Functional and evolutionary analysis of sugar transporters in Kluyveromyces marxianus

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Varela, Javier A.
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University College Cork
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Kluyveromyces marxianus is a yeast species traditionally found in fermented dairy products such as yogurt and kefir. Because of its association with these products, this yeast has a QPS/GRAS status that facilitates applications in the food industry. K. marxianus is also known for possessing remarkable physiological traits: the yeast is thermotolerant, has a rapid growth rate and is able to utilise a wide range of substrates. Since these traits are desirable from an industrial standpoint, K. marxianus has been exploited for several biotechnological applications including bioethanol, flavour molecule and organic acid production. Perhaps the best-known application involving this yeast is the production of ethanol from whey, a by-product of the dairy industry. The capacity of the yeast to utilise lactose, essential in this environment, was previously shown to be a variable trait with some strains being able to utilise the sugar better than others. To understand the genetic basis that accounts for these differences, a detailed analysis of lactose utilisation was performed. The K. marxianus genome was found to encode four copies of the lactose permease (LAC12) gene, which are present in both good and poor lactose utilising strains. To determine if these genes were responsible for the growth differences observed, the LAC12 genes from a good and a poor lactose utiliser were cloned into a replicative plasmid and expressed in S. cerevisiae. Expression of one of these gene copies (KmLAC12) conferred growth on lactose, indicating that this is the main lactose transporter in K. marxianus. However, growth on lactose was only observed when expressing the KmLAC12 gene from a good lactose utiliser, suggesting that good and poor utilisers carry a functional (KmLAC12-B) and non-functional (KmLAC12-A) version of the lactose permease gene, respectively. Overexpression of KmLAC12-B and not KmLAC12-A conferred growth on a poor lactose utiliser, supporting this idea. A comparison of KmLac12p sequences from 12 K. marxianus strains confirmed that poor and good lactose utilisers carry distinct versions of the lactose transporter. Through this analysis it was established that the KmLac12p-B and KmLac12p-A differ in 16 amino acid positions. Altogether, these results showed unequivocally that differences in the KmLAC12 sequence are the main factor accounting for variability in lactose utilisation in K. marxianus. Lactose utilisation is not the only variable trait in K. marxianus. In fact, several studies have shown that the yeast displays a high level of diversity in a range of phenotypes including thermotolerance, stress tolerance, glucose repression and sugar utilisation. While several K. marxianus genome sequences are currently available, a comparative analysis aiming to determine the level of genetic diversity between these sequences had not previously been performed. To investigate this aspect, the genomes of nine K. marxianus strains from different origins were sequenced and compared. Also, five previously published genomes were included in this analysis. Single Nucleotide Polymorphism (SNP) analyses revealed a relatively high level of genetic diversity in the species, with up to 3 % DNA sequence divergence between alleles. These data also showed that K. marxianus strains can be found in different ploidy states (i.e. haploid, diploid or triploid), a finding that was confirmed by flow cytometry experiments. Diploid and triploid strains showed large regions of loss of heterozygosity in several chromosomes. Also, sequence coverage analyses revealed differences in copy number of large regions of the genome, indicating that some of these isolates are partially aneuploid. Remarkably, ploidy was found to correlate with strain origin: all the strains isolated from dairy environments were diploid or triploid while non-dairy strains were haploid. A phylogenetic tree based on SNP data was constructed to further investigate the relationship between these isolates. The haplotype tree showed that dairy and nondairy strains form two distinct clades. In addition, all of the dairy strains utilised lactose efficiently and were found to carry the KmLAC12-B gene, encoding the functional lactose transporter. The SNP data also allowed us to determine that the diploid strains in this study were hybrids between a dairy and non-dairy haplotype. On the other hand, triploid strains contained three copies of the dairy haplotype. The data obtained in this study reveals that K. marxianus displays a high level of genetic diversity and shows that there is a relationship between genetic diversity and the environment from where the strains were isolated. The role of KmLAC12-B in lactose utilisation was clear, but the function of the rest of the LAC12 genes was not known. Since the LAC12 gene was previously shown to encode a galactose transporter in K. lactis, we tested whether any of the LAC12 genes from K. marxianus encoded a similar function. Heterologous expression of these genes revealed that three out of the four copies encoded galactose transporters – no function was identified for LAC12-3. However, single or multiple disruptions of these genes in K. marxianus did not abolish growth on galactose, indicating the presence of additional galactose transporters. A second galactose transport system, encoded by the HGT1 gene, was previously described in K. lactis. To examine if K. marxianus genome encoded any orthologues of this gene, a bioinformatics analysis was carried out. Several tandem copies of the HGT1 gene were identified in K. marxianus genome. Also, multiple tandem copies of the glucose transporters KHT1 and KHT2 were discovered through this analysis. The expansion of these sugar transporters was not found in other Kluyveromyces genomes examined, indicating that these gene expansions are specific to K. marxianus. Interestingly, copy number of the KHT and HGT1 genes was variable among K. marxianusstrain; the strains were found to encode either 6 or 5 copies of KHT and either 5 or 3 copies of HGT1. To assess the functionality of these genes, heterologous expression experiments were carried out in S. cerevisiae EBY.VW4000, a strain deleted for all hexose transporters. Expression of the K. marxianus transporters identified additional galactose transporters; 4 encoded by HGT1 orthologues and 1 encoded by a KHT orthologue. K. marxianus strains carrying mutations in these genes were constructed and tested on galactose medium. Only a mutant carrying mutations in all eight of the identified galactose transporters showed a significant growth defect on 2 % galactose medium whereas a mutant in the HGT1 locus and the three functional LAC genes failed to grow on 0.1 % galactose. These results demonstrate that galactose transport is highly redundant in K. marxianus, that different proteins appear to be involved in low or high-affinity transport and highlight important differences between the K. lactis and K. marxianus galactose transport systems. In addition to studying the expansion of sugar transporter families in K. marxianus, molecular tools to genome engineer this yeast were also developed during this PhD. While some tools to genetically manipulate this yeast are currently available, there is a need to develop new techniques that allow simple and efficient gene targeting for construction of mutants and for allelic replacement. To tackle this issue, the CRISPRCas9 system was implemented in K. marxianus. To establish the methodology, a single-plasmid system, comprising Cas9 and an sgRNA, was constructed and successfully used to target the ADE2 gene at high efficiency (> 96%) in this yeast. The system was also shown to function in K. lactis, where the ADE2 gene was targeted at similar efficiency. Mutants were obtained in both yeasts both by non-homologous end- joining (NHEJ) and homology-dependent repair (HDR) methods, though the capacity to carry out allelic replacement by homologous recombination using repair fragments was of variable efficiency in different strains. To improve gene targeting in K. marxianus, the KU80 gene involved in the NHEJ DNA repair system was disrupted using the CRISPR-Cas9 system. Homologous recombination frequency was greatly enhanced in a ku80 mutant, suggesting that the NHEJ had been inactivated. Finally, to further improve homologous recombination repair, the RAD52 gene, involved in homologous recombination in S. cerevisiae, was expressed in K. marxianus. A strain expressing this gene showed a seven-fold increase in homologous recombination-mediated repair, albeit from a low base. These tools were used throughout the thesis to facilitate the functional analysis of sugar transporters. The results presented in this thesis contribute to better understanding the function and evolution of sugar transporters in K. marxianus. Also, the tools developed in this work will help to further develop this yeast for cell factory applications.
K. marxianus , Sugar transporters , Gene duplications , Genome evolution
Varela, J. A. 2018. Functional and evolutionary analysis of sugar transporters in Kluyveromyces marxianus. PhD Thesis, University College Cork.