INTRODUCTION

Pectin is a plant polysaccharide consisting of galacturonic acid residues partially esterifed with methanol and linked by the α(1–4)-glycosidic bonds. Pectin compounds, as structural elements of plant tissues, are contained in all higher plants and contribute to their tolerance to both biotic and abiotic environmental factors. The greatest amount of pectin is characteristic of berries, fruits and vegetables. As an example, in grapes, this is 0.5–5.0 g/L depending on the cultivar. Mechanical grinding of fruits rich in pectin results in obtaining a highly viscous juice, which remains bound to the pulp and forms a jelly-like mass. Even a small content of pectins in wine may cause colloidal turbidities and filter clogging [1].

Hydrolysis of high-molecular plant compounds is a complex process involving several enzymes; the main one is pectinase (endo-polygalacturonase, EC 3.2.1.15). Pectinolytic enzymes are widely used in biotechnology for the clarification of fruit juices and wines, fermentation of tea and coffee, purification of plant fibers and waste water, bleaching paper, etc. [2–7]. Pectinases account for approximately 25% of the global food enzyme purchases and their production is constantly growing [8]. The pectinolytic enzymes are used most actively in winemaking and fruit juice manufacture. The treatment of pulp with pectinases improves the separation and clarification of the wort, reduces excessive foaming during fermentation and increases the organoleptic characteristics of the final product [9].

Almost all commercial enzyme preparations are obtained from the Aspergillus and Trichoderma filamentous fungi. Apart from endo-polygalacturonases, these fungal preparations also contain admixtures and enzymes with undesirable side activities, for instance, pectinesterase, which leads to an enhanced content of toxic methanol in wine [10]. Yeast usually do not secrete pectinesterase; therefore, their pectinases are safe for wine and juice clarification [11, 12]. It has been shown that the use of Saccharomyces cerevisiae strains with endo-polygalacturonase activity in winemaking results in an efficient wine clarification and a two-fold decrease in its filtration time [9].

The Saccharomyces genus includes eight species: S. cerevisiae, S. arboricola, S. cariocanus, S. bayanus, S. kudriavzevii, S. jurei, S. mikatae and S. paradoxus [13–17]. S. arboricola, S. kudriavzevii and S. mikatae have low pectinolytic activity, while some strains of S. cariocanus, S. paradoxus and S. jurei are able to readily degrade pectin compounds [18]. The above yeasts are not associated with human economic activities; usually, they are isolated from the exudate and bark of broad-leaved trees, various insects, soils, etc. Two Saccharomyces species, S. cerevisiae and S. bayanus, are involved in winemaking together with their interspecific hybrids: S. cerevisiae × S. bayanus, S. cerevisiae × S. kudriavzevii and S. cerevisiae × S. bayanus × S. kudriavzevii [19–24].

As a rule, the S. cerevisiae yeast is characterized by extremely low pectinolytic activity or is even unable to degrade pectin compounds [11, 25, 26]. At the same time, some strains of the related species S. bayanus actively secrete endo-polygalacturonase [18, 27, 28]. The S. bayanus species has a complex composition, including two varieties, S. bayanus var. bayanus and S. bayanus var. uvarum [21, 29]. The latter occupies the specific ecological niche in viticulture and winemaking at low temperatures [30]. These yeasts are associated with the production of white, sweet, and sparkling wines, as well as cider [19, 31–36]. S. bayanus var. bayanus is mainly represented by brewery contaminating strains [21]. In 2011, the related yeast S. eubayanus was isolated from the bark of beeches in Argentina [37]. Later, this species was found in China [38], the United States, and Canada [38, 40]. The yeast has been partially genetically isolated; its hybrids are characterized by low survival of ascospores [41]. The European bottom fermenting brewer’s yeast S. pastorianus (syn. S. carlsbergensis) is an interspecific hybrid of S. cerevisiae and S. bayanus, and its cold resistance is inherited from S. bayanus. Whole genome sequencing of several S. eubayanus strains revealed their high similarity with the cold-resistant parent of hybrid yeast S. pastorianus [40, 42, 43].

The goal of this work was to study the pectinolytic activity of the S. bayanus var. bayanus, S. bayanus var. uvarum and S. eubayanus yeasts using strains of different ecological and geographical origin, as well as to select strains with high pectinolytic activity that are of interest for the wine yeast selection.

MATERIALS AND METHODS

The origin of the studied S. bayanus strains is indicated in Table 1. The yeast was cultivated at 28°C on a complete YPD nutrient medium containing, g/L: yeast extract, 10; peptone, 20; glucose, 20; and agar, 20. To prepare native chromosomal DNA, the yeast was grown in 15 mL of liquid YPD at 28°С for 12–16 h.

Table 1. The origin and pectinolytic activity of Saccharomyces strains
Full size table

Yeast Pectinolytic Activity

Screening for pectinolytic activity was carried out by the Louw et al. method [26] with some modifications. Yeasts were grown on YPD solid medium. Overnight yeast cultures were inoculated with a loop onto a minimal medium with polygalacturonic acid, containing, g/L: yeast nitrogen base with amino acids (Difco, United States), 6.7; polygalacturonic acid (Sigma, United States), 12.5; glucose, 10; agar (Difco), 20; and Na2HPO4, 6.8 (pH 4.0). Cells were grown for 3 days at 28°С; the formed yeast colonies were washed off with distilled water, after which Petri dishes were filled with 6 M HCl for 5–10 min. Enzymatic activity was evaluated by clear hydrolysis zones (halos) of polygalacturonic acid around the yeast colonies. Petri dishes were photographed and the size of the halos was determined using the IC Measure_2.0.0.272 software (www.helicon.ru). Two independent experiments were carried out for each strain. The patented S. cerevisiae VKPM Y-718 strain (a polyploid of the Kokur-3 wine strain) with high pectinolytic activity [44] was used as a control.

