Evolution and functional diversification of yeast sugar transporters

Abstract

While simple sugars such as monosaccharides and disaccharide are the typical carbon source for most yeasts, whether a species can grow on a particular sugar is generally a consequence of presence or absence of a suitable transporter to enable its uptake. The most common transporters that mediate sugar import in yeasts belong to the major facilitator superfamily (MFS). Some of these, for example the Saccharomyces cerevisiae Hxt proteins have been extensively studied, but detailed information on many others is sparce. In part, this is because there are many lineages of MFS transporters that are either absent from, or poorly represented in, the model S. cerevisiae, which actually has quite a restricted substrate range. It is important to address this knowledge gap to gain better understanding of the evolution of yeasts and to take advantage of sugar transporters to exploit or engineer yeasts for biotechnological applications. This article examines the full repertoire of MFS proteins in representative budding yeasts (Saccharomycotina). A comprehensive analysis of 139 putative sugar transporters retrieved from 10 complete genomes sheds new light on the diversity and evolution of this family. Using the phylogenetic lens, it is apparent that proteins have often been misassigned putative functions and this can now be corrected. It is also often seen that patterns of expansion of particular genes reflects the differential importance of transport of specific sugars (and related molecules) in different yeasts, and this knowledge also provides an improved resource for the selection or design of tailored transporters.

Keywords: Major Facilitator Superfamily; Saccharomyces cerevisiae; biotechnology; glucose transport; transport.

Figure 1. Topology of yeast sugar transporters belonging to the MFS

(A) The 12 transmembrane domains are represented as cylinders. The secondary structural prediction of the structure of a MFS transporter was made using the Protter program. (B) 3D-structure of a sugar transporter bound to the ligand (in pink) was obtained using the I-Tasser online platform and drawn using Pymol v2.3 [18].

Figure 2. Phylogenetic relationship of the yeasts included in this study

A phylogenetic tree to show the relationship between the yeast lineages from which the transporters described in detail in this study originate is presented. The star indicates the Whole Genome Duplication (WGD) event, which is now believed to be a hybridization between parents that were basal in the Zygosaccharomyces/Torulaspora (ZT) and the Kluyveromyces/Lachancea/Eremothecium (KLE) lineages. Figure adapted from Brown et al. 2010 [18] and Gabaldón et al. 2013 [23].

Figure 3. Phylogenetic tree of 139 putative sugar transporters belonging to different yeast species

S. cerevisiae (Sc, orange), C. glabrata (Cg, pink), S. pombe (Sp, fuchsia), T. delbrueckii (Td, ochre), K. lactis (Kl, teal), K. marxianus (Km, green), A. gossypii (Ag, brown), D. hansenii (Dh, black), S. stipitis (Ps, blue), Y. lipolytica (Yl, light blue). Transporters with putative similar function are put in coloured squares. S. cerevisiae strain EC1118 fructose-symporter Fsy1 is included in the tree (marked with *) as reference for the specific fructose transporter cluster. Proteomes of S. cerevisiae, C. glabrata, S. pombe, T. delbrueckii, K. lactis, K. marxianus, A. gossypii, D. hansenii, P. stipitis and Y. lipolytica were retrieved from Uniprot and putative sugar transporters were selected using the Gene Ontology classification inferred by electronic annotation using ‘sugar transporter’ and ‘MFS’ as key words. The resulting sequences were submitted to the TMHMM server for the prediction of transmembrane domains. All proteins containing between 10 and 12 transmembrane domains were considered potential sugar transporters, with the exception of maltose transporters which are predicted to comprise only 8 transmembrane spans. Syntenic arrangements were inspected using the Yeast Gene Browser (YGOB, www.ygob.ucd.ie) and used to assist in identifying orthologues [24]. Sequence alignment was performed using MUSCLE algorithm 3.8 [25]. Neighbour joining was used for the complete phylogenetic tree comprising 139 sugar transporters. The phylogenetic analysis was performed using MEGA6/MUSCLE [26] and the trees were viewed with FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Accession details for the proteins are available in Supplementary File S1.

Figure 4. Relationship of Hxt proteins among yeast species

The colour-code is the same as the one used in Figure 2. The phylogenetic analysis was performed using MEGA6/MUSCLE and is maximum-likelihood tree with 500 times bootstrapping [26].

Figure 5. Evolutionary reconstruction of the HXT gene family at the ancestral locus in the Saccharomycetaceae

In Saccharomycetaceae, HXT-like genes are present in a variable number at a conserved locus. This tree reconstructs their evolution considering gene duplications, losses and translocations, all of which are indicated. It is proposed that a duplication of a single ancestral gene occurred prior to the divergence of the KLE and ZT lineages giving rise to the ancestors of the KHT1 (blue) and KHT2 (green) families. Representative extant species using recognised gene names are shown. As discussed in the main text, in multiple species, there are other Hxt-encoding genes in other loci that arose by duplication of genes from the ancestral lineage shown here.

Figure 6. Phylogenetic relationship of Fsy1/Frt1 in the Saccharomycetaceae

A phylogeny of this fructose/H⁺ symporter was created using the Maximum-likelihood method. A more exhaustive phylogeny that includes representatives from other yeast and fungi can be found in Coelho et al. 2013 [40].

Figure 7. Synteny analysis of FRT1/ FSY1

Orthologous genes, whether of known or unknown function, are depicted in the same colour. Genes shown in white encode proteins of unknown function and are not orthologous. Panels show the location of FSY1/FRT1 in the specified yeast(s) and include the syntenic location in other yeasts for comparison. Further details of these loci, including in other yeasts, can be found on the Yeast Gene Order Browser (http://ygob.ucd.ie). With the exception of Kluyveromyces and Lachancea (B), all other loci are telomeric or sub-telomeric. (A) FSY1 in Zygosaccharomyces and Torulaspora. (B) FRT1 in K. lactis and L. kluyveri. The same syntenic arrangement is seen in K. marxianus but, in L. thermotolerans, there has been an intrachromosomal translocation immediately to the left of FRT1 (not shown). (C) FSY1 S. uvarum. The synteny is identical in S. eubayanus and S. pastorianus. (D) FSY1 in S. cerevisiae EC1118. Here, these genes are part of the ‘region C’ proposed to have been acquired horizontally and there is not precise synteny with other strain of S. cerevisiae, or other species shown here.

Figure 8. LAC and CEL gene clusters in K. marxianus, K. lactis, S. stipitis and D. hansenii

In pink and purple colours are reported the enzymes that hydrolyse glycosidic bonds in di- or polysaccharides: LAC4, β-galactosidase hydrolyses lactose in β-D-galactose and β-D-glucose; CEL2, cellobiase cleaves cellobiose in two glucose molecules; BMS, β-galactosidase/mannosidase; BGL, β-galactosidase; EGC, endo- β-1,4-glucanase), in light blue, green and blue are, respectively, indicated the lactose transporters, cellobiose transporters and hexose transporters.