Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside maltotriose, a major carbohydrate in brewer’s wort. In Saccharomyces yeasts, utilization of maltotriose is encoded by the subtelomeric MAL gene family, and requires transporters for maltotriose uptake. While S. eubayanus strain CBS 12357T harbors four SeMALT genes which enable uptake of the α-di-glucoside maltose, it lacks maltotriose transporter genes. In S. cerevisiae, sequence identity indicates that maltotriose and maltose transporters likely evolved from a shared ancestral gene. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357T, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation in 7 L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalT413 was comparable to the original SeMalT transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has high sequences identity to alternatingly S. cerevisiae MALx1, S. paradoxus MALx1 and S. eubayanus SeMALT3. Evolution of the maltotriose transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.
Saccharomyces eubayanus was discovered in Patagonia and identified as the non-S. cerevisiae parental species of hybrid S. pastorianus lager-type beer brewing yeasts [1,2]. While S. cerevisiae is strongly associated with biotechnological processes, including dough leavening, beer brewing and wine fermentation [3], S. eubayanus has only been isolated from the wild [4–6]. Beer brewing is performed with wort, a complex substrate containing a fermentable sugar mixture of 15% of the monosaccharide glucose, 60% of the α-di-glucoside maltose and 25% of the α-tri-glucoside maltotriose [7]. While many S. cerevisiae and S. pastorianus strains utilize all three sugars, S. eubayanus isolates do not utilize maltotriose [8–10]. In Saccharomyces, the ability to utilize maltotriose requires its uptake into the cell and subsequent hydrolysis into glucose [11,12]. Maltose and maltotriose utilization are encoded by genes clustered in the MAL loci, which can be present on up to five different chromosomes [13]. MAL loci typically harbor genes from up to three gene families (Fig 1): a MALT polysaccharide proton-symporter gene, a MALS α-glucosidase gene which hydrolyses α-oligo-glucosides into glucose, and a MALR regulator gene that induces the transcription of MALT and MALS genes in the presence of maltose [14]. While MALS genes enable hydrolysis of both maltose and maltotriose, the MALT gene family comprises transporters with diverse substrate specificities [11,15]. In S. cerevisiae, most MAL loci harbor an ScMalx1 transporter (Fig 1), which transports maltose and other disaccharides, such as turanose and sucrose [12,16], but cannot import maltotriose [11]. In contrast, the MAL1 locus located on chromosome VII of S. cerevisiae contains ScAGT1, a transporter gene with only 57% nucleotide identity with ScMALx1 transporter genes. ScAGT1 encodes a broad-substrate-specificity sugar-proton symporter that enables maltotriose uptake [11,17,18]. The S. eubayanus type strain CBS 12357T is able to utilize maltose, but cannot utilize maltotriose, suggesting that it expresses a functional maltase and a functional maltose transporter, but no maltotriose transporter [9]. Indeed, the four MAL loci in CBS 12357T harbor a total of two MALS genes and four MALT genes with high homology to ScMALx1: SeMALT1, SeMALT2, SeMALT3 and SeMALT4 (Fig 1) [20]. Deletion of these genes in S. eubayanus type strain CBS 12357T indicated that its growth on maltose relies on expression of SeMALT2 and SeMALT4 [9]. SeMALT1 and SeMALT3 were found to be poorly expressed in the presence of maltose in this strain, supposedly due to incompleteness of the MAL loci which harbor them. However, no homolog of ScAGT1 was found in the genome of CBS 12357T, and neither CBS 12357T nor its derivatives overexpressing SeMALT genes were able to utilize maltotriose [9]. The MALT transporter genes in Saccharomyces yeasts are localized to the subtelomeric regions (Fig 1) [9,11,12,20–22], which are gene-poor and repeat-rich sequences adjacent to the telomeres [23–25]. The presence of repeated sequences makes subtelomeric regions genetically unstable by promoting recombination [26,27]. As a result, subtelomeric gene families are hotspots of genetic diversity [28–30]. In S. cerevisiae, subtelomeric gene families contain more genes than non-subtelomeric gene families, reflecting a higher incidence of gene duplications [28]. As previously shown in Candida albicans submitted to long term laboratory evolution, the gene repertoire of the subtelomeric TLO family can be extensively altered due to ectopic recombinations between subtelomeric regions of different chromosomes, resulting in copy number expansion, in gene disappearance and in formation of new chimeric genes [31]. Despite their common origin, genes within one family can have different functions, due to the accumulation of mutations [32,33]. In silico analysis of the sequences and functions of genes from the MALT, MALS and MALR gene families indicated functional diversification through gene duplication and mutation [28]. Gene duplication is a critical step for the evolution of new gene functions [34,35]. Indeed, the presence of multiple gene copies can facilitate the emergence of advantageous mutations mainly by one of three mechanisms: (i) neofunctionalization, corresponding to the emergence of a novel function which was previously absent in the gene family [36], (ii) subfunctionalization, corresponding to the specialization of gene copies for part of the function of the parental gene [37] and (iii) altered expression due to gene dosage effects resulting from the increased copy number [38]. While the different functions of MALS genes were assigned to subfunctionalization of the ancestral MALS gene [15], it is not known