Abstract
In yeasts, proteins of the Major Superfamily Transporter selectively bind and allow the uptake of sugars to permit growth on varied substrates. The genome of brewer’s yeast, Saccharomyces cerevisiae, encodes multiple hexose transporters (Hxt) to transport glucose and other MFS proteins for maltose, galactose, and other monomers. For sugar uptake, the dairy yeast, Kluyveromyces lactis, uses Rag1p for glucose, Hgt1 for glucose and galactose, and Lac12 for lactose. In the related industrial species Kluyveromyces marxianus, there are four genes encoding Lac12-like proteins but only one of them, Lac12, can transport lactose. In this study, which initiated with efforts to investigate possible functions encoded by the additional LAC12 genes in K. marxianus, a genome-wide survey of putative MFS sugar transporters was performed. Unexpectedly, it was found that the KHT and the HGT genes are present as tandem arrays of five to six copies, with the precise number varying between isolates. Heterologous expression of individual genes in S. cerevisiae and mutagenesis of single and multiple genes in K. marxianus was performed to establish possible substrates for these transporters. The focus was on the sugar galactose since it was already reported in K. lactis that this hexose was a substrate for both Lac12 and Hgt1. It emerged that three of the four copies of Lac12, four Hgt-like proteins and one Kht-like protein have some capacity to transport galactose when expressed in S. cerevisiae and inactivation of all eight genes was required to completely abolish galactose uptake in K. marxianus. Analysis of the amino acid sequence of all known yeast galactose transporters failed to identify common residues that explain the selectivity for galactose. Instead, the capacity to transport galactose has arisen three different times in K. marxianus via polymorphisms in proteins that are probably ancestral glucose transporters. Although, this is analogous to S. cerevisiae, in which Gal2 is related to glucose transporters, there are not conserved amino acid changes, either with Gal2, or among the K. marxianus galactose transporters. The data highlight how gene duplication and functional diversification has provided K. marxianus with versatile capacity to utilise sugars for growth.
Highlights
Kluyveromyces marxianus is a yeast traditionally found in fermented dairy products such as cheese and kefir (Gethins et al, 2016; Coloretti et al, 2017; Tittarelli et al, 2018)
Lactose transport by Lac12p appears to be a trait that evolved in a particular dairy lineage of K. marxianus (Ortiz-Merino et al, 2018) and it was hypothesised that the proteins encoded by the LAC12 genes might have other substrates
Because the K. lactis Lac12p protein is known to transport lactose and galactose (Riley et al, 1987), it was decided to test whether the additional K. marxianus Lac12 proteins were galactose transporters
Summary
Kluyveromyces marxianus is a yeast traditionally found in fermented dairy products such as cheese and kefir (Gethins et al, 2016; Coloretti et al, 2017; Tittarelli et al, 2018). Cocoa bean fermentation, an essential step in chocolate production, is generally carried out by a consortium of indigenous species that includes K. marxianus (De Vuyst and Weckx, 2016) In this environment the pectinase enzyme produced by K. marxianus degrades the pectin present in the cocoa pulp allowing the establishment of other yeast species required in the fermentation process (Schwan and Wheals, 2004). It is able to grow at high temperatures (up to 50◦C), exhibits the fastest growth rate among eukaryotes and naturally produces significant amounts of flavour molecules such as esters and fusel alcohols (Morrissey et al, 2015) This species is used for some commercial applications, for example, the production of bioethanol from cheese whey and production of the flavour molecule 2-phenylethanol (Fonseca et al, 2008; Morrissey et al, 2015; Varela et al, 2017a)
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