INTRODUCTION

Until very recently, the role of pectin as the source of carbon in microorganisms has obviously been underestimated. It was only the presence of the methyl group in the pectin molecule that allowed researchers to get an idea of the truly planetary scale of methanol release by plants and the role of pectin in this process. It should also be noted that a large number of methanol-utilizing yeast species associated with plants have been described. The polygalacturonic component of pectin is an important part of carbohydrate nutrition for various microorganisms, including yeasts. It is not without reason that the yeast identification guide [1] would include a differentiating test for the utilization of galacturonic acid, which is the product of the enzymatic destruction of pectin.

Pectin, one of the key plant polysaccharides, is a polymer that consists, to varying degrees, of methylated galacturonic acid residues linked by the α-1,4 glycosidic bond. The molecular weight of pectin varies from 20 000 to 50 000. The biochemical destruction of pectin is a complex process that involves many different enzymes [2]. The most thoroughly studied enzyme from the genetic point of view is the pectinase from the Saccharomyces yeasts [3]. This endopolygalacturonase (EC 3.2.1.15) catalyzes the hydrolysis of α-1,4 glycosidic bonds between the galacturonic acid residues lacking methoxyl groups with the formation of oligogalactouronates. This enzyme is used for tea and coffee processing, the cleaning of plant fiber (e.g., flax), and the clarification and filtration of fruit and berry juices, as well as grape must and wine [2].

Although the pectinolytic activity in yeasts has drawn scientists’ attention since the middle of the last century [4–6], there are only a few works reporting the study of pectinase-producing yeasts [7–9]. The only exception, as mentioned above, is the group of pectinolytic yeasts from the Saccharomyces genus [3]. It should be noted that Russian research prioritizes the selection of wine yeasts with pectinase activity from the Saccharomyces genus [10, 11].

The present work continues the previously published work on the degree of homology between the nucleotide sequence of the PGU1 gene from Saccharomyces cerevisiae S288c and the superfamily of the pectinase PGU genes in the Saccharomyces yeasts [3].

The goal of the current work was to find the nucleotide sequences of PGU genes in the genomes of ascomycetous yeasts deposited in the genomic databases on the basis of their similarity with the known nucleotide sequence of the PGU1 gene and the corresponding amino acid sequence of the endopolygalacturonase from S. cerevisiae S288c and to determine the level of the intraspecies and the interspecies divergence between the identified genes.

MATERIALS AND METHODS

The characteristics of the 26 analyzed ascomycetous yeasts, including their origin, are listed in the table.

BLAST was used to search for sequences homologous to the open reading frame (1086 bp) of the known nucleotide sequence of the PGU1 gene from the S. cerevisiae S288c yeast (GenBank accession no. BK0069431) in the GenBank ((http://www.ncbi. nlm.nih.gov/genbank/) and Sanger Institute (http:// www.sanger.ac.uk) databases. Nucleotide and amino acid sequences were aligned manually with the aid of the BioEdit software (http://www.mbio.ncsu.edu/ BiEdit/bioedit.html). Phylogenetic trees were reconstructed by the neighbor-joining method with the MEGA6 software [31].

RESULTS AND DISCUSSION

In our previous works [3, 32, 23], we described a superfamily of divergent species-specific PGU genes in 115 Saccharomyces strains belonging to the following yeast species: S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus, as well as to the hybrid taxon S. pastorianus (syn. S. carlsbergensis). We also demonstrated the naturally occurring interspecies PGU gene transfer from S. cerevisisae to S. bayanus and from S. paradoxus to S. cerevisiae.

The phylogenetic analysis performed in the current work revealed a number of distinct clusters of PGU genes, which strictly follow the subdivision of the analyzed ascomycetous yeasts into the genera (Fig. 1).

Fig. 1.
figure 1

Phylogenetic tree of ascomycetous yeasts reconstructed on the basis of the nucleotide sequences of the PGU genes. Bootstrap values greater than 70% are shown. Scale bar corresponds to 100 substitutions per 1000 nucleotides. Endopolygalacturonase genes from the E. gossypii strains FDAG1 and ATCC10895 are identical. In certain strains, the names for the identified PGU genes are provided according to the original classification in slant brackets.

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The first gene cluster found in Saccharomyces yeasts was analyzed in detail earlier (see above). For this reason, for comparison, the phylogenetic tree presented in Fig. 1 contains only the PGU genes from the type strains and reference strains of the species belonging to this genus.