how the different functions of the maltotriose transporter gene ScAGT1 and of other maltose transporter genes of the MALT family evolved from a common ancestor gene [28]. In general, the emergence of a large array of gene functions was attributed to subfunctionalization and neofunctionalization [15,28,31,39–42]. However, current evidence for neofunctionalization within subtelomeric gene families is based on a posteriori analysis and rationalization of existing sequence diversity. While in some cases the genetic process leading to neofunctionalization could be reconstructed at the molecular level [42–44], the emergence of a completely new function within a subtelomeric gene family was never observed within the timespan of an experiment to the best of our knowledge. However, the genetic diversity within Saccharomyces MALT transporters suggests that evolution of SeMalT transporters could lead to the emergence of a maltotriose transporter by neofunctionalization [28]. Therefore, laboratory evolution may be sufficient to obtain maltotriose utilization in S. eubayanus strain CBS 12357T. Laboratory evolution is a commonly-used method for obtaining desired properties by prolonged growth and selection under conditions favoring cells which develop the desired phenotype [45,46]. Similarly as in Darwinian natural evolution, the conditions under which laboratory evolution is conducted shape the phenotypes acquired by evolved progeny following the process of survival of the fittest [47]. In Saccharomyces yeasts, selectable properties include complex and diverse phenotypes such as high temperature tolerance, efficient nutrient utilization and inhibitor tolerance [48–51]. Laboratory evolution was successfully applied to improve sugar utilization for arabinose, galactose, glucose and xylose [49,52–54]. In S. pastorianus, improved maltotriose uptake was successfully selected for in a prolonged chemostat cultivation on medium enriched with maltotriose [55]. Theoretically, laboratory evolution under similar conditions could select S. eubayanus mutants which develop the ability to utilize maltotriose. In this study, we submitted S. eubayanus strain CBS 12357T to UV-mutagenesis and laboratory evolution in order to obtain maltotriose utilization under beer brewing conditions. While obtaining a non-GMO maltotriose-consuming S. eubayanus strain was a goal in itself for industrial beer brewing, we were particularly interested in the possible genetic mechanisms leading to the emergence of maltotriose utilization. Indeed, we hypothesized that the genetic plasticity and functional redundancy of the four subtelomeric SeMALT genes of CBS 12357T could facilitate the emergence of maltotriose transport by neofunctionalization. The evolution process leading to maltotriose utilization in a strain with only maltose transporters, such as CBS 12357T, may provide insight in the emergence of maltotriose utilization in general.
To obtain maltotriose-consuming mutants, CBS 12357T was grown on synthetic medium containing 20 g L-1 glucose (SMG) until stationary phase, and approximately 108 cells were used to inoculate synthetic medium containing 20 g L-1 maltotriose (SMMt) as sole carbon source. After incubation at 20°C for three months, neither growth nor maltotriose utilization was observed. Exposure to UV-radiation can cause DNA damage, resulting in the emergence of cells with diverse mutations due to error-prone repair. Therefore, UV-mutagenesis was applied to increase the likelihood of obtaining a mutation enabling maltotriose utilization. To this end, CBS 12357T was grown in SMG medium, sporulated, submitted to mild UV-mutagenesis (46% survival rate) and approximately 108 cells of the mutagenized population were used to inoculate SMMt containing 20 g L-1 maltotriose. After two weeks at 20°C, growth was observed and, after 3 weeks, the maltotriose concentration had decreased to 10. 5 g L-1. After two subsequent transfers in fresh SMMt, 96 single cells were sorted into a 96 well plate containing YPD medium by fluorescence-activated cell sorting (FACS). The resulting single-cell cultures were transferred to a 96 well plate containing SMMt, in which growth was monitored by OD660 measurements. The seven single-cell isolates with the highest final OD660 were selected and named IMS0637-IMS0643. To characterize growth on maltotriose, the strain CBS 12357T, the single-cell isolates IMS0637-IMS0643 and the maltotriose-consuming S. pastorianus strain CBS 1483 were grown in shake flasks on SMMt (Fig 2A and S1 Fig). After 187 h, S. eubayanus CBS 12357T did not show any maltotriose consumption. Conversely, isolates IMS0637-IMS0643, all showed over 50% maltotriose consumption after 91 h (as compared to 43 h for CBS 1483). Upon reaching stationary phase, isolates IMS0637-IMS0643 had consumed 93 ± 2% of the initial maltotriose concentration, which was similar to the 92% conversion reached by S. pastorianus CBS 1483. While these results indicated that the single cell isolates IMS0637-IMS0643 utilized maltotriose in synthetic medium, they did not consume maltotriose in presence of glucose and maltose after 145 h of incubation (Fig 2B). Under the same conditions, S. pastorianus CBS 1483 consumed 50% of the maltotriose after 145 h (Fig 2B). Nutrient-limited growth confers a selective advantage to spontaneous mutants with a higher nutrient affinity [45,55]. Therefore, to improve maltotriose utilization under industrially-relevant conditions, the pooled isolates IMS0637-IMS0643 were subjected to laboratory evolution in a chemostat culture on modified brewer’s wort. To avoid the presence of residual maltose, which would prevent selection for maltotriose utilization, brewer’s wort was diluted 6-fold. To strengthen the selective advantage for maltotriose-consuming cells, the diluted wort was complemented with 10 g L-1 maltotriose, yielding concentrations of 2 g L-1 glucose, 15 g L-1 maltose and 15 g L-1 maltotriose in the medium feed. To prevent growth limitation due to the availability of limited oxygen or nitrogen, the medium was supplemented with 10 mg L-1 ergosterol, 420 mg L-1 Tween 80 and 5 g L-1 ammonium sulfate [56]. During the batch cultivation phase that preceded continuous chemostat cultivation, glucose and maltose were completely consumed, leaving maltotriose as the only carbon source. After initiation of continuous cultivation at a dilution rate of 0. 