Pulsed-Field Gel Electrophoresis of Native Chromosomal DNA (Molecular Karyotyping) and Southern Hybridization

Chromosomal DNA was isolated as described in [31] and separated on a CHEF-DR III device (Bio-Rad, United States). Samples were placed in the wells of 1% agarose gel. Pulsed-field gel electrophoresis was performed at 200 V for 15 h at a field switching time of 60 s and for 9 h at a field switching time of 90 s. 0.5 × TBE (45 mM Tris, 10 mM EDTA, and 45 mM boric acid, pH 8.0) cooled to 14°C was used as electrophoresis buffer. The S. cerevisiae YNN 295 strain (Bio-Rad) with a known order and size of chromosomes served as a reference karyotypic. After electrophoresis, the gel was stained with ethidium bromide for 2–3 h, then washed in distilled water for 2 h and photographed in UV light.

Chromosomal DNA was transferred onto a nitrocellulose membrane using a Vacuum blotter (Bio-Rad). DNA was fixed on the membrane by annealing at 80°C for 2 h. A PCR-amplified fragment covering the main portion of the gene PGU1b coding region of S. bayanus var. uvarum CBS 7001 served as a probe. The label was introduced by a nonradioactive method using digoxigenin-labeled dUTP (dig-II-dUTP) from the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche, Switzerland), according to the producer’s instructions. Hybridization and development of hybridization bands were also carried out according to these instructions.

Sequencing and Phylogenetic Analysis

PCR was performed using a DNA amplifier (Bio-Rad). Yeast DNA was isolated by the Lõoke et al. method [45]. S. bayanus pectinase genes PGU1b, PGU2b and PGU3b were amplified with the following primer pairs: PGU13/PGU14 (5'-CCACCAAACGCAATGATTT-3'/5'-ATGATGCACCTGAGCCAGAT-3'); PGE11/PGE12 (5'-GCTTTATGCGCTTTTGCTGT-3'/5'-AACCAGATGGGATTCCAGAA-3') and PGB51/-PGB52 (5'-TTTTGCTGTCTCAGCAGCTC-3'/5'-TTCCAGAACAGCCAGAAAAGG-3'). Primers PGU11 (5'-CACATTGATGGACAAACGCA-3')/PGU12 (5'-AGGATTAACAGCTTGCACCA-3') were used to amplify the S. cerevisiae PGU1 gene.

PCR was carried out in a buffer (30 μL) containing 2.5 mM MgCl2, 0.1 mmol of each dNTP, 50 pmol of each primer, 2.5 U of Taq polymerase (Syntol, Russia) and 20–200 ng of DNA. The reaction was performed according to this format: initial denaturation, 94°C for 4 min; 35 cycles [denaturation at 94°C for 60 s, primer annealing at 55°C for 60 s, DNA synthesis at 72°C for 120 s], and final elongation at 72°C for 10 min. The amplification products were separated by electrophoresis in 1% agarose gel at 60–65 V in 0.5× TBE buffer for 2–3 h. The gel was stained with ethidium bromide, washed in distilled water and photographed in UV light on a Vilber Lourmat transilluminator (Vilber, France). The 1 kb DNA Ladder kit (Fermentas, Thermo Fisher Scientific, United States) was used as a MW marker.

The amplified fragments were eluted from the gel using a GeneJET Gel Extraction Kit (Thermo Fisher Scientific) according to the producer’s protocol. The nucleotide sequences of the PGU genes were identified by two strands by direct Sanger sequencing on an Applied Biosystems 3730 automatic sequencer (Applied Biosystems, United States). Similarity of the obtained and the known nucleotide sequences was determined using the BLAST program in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and SGD (http://www.yeastgenome.org/). Multiple alignments of the known and hypothetical amino-acid sequences were carried out using the BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogenetic trees were built by the neighbor-joining method in the MEGA 7 program [46]. Kluyveromyces marxianus CBS 6566 yeast endo-polygalacturonase (Epg1) was used as an outgroup.

RESULTS AND DISCUSSION

Pectinolytic Activity

Pectinolytic activity was studied in the type S. pastorianus CBS 1538 culture and in 86 S. bayanus strains isolated from various wines, grapes, fruit and berry juices, and natural sources (exudate and bark of broad-leaved trees, insects, soil, and others) (Table 1). The S. cerevisiae VKPM Y-718 polyploid strain with high endo-polygalacturonase activity was used as a control. The type cultures of S. cerevisiae CBS 1171, S. arboricola CBS 10644, S. cariocanus UFRJ 50816, S. kudriavzevii NBRC 1802, S. jurei NCYC 3947, S. mikatae NBRC 1815 and S. paradoxus CBS 432 were also used for comparison.

The strains under study were divided into four groups depending on the diameter of the polygalacturonic acid hydrolysis area; (1), less than 10 mm; (2), 10–15 mm; (3), 15–20 mm; and (4), more than 20 mm (Table 1). Group 1 was represented by the S. bayanus var. uvarum LC1.95 (halo diameter 9.1 mm) and SRC410 (8.6 mm) strains isolated in France from fermenting pulp and apple juice, respectively (Table 1). Pectinolytic activity was completely absent only in one wine strain, PJS1.94, isolated from fermenting pulp in France. Most of the studied strains exhibited a fairly high pectinolytic activity: 44 strains were assigned to group 2 