The individual branch representing the PGU gene from Lachancea kluyveri NRRL Y-12651 adjoins the first cluster. It seems possible that, if additional species from this polytypic genus [1] are taken into analysis, a full-fledged second cluster could be obtained.

The third cluster contains the PGU genes from the two Kluyveromyces genus species, namely K. marxianus and K. wickerhamii. We should note the high similarity level (97.7–98.2%) between the PGU alleles in five K. marxianuis strains, while the level of similarity within the entire genus is much lower (64.5%).

The fourth cluster is represented by the yeast-like fungi from the Galactomyces and Geotrichum genera, with the identity level between the corresponding PGU genes reaching 72.7–81.3%. Here, we should point out the appearance of divergent PGU genes (the presence of superfamilies) within the Galactomyces geotrichum, Galactomyces citi-aurantii, and Galactomyces candidus species, as well as the high similarity of the PGU genes observed in the following species pairs: Galactomyces citi-aurentii and Geotrichum klebahnii and Galactomyces citi-aurentii and Galactomyces candidus. The latter observation indicates the possible interspecies transfer of the PGU genes within the Galactomyces/Geotrichum genera, similar to that shown by us for Saccharomyces species. It should be mentioned here that Galactomyces yeasts basically meet the genetic concept of the genus [34, 35], according to which the species of the same genus share a common mating-type system allowing them to cross in any interspecific combinations [36, 37]. This apparently may result in the interspecies transfer of the PGU genes within the Galactomyces genus and the anamorphic Geotrichum species.

The fifth cluster is composed by the PGU genes (69–87.9% similarity) of different species of the recently expanded Eremothecium genus [1], namely E. cymbalariae, E. gossypii, and Eremothecim sp.

Figure 2 presents the results of phylogenetic analysis of the amino acid sequences of pectinases from the analyzed ascomycetous yeasts; the obtained phylogenetic tree corresponds well with that obtained on the basis of the nucleotide sequences of the PGU genes (see Fig. 1).

Fig. 2.
figure 2

Phylogenetic tree of ascomycetous yeasts reconstructed on the basis of the amino acid sequences of their Pgu endopolygalacturonases. Bootstrap values greater than 70% are shown. Scale bar corresponds to 50 substitutions per 1000 amino acid residues. Endopolygalacturonases from the E. gossypii strains FDAG1 and ATCC10895 are identical. For certain strains, the abbreviated names for different Pgu endopolygalacturonases are provided in slant brackets.

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We have already mentioned above the taxonomic importance of PGU genes, at least for the identification of the yeast genera. The evolutionary tree of the analyzed ascomycetous yeasts based on the D1/D2 ribosome sequences presented in Fig. 3 shows good correspondence with the divergence of the PGU genes in these yeast strains. When yeasts are identified based on the phenotype, it seems reasonable, along with an assessment of their ability to utilize galacturonic acid, to use Petri dish test for the presence of active pectinase, especially since it would allow primary selection of producers of this enzyme.

Fig. 3.
figure 3

Phylogenetic tree of the ascomycetous yeasts based on the nucleotide sequences of the D1/D2 region of the 26S large ribosome subunit. Bootstrap values greater than 70% are shown. Scale bar corresponds to 50 substitutions per 1000 nucleotides. The following strains are characterized by the identical D1/D2 nucleotide sequences: (1) S. bayanus var. uvarum MCYC623 and CBS 395; (2) K. marxianus KCTC 17555, NBRC 1777, VKM Y-719, CECT 1043, and CCT 7735.

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Another conclusion that can be made from our study, in addition to the ascertainment of the taxonomic value of PGU genes, is the importance of the identification of PGU genes. From our point of view, the analysis of pectinase genes will also allow primary screening for producers of this enzyme. It was demonstrated previously that, in filamentous fungi with high pectinase activity, such as for example Aspergillus niger [38] and Sclerotina sclerotiorum [39], as well as in the yeasts G. citri-aurantii [24], G. geotrichum [25], and S. bayanus var. uvarum [3], the genome of a single strain may contain not a single, but several, polymeric PGU genes. Polymeric genes normally show a cumulative effect that enhances the corresponding trait. On the other hand, only one PGU gene in the highly efficient pectinolytic strains, such as K. marxianus, may indicate that the overexpression of this gene is the result of some regulatory mechanisms, in particular, the strong promoter. The identification of such strains is important for subsequent genetic engineering work.

Table 1.   Genetic characteristics and origin of the ascomycetous yeasts studied in this work
Full size table

CONCLUSIONS

To summarize, phylogenetic analysis of the pectinase genes has great importance for the evolutionary genetics and selection of ascomycetous yeasts.