03 h-1, the medium outflow initially contained 13. 2 g L-1 of maltotriose. After 121 days of chemostat cultivation, the maltotriose concentration had progressively decreased to 7. 0 g L-1 (Fig 2C). At that point, which corresponded to ca. 125 generations, 10 single colonies were isolated from the culture on SMMt agar plates and incubated at 20°C. Three single-cell lines were named IMS0750, IMS0751 and IMS0752 and selected for further characterization in micro-aerobic cultures, grown at 12°C on 3-fold diluted wort, along with S. eubayanus CBS 12357T and S. pastorianus CBS 1483 (Fig 2D). In these cultures, strains CBS 12357T and IMS0751 only consumed glucose and maltose, while S. pastorianus CBS 1483, as well as the evolved isolates IMS0750 and IMS0752, also consumed maltotriose (S2 Fig). After 263 h, maltotriose concentrations in cultures of strains IMS0750 and IMS0752 had decreased from 10. 7 to 4. 3 g L-1 maltotriose as compared to 2. 0 g L-1 in cultures of strain CBS 1483. We sequenced the genomes of the S. eubayanus strain CBS 12357T, of the UV-mutagenized isolates IMS0637-IMS0643 and of the strains isolated after subsequent chemostat evolution IMS0750-IMS0752 using paired-end Illumina sequencing. Sequencing data were mapped to a chromosome-level assembly of strain CBS 12357T [9] to identify SNPs, INDELs and copy number changes. The genomes of the UV-mutants IMS0637, IMS0640, IMS0641 and IMS0642 shared a set of 116 SNPs, 5 INDELs and 1 copy number variation (S1 Data File). In addition to these shared mutations, isolates IMS0638, IMS0639 and IMS0643 carried three identical SNPs. Overall, 97% of SNPs and INDELs of IMS0637-IMS0643 were heterozygous, indicating that the haploid spores of CBS 12357T diploidized by mating after mutagenesis (S1 Data File). Of the mutations present in all isolates, 34 SNPs and all 5 INDELs affected intergenic regions, 30 SNPs were synonymous, 48 SNPs resulted in amino acid substitutions and 4 SNPs resulted in a premature stop codon (S1 Data File). None of the 52 non-synonymous SNPs affected genes previously linked to maltotriose utilization. The only copy number variation concerned a duplication of the right subtelomeric region of CHRVIII. Read mate-pairing indicated that the duplicated region was attached to the left arm of CHRII, causing the replacement of left subtelomeric region of CHRII by a non-reciprocal translocation. The recombination resulted in loss of one of the SeMALT1 allele, which is not expressed in CBS 12357T [9]. Since the ability to utilize maltotriose in wort emerged only after laboratory evolution during chemostat cultivation, mutations present in the chemostat-evolved strains IMS0750 and IMS0752 were studied in more detail. With the exception of one synonymous SNP, IMS0750 and IMS0752 were identical and shared 100 SNPs, 3 INDELs and 5 copy number changes (S1 Data File). The non-maltotriose utilizing strain IMS0751 shared only 63 SNPs and 3 INDELs with IMS0750 and IMS0752, of which 98% were homozygous, indicating a recent loss of heterozygosity event affected its entire genome. Of the mutations in maltotriose-utilizing strains IMS0750 and IMS0752, only 5 SNPs and 4 copy number changes were absent in IMS0637-IMS0643, and could therefore explain the ability to utilize maltotriose in wort (Fig 3A). The 5 SNPs consisted of two intergenic SNPs and three non-synonymous SNPs in genes with no link to maltotriose. However, the changes in copy number affected several regions harboring SeMALT genes: a duplication of 550 bp of CHRII including SeMALT1 (coordinates 8,950 to 9,500), a duplication of the left arm of CHRXIII including SeMALT3 (coordinates 1–10,275), loss of the left arm of CHRXVI (coordinates 1–15,350), and loss of 5. 5 kb of CHRXVI including SeMALT4 (coordinates 16,850–22,300). Analysis of read mate pairing indicated that the copy number variation resulted from a complex set of recombinations between chromosomes II, XIII and XVI. The high degree of sequence identity of the affected MAL loci and their localization in the subtelomeric regions made exact reconstruction of the mutations difficult. Therefore, IMS0637 and IMS0750 were sequenced using long-read sequencing on ONT’s MinION platform, and a de novo genome assembly was made for each strain. Comparison of the resulting assemblies to the chromosome-level assembly of CBS 12357T indicated that two recombinations had occurred. Both in IMS0637 and IMS0750, an additional copy of the terminal 11. 5 kb of the right arm of chromosome VIII had replaced the terminal 11. 4 kb of one of the two copies of the left arm of chromosome II (Fig 3B). This recombination was consistent with the copy number changes of the affected regions in IMS0637-IMS0643, IMS0750 and IMS0752 and resulted in the loss of one copy of the MAL locus harboring SeMALT1. In addition, the genome assembly of IMS0750 indicated the replacement of both copies of the first 22. 