pathway in S. bacillaris [30], as well as other metabolic pathways, as shown in Starmerella bombicola [31,32]. For example, most of the genes in the thiamine salvage pathway in distinct subclades within the W/S lineage were originally acquired from bacteria by either horizontal gene transfer (HGT) or horizontal operon transfer (HOT) events [32].…”