3 kb of CHRXVI by complexly rearranged sequences from CHRII, CHRXVIII and CHRXVI. The recombined region comprised the terminal 10,273 nucleotides of the left arm of CHRIII, followed by 693 nucleotides from CHRII, 1,468 nucleotides from CHRXVI and 237 nucleotides from CHRXIII (Fig 3B). The recombinations were non reciprocal, as the regions present on the recombined chromosome showed increased sequencing coverage while surrounding regions were unaltered. This recombination resulted in the loss of the canonical MAL locus harboring SeMALT4 on chromosome XVI. However, the recombined sequence contained a chimeric open reading frame consisting of the 5’ part of SeMALT4 from CHRXVI, the middle of SeMALT1 from CHRII and the 3’ part of SeMALT3 from CHRXIII (Fig 3C, S3 Fig). To verify this recombination, the ORF was PCR amplified using primers binding on the promotor of SeMALT4 and the terminator of SeMALT3, yielding a fragment for strain IMS0750, but not for CBS 12357T and IMS0637. Sanger sequencing of the fragment amplified from strain IMS0750 confirmed the chimeric organization of the ORF, which we named SeMALT413. The sequence of SeMALT413 showed 100% identity to SeMALT4 for nucleotides 1–434 and 1113–1145,100% identity to SeMALT1 for nucleotides 430–1122 and 100% identity to SeMALT3 for nucleotides 1141–1842 (Fig 3C). Nucleotides 1123–1140, which showed only 72% identity with SeMALT1 and 61% identity with SeMALT3, were found to represent an additional introgression (Fig 3B). While the first 434 nucleotides can be unequivocally attributed to SeMALT4 due to a nucleotide difference with SeMALT2, the nucleotides 1123–1140 are identical in SeMALT2 and SeMALT4. Therefore, this part of the sequence of SeMALT413 might have come from SeMALT2 on CHRV or from SeMALT4 on CHRXVI. Overall, SeMALT413 showed a sequence identity of only 85 to 87% with the original SeMALT genes, with the corresponding protein sequence exhibiting between 52 and 88% similarity. We therefore hypothesized that the recombined SeMalT413 transporter might have an altered substrate specificity and thereby enable maltotriose utilization. The tertiary protein structure of the chimeric SeMALT413 gene was predicted with SWISS-MODEL (https: //swissmodel. expasy. org/), based on structural homology with the Escherichia coli xylose-proton symporter XylE [57], which has previously been used as a reference to model the structure of the maltotriose transporter ScAgt1 [58]. Similarly to the maltose transporters in Saccharomyces, XylE is a proton symporter belonging to the major facilitator superfamily with a transmembrane domain composed of 12 α-helixes (S4 Fig). The same structure was predicted for SeMalT413, with 1 α-helix formed exclusively by residues from SeMalT4,4 α-helixes formed exclusively by residues from SeMalT1 and 5 α-helixes formed exclusively by residues from SeMalT3 (Fig 3D). The remaining two α-helixes were composed of residues from more than one transporter. Since the first 100 amino acids were excluded from the model due to absence of similar residues in the xylose symporter reference model, the structure prediction underestimated the contribution of SeMalT4. The three-dimensional arrangement of the α-helixes of SeMalT413 was almost identical to SeMalT1, SeMalT3 and SeMalT4, indicating that it retained the general structure of a functional maltose transporter (S5 Fig). The small structural differences identified between SeMalT413 and the wild-type S. eubayanus SeMalT transporters could not be used to predict the ability of SeMalT413 to transport maltotriose [58]. Therefore, to investigate its role in maltotriose transport, SeMALT413 and, as a control, SeMALT2 were overexpressed in the wild-type strain S. eubayanus CBS 12357T (Fig 4A and S6 Fig). Consistently with previous gene editing in CBS 12357T [9], the expression cassettes were inserted at the SeSGA1 locus, encoding an intracellular sporulation-specific glucoamylase which is not expressed during vegetative growth [59,60]. Growth of the resulting strains S. eubayanus IMX1941 (SeSGA1Δ: : ScTEF1p-SeMALT2-ScCYC1t) and IMX1942 (SeSGA1Δ: : ScTEF1p-SeMALT413-ScCYC1t), as well as the wild-type strain CBS 12357T and the evolved isolate IMS0750 was tested on SM supplemented with different carbon sources (S7 Fig). On glucose, strains IMX1941 and IMX1942 exhibited the same specific growth rate of 0. 25 ± 0. 01 h-1 as CBS 12357T, while IMS0750 grew faster with a growth rate of 0. 28 ± 0. 01 h-1. Glucose was completely consumed after 33 h (Fig 4B). On maltose, the specific growth rates of CBS 12357T, IMX1941, IMX1942 and IMS0750 ranged between 0. 17 and 0. 19 h-1 and did not differ significantly. Maltose was completely consumed after 43 h (Fig 4C). On maltotriose, only the evolved mutant IMS0750 and reverse engineered strain IMX1942 (ScTEF1p-SeMALT413-ScCYC1t) showed growth. IMS0750 grew with a specific growth rate of 0. 19 ± 0. 01 h-1 and consumed 55% of maltotriose within 172 h. Over the same period, IMX1942 grew at 0. 03 ± 0. 00 h-1 and consumed 45% of the maltotriose after 172 h (Fig 4D), demonstrating the capacity of SeMALT413 to transport maltotriose. The emergence of the maltotriose transporter SeMalT413 by recombination between different MALT genes during laboratory evolution demonstrates that MALT gene neofunctionalization can contribute to the emergence of maltotriose utilization. To investigate if such neofunctionalization could have played a role in the emergence of maltotriose transporter genes in Saccharomyces yeasts, we analyzed the sequences of existing maltotriose transporter genes in S. cerevisiae and S. pastorianus genomes. In S. cerevisiae strains, maltotriose transport is encoded by the ScAGT1 gene [11,17,18]. However, ScAGT1 is truncated and non-functional in S. pastorianus [61]. Instead, maltotriose utilization has been attributed to two S. pastorianus-specific genes: LgAGT1 and SpMTY1. The maltotriose transporter gene LgAGT1 was identified on S. eubayanus chromosome XV of S. pastorianus and shares 85% sequence identity with ScAGT1 [62,63]. Although it is absent in CBS 12357T [20], LgAGT1 was found to enable maltotriose transport in the north-American S. eubayanus isolate yHRVM108 [19,20]. The SpMTY1 gene, also referred to as MTT1, was found in the MAL1 locus of S. pastorianus [21,22]. In addition to S. cerevisiae chromosome VII, SpMTY1 was also found on S. eubayanus chromosome VII of S. pastorianus, of which the right arm originates from S. cerevisiae due to a recombination [62]. SpMTY1 shows 90% sequence identity with ScMALx1 genes [21,22], but also displays segmental sequence identity with SeMALT genes [64,65]. The relatively low homology of ScAGT1 and LgAGT1 genes indicates that they are less related to maltose transporter genes such as ScMALx1 and SeMALT than SpMTY1. Moreover, their sequence identity to maltose transporters from the MALT family such as ScMAL31 is roughly homogenous over their coding region. Therefore there is no evidence that they resulted from recombinations between other MALT genes. In contrast, the identity of some segments of SpMTY1 relative to ScMAL31 deviates strongly from the average identity of 89% [21]. Indeed, sequence identity with ScMAL31 of S. cerevisiae S288C [66] is above 98% for nucleotides 1–439,627–776,796–845,860–968 and 1,640–1,844, while it is only 79% for nucleotides 440–626,65% for nucleotides 777–795,50% for nucleotides 846–859 and 82% for nucleotides 969–1,639 (S8 Fig). Alignment of the sequences of S. eubayanus CBS 12357T SeMALT genes [9] to SpMTY1 showed high sequence identity with SeMALT3 across several regions that showed significant divergence from the corresponding ScMAL31 sequences: 91% identity for nucleotides 478–533,94% identity for nucleotides 577–626 and 94% identity for nucleotides 778–794 (S8 Fig). These observations would indicate that the evolution of SpMTY1 might have involved introgression events similar to those responsible for the SeMALT413 neofunctionalization described in the present study. However, introgressions from SeMALT genes cannot explain the entire SpMTY1 gene structure. Its evolution may therefore have involved multiple introgressions, similarly as for SeMALT413. While most regions with low identity to ScMAL31 and SeMALT3 were too short to identify their provenance, the sequence corresponding to the 969th to 1,639th nucleotide of SpMTY1 could be blasted on NCBI. In the S288C genome, ScMAL31 was the closest hit with 82% identity. However, when blasting the sequence against the full repository excluding S. pastorianus genomes, the closest hit was the orthologue of ScMAL31 on chromosome VII of S. paradoxus strain YPS138. In addition to an 89% identity to nucleotides 969–1,639 of SpMTY1, SparMAL31 had an identity of 94% for nucleotides 544–575 and of 93% for nucleotides 846–859 (S8 Fig). Therefore, the sequence of SpMTY1 may have resulted from recombination between different MALT genes, involving ScMALx1 and other MALT genes such as SeMALT3 and SparMAL31. While the chimeric SeMALT413 ORF can be fully explained by recombination between SeMALT genes, SpMTY1 probably accumulated additional mutations during its evolution. S. eubayanus strains are currently used for industrial lager beer brewing [9]. To test the evolved strain IMS0750 under laboratory-scale brewing conditions, its performance was compared with that of its parental strain CBS 12357T in 7 L cultures grown on high-gravity (16. 6° Plato) wort (Fig 5). After 333 h, IMS0750 had completely consumed all glucose and maltose, and the concentration of maltotriose had dropped from 19. 3 to 4. 7 g L-1 (Fig 5). In contrast, CBS 12357T did not utilize any maltotriose. In addition to its improved maltotriose utilization, IMS0750 also showed improved maltose consumption: maltose was completely consumed within 200 h, while complete maltose consumption by strain CBS 12357T took ca. 330 h (Fig 5). Consistent with its improved sugar utilization, the final ethanol concentration in cultures of strain IMS0750 was 18. 5% higher than in corresponding cultures of strain CBS 12357T (Fig 5). Brewing-related characteristics of IMS0750 were further explored by analyzing production of aroma-defining esters, higher alcohols and diacetyl. Final concentrations of esters and higher alcohols were not significantly different in cultures of the two strains, with the exception of isoamylacetate, which showed a 240% higher concentration in strain IMS0750 (Table 1). In addition, while the concentration of the off-flavour diacetyl remained above its taste threshold of 25 μg L-1 after 333h for CBS 12357T, it dropped below 10 μg L-1 for IMS0750 (Table 1).
UV-mutagenesis and subsequent laboratory evolution yielded mutants which were able to utilize maltotriose in synthetic medium and in brewer’s wort. In the resulting isolates IMS0750 and IMS0752, several recombinations affecting subtelomeric regions were identified. All four maltose transporter genes in S. eubayanus CBS 12357T are localized in subtelomeric MAL loci: SeMALT1 on chromosome II, SeMALT2 on chromosome V, SeMALT3 on chromosome XIII and SeMALT4 on chromosome XVI [9,20]. In the evolved strain IMS0750, a complex recombination between the subtelomeric regions of chromosomes II, XIII and XVI involved at least three of these MAL loci. Long-read nanopore sequencing enabled complete reconstruction of the recombined left arm of chromosome XVI, revealing recombinations between the ORFs of at least SeMALT1, SeMALT3 and SeMALT4. These recombinations occurred within the open reading frame of SeMALT4 and the newly-formed chimeric ORF SeMALT413 encoded a full length protein with a structure comparable to that of SeMalT transporters. In contrast to the original SeMALT genes, overexpression of SeMALT413 enabled growth on maltotriose, indicating that SeMalT413 acquired the ability to import maltotriose. The predicted structure of SeMalT413 was highly comparable to the structure of other transporters from the major facilitator superfamily [67] and to the structure of SeMalT1, SeMalT3 and SeMalT4. While nothing is known about the amino acid residues responsible for substrate specificity in SeMalT transporters, the threonine and serine residues at the 505th and 557th position respectively of ScAgt1 were identified as critical for maltotriose transport [68]. In SeMalT413, the corresponding amino acids originate from SeMALT3. However, since SeMALT3 itself is unable to utilize maltotriose, the ability of SeMalT413 to transport maltotriose likely depends on the interaction of residues from the different parental transporters, rather than from the residues of one of the transporters. Interestingly, CBS 12357T was recently evolved for maltotriose utilization in another study, resulting in a chimeric SeMALT434 transporter which enabled maltotriose uptake [19]. In this study, a 230-bp introgression of SeMALT3 into the ORF of SeMALT4 was found, including the 505th and 557th residues. While the shorter α-helixes of SeMALT434 could lead to broader substrate-specificity by increasing structural flexibility, the length of these helixes was not affected in SeMALT413. As a result, we hypothesize that the acquired maltotriose utilization does not depend solely on specific residues, but rather on the interaction of the residues from the different parental transporters, either by increasing structural flexibility, or by the properties of several critical residues from different α-helixes. The 230-bp from SeMALT3 which were present in SeMALT413 and in SeMALT434 may be of particular importance. However, the specific combination of sequences from SeMALT4, SeMALT1 and SeMALT3 in SeMALT413 may further contribute to the maltotriose specificity. Recombinations are an important driver of evolution, as illustrated by the emergence of aerobic growth on citrate during laboratory evolution of Escherichia coli [69]. Indeed, a tandem repeat of the citrate/succinate antiporter citT placed under the constitutive rna promoter enabled aerobic growth on citrate. Moreover, the emergence of a new ORF by recombination has been observed previously between the TLO genes of C. albicans, although it was not associated with a new gene function [31]. In contrast, the emergence of SeMALT413 is an example of gene neofunctionalization, which occurred by recombination within genes of the subtelomeric MALT family. Neofunctionalization by in vivo formation of chimeric sequences is reminiscent of the mechanism used by the pathogen Trypanosoma brucei to evade its host’s immune system [70]. T. brucei expresses a single variant surface glycoprotein (VSG) gene from a subtelomeric location and its genome contains many VSG pseudogenes [71]. Due to 70 bp repetitive elements, the actively expressed VSG gene can be altered by gene conversion from pseudogenes, resulting in a chimeric VSG gene [72–74]. While antigen switching may not qualify as neofunctionalization, it demonstrates the ability of recombinations to diversify gene functions by creating chimeric ORFs. This ability has also been exploited for in vitro protein engineering, a strategy known as gene shuffling or gene fusion [75,76]. Gene shuffling involves randomized assembly of diverse DNA sequences into chimeric genes, followed by screening for novel or improved functions. Analogous to in vitro gene shuffling, the complex protein remodeling caused by in vivo formation of chimeric sequences may be particularly potent for protein neofunctionalization [77]. The demonstration of neofunctionalization of a sugar transporter in S. eubayanus by in vivo gene shuffling supports the notion that gene fusion is an essential driver of evolution by accelerating the emergence of new enzymatic functions [78]. Moreover, analysis of the SpMTY1 maltotriose transporter gene revealed a chimeric structure similar to that of SeMALT413, albeit with alternating sequence identity with ScMAL31, SeMALT and SparMAL31. While sequences from S. cerevisiae and S. eubayanus were already present in the genome of S. pastorianus, the presence of sequences from S. paradoxus is plausible as introgressions from S. paradoxus were commonly found in a wide array of S. cerevisiae strains [79]. Therefore, the sequence of SpMTY1 could have resulted from in vivo gene shuffling between genes from the MALT family, followed by accumulation of mutations. The emergence of SeMALT413 could therefore be representative of the emergence of maltotriose utilization during the evolution of S. pastorianus. Moreover, the emergence of a maltotriose transporter after laboratory evolution of CBS 12357T, which was discovered at the same time as SeMALT413 provides further credibility to the evolutionary importance of in vivo gene shuffling for gene neofunctionalization [19]. No evidence of reciprocal translocations between SeMALT1, SeMALT3 and SeMALT4 was found in the genome of IMS0750, indicating genetic introgression via non-conservative recombinations. Such introgressions can occur during repair of double strand breaks by strand invasion of a homologous sequence provided by another chromosome and resection [80], leading to localized gene conversion and loss of heterozygosity. This model, which was proposed to explain local loss of heterozygosity of two orthologous genes in an S. cerevisiae x S. uvarum hybrid [80], provides a plausible explanation of the emergence of SeMALT413 through non-reciprocal recombination between paralogous SeMALT genes in S. eubayanus. The mosaic sequence composition of the resulting transporter gene suggests that neofunctionalization required multiple successive introgression events. As a result of these genetic introgressions, the SeMALT4 gene was lost. The fact that IMS0750 harbored two copies of SeMALT413 and no copy of SeMALT4 indicates a duplication of the newly-formed ORF at the expense of SeMALT4 via loss of heterozygosity. As functional-redundancy enables the accumulation of mutation without losing original functions [28,31,32,81], the loss of SeMALT4 was likely facilitated by the presence of the functionally-redundant maltose transporter SeMALT2 [9]. The observation that introgressions were only found at SeMALT4 may be due to the low number of tested mutants. However, it should be noted that introgressions in the SeMALT1 and SeMALT3 ORF’s would have been unlikely to be beneficial, since these genes are not expressed in CBS 12357T [9]. This study illustrates the role of the rapid evolution of subtelomeric genes in adaptation to environmental changes. In Saccharomyces yeasts, subtelomeric regions contain a large number of gene families encoding functions critical to the interaction of a cell with its environment, such as nutrient uptake, sugar utilization, inhibitor tolerance and flocculation [28,82–87]. The high number of genes within subtelomeric families results in functional redundancy and therefore in mutational freedom [28,31,32,81]. In Saccharomyces species, many industrially-relevant brewing traits are encoded by subtelomeric gene families, such as the MAL genes encoding maltose utilization and the FLO genes encoding flocculation [88]. While subtelomeric regions are difficult to reconstruct due to their repetitive nature, they encode much of the genetic diversity between genomes [89,90]. A posteriori sequence analysis of existing gene families can elucidate their evolutionary history. For example, the α-glucosidase genes from the MALS family emerged by expansion of an ancestral pre-duplication gene with maltose-hydrolase activity and trace isomaltose-hydrolase activity [15]. The evolution of MALS isomaltase genes from this ancestral gene is an example of subfunctionalization: the divergent evolution of two gene copies culminating in their specialization for distinct functions which were previously present to a lesser extent in the ancestral gene. The generation of functional redundancy by gene duplication is critical to this process as it enables mutations to occur which result in loss of the original gene function without engendering a selective disadvantage [28,31,32,36,37,81]. In contrast to subfunctionalization, neofunctionalization consists of the emergence of a function which was completely absent in the ancestral gene [40]. While the emergence of many genes from a large array of organisms has been ascribed to subfunctionalization and to neofunctionalization, these conclusions were based on a posteriori analysis of processes which had already occurred, and not on their experimental observation [15,28,31,39–42]. Ex-vivo engineering of the subtelomeric FLO genes demonstrated that recombinations within subtelomeric gene families can alter their function [39]. However, in vivo neofunctionalization within a subtelomeric gene family was never observed in real time. Here we present clear experimental evidence of neofunctionalization within a laboratory evolution experiment. The ability of SeMALT413 to transport maltotriose proves that such in vivo gene shuffling is relevant for evolutionary biology. Given their high genetic redundancy of subtelomeric gene families, and the large body of evidence of gene sub- and neofunctionalization in their evolutionary history, it is likely that subtelomeric localization of genes facilitates the emergence of new functions. As a result, subtelomeric regions would not only be a hotspot of genetic diversity between different genomes, but also a preferred location for the birth of new genes and new gene functions. While SeMALT413 was shown to enable maltotriose utilization, it remained unclear how the UV-mutagenized cells acquired the ability to utilize maltotriose and why these mutations were insufficient to enable maltotriose utilization in wort. Since maltotriose-consuming mutants did not arise in the absence of UV-mutagenesis, the ability to utilize maltotriose likely emerged as a result of genetic evolution rather than due to epigenetic adaptation. Moreover, the introduction of SeMALT413 in CBS 12357T resulted in slower maltotriose utilization than IMS0750, suggesting that other mutations may contribute to the maltotriose-utilization phenotype of IMS0750. While whole genome sequencing of IMS0637-IMS0643 revealed a wide array of mutations, none affected genes which were previously linked to maltotriose utilization. The fact that SeMALT1, SeMALT2, SeMALT3 and SeMALT4 could be PCR amplified from the IMS0637 genome while SeMALT413 could not, indicates that maltotriose utilization was not due to an undetected SeMALT413 gene. In addition, alignment of short-read data to the reference genome and de novo genome assembly based on long-read data did not reveal any mutations affecting MAL genes, except a recombination between CHRII and CHRVII, which resulted in the loss of one of the two copies of SeMALT1. Since deletion of SeMALT1 does not enable maltotriose utilization in CBS 12357T [9], this mutation is unlikely to be causal. While one of the 122 mutations affecting the UV-mutagenized strains or additional undetected mutations may have contributed to maltotriose utilization, their elucidation is beyond the scope of this study. Moreover, while overexpression of SeMALT413 in CBS 12357T resulted in lesser maltotriose utilization than the evolved strain IMS0750, the maltotriose transporter SeMalT413 is not necessarily suboptimal. Indeed, when overexpressing transporters suboptimal growth is commonly observed and has been attributed to imbalances between transporter activity and the subsequent metabolic steps [91,92]. Overall, regardless of the presence of other mutations contributing to maltotriose utilization, the emergence of the maltotriose transporter gene SeMAL413 from parental genes which do not enable maltotriose transport demonstrates that gene neofunctionalization occurred. While the introduction of SeMALT413 in CBS 12357T via genetic engineering demonstrated its neofunctionalization, the use of GMO-strains is limited in the brewing industry by customer acceptance issues [93]. However, the non-GMO evolved S. eubayanus isolate IMS0750 could be tested on industrial brewing wort at 7 L scale. In addition to near-complete maltotriose conversion, the maltose consumption, isoamylacetate production and diacetyl degradation of IMS0750 were superior to CBS 12357T. While the increased maltotriose consumption could be at least partially attributed to the emergence of the SeMALT413, it remained unclear if and what mutations could underlay the other changes. However, efficient maltose and maltotriose consumption, as well as the concomitantly increased ethanol production, are important factors determining the economic profitability of beer brewing processes [94]. In addition, low residual sugar concentration, low concentrations of diacetyl and high concentrations of isoamylacetate are desirable for the flavor profile of beer [95,96]. S. eubayanus strains typically generate high concentrations of 4-vinyl guaiacol, a clove-like off-flavor [97,98], a strategy to eliminate this production in S. eubayanus have recently been described [97]. Therefore, expansion of phenotypic landscape of S. eubayanus might be accelerated by combining these domesticated traits. In terms of application, the laboratory evolution approach for conferring maltotriose utilization into S. eubayanus presented in this paper is highly relevant in view of the recent introduction of this species in industrial-scale brewing processes [9,99]. The ability to ferment maltotriose can be introduced into other natural isolates of S. eubayanus, either by laboratory evolution or by crossing with evolved strains such as S. eubayanus IMS0750. Besides their direct application for brewing, maltotriose-consuming S. eubayanus strains are of value for the generation of laboratory-made hybrid Saccharomyces strains for brewing and other industrial applications [8,100–102].
All yeast strains used and generated in this study are listed in Table 2. S. eubayanus type strain CBS 12357T [1] and S. pastorianus strain CBS 1483 [55,103] were obtained from the Westerdijk Fungal Biodiversity Institute (Utrecht, the Netherlands). Stock cultures were grown in YPD, containing 10 g L−1 yeast extract, 20 g L−1 peptone and 20 g L−1 glucose, at 20°C until late exponential phase, complemented with sterile glycerol to a final concentration of 30% (v/v) and stored at -80°C until further use. Plasmids were propagated overnight in Escherichia coli XL1-Blue cells in 10 mL LB medium containing 10 g L-1 peptone, 5 g L-1 Bacto Yeast extract, 5 g L-1 NaCl and 100 mg L-1 ampicillin at 37°C. YPD medium was prepared using 10 g L-1 yeast extract, 20 g L-1 peptone and 20 g L-1 glucose. Synthetic medium (SM) contained 3. 0 g L-1 KH2PO4,5. 0 g L-1 (NH4) 2SO4,0. 5 g L-1 MgSO4. , 7 H2O, 1 mL L-1 trace element solution, and 1 mL L-1 vitamin solution [56], and was supplemented with 20 g L-1 glucose (SMG), maltose (SMM) or maltotriose (SMMt) by addition of autoclaved 50% w/v sugar solutions. Maltotriose (95. 8% purity) was obtained from Glentham Life Sciences, Corsham, United Kingdom. Industrial wort was provided by HEINEKEN Supply Chain B. V. , Zoeterwoude, the Netherlands. The wort was supplemented with 1. 5 mg L-1 of Zn2+ by addition of ZnSO4·7H2O, autoclaved for 30 min at 121ᵒC and filtered using Nalgene 0. 2 μm SFCA bottle top filters (Thermo Scientific, Waltham, MA) prior to use. Where indicated, filtered wort was diluted with sterile demineralized water. Solid media were supplemented with 20 g L-1 of Bacto agar (Becton Dickinson, Breda, The Netherlands). S. eubayanus strains transformed with plasmids pUDP052 (gRNASeSGA1) were selected on medium in which (NH4) 2SO4 was replaced by 5 g L-1 K2SO4 and 10 mM acetamide (SMAceG) [104].
Fermentation of the wort sugar maltotriose is critical for the flavor profile obtained during beer brewing. The recently discovered yeast Saccharomyces eubayanus is gaining popularity as an alternative to S. pastorianus and S. cerevisiae for brewing, however it is unable to utilize maltotriose. Here, a combination of non-GMO mutagenesis and laboratory evolution of the S. eubayanus type strain CBS 12357T was used to enable maltotriose fermentation and improve brewing performance. The improved strain expressed a novel transporter gene, SeMALT413, which was formed by recombination between three different SeMALT maltose-transporter genes. Overexpression of SeMALT413 in CBS 12357T confirmed its neofunctionalization as a maltotriose transporter. As the S. pastorianus maltotriose transporter SpMty1 has a mosaic structure similar to SeMalT413, maltotriose utilization likely involved similar recombination events during the domestication of current lager brewing strains. Based on a posteriori sequence analysis, the emergence of gene functions has been attributed to gene neofunctionalization in a broad range of organisms. The real-time observation of neofunctionalization during laboratory evolution constitutes an important validation of the relevance and importance of this mechanism for Darwinian evolution.
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In vivo recombination of Saccharomyces eubayanus maltose-transporter genes yields a chimeric transporter that enables maltotriose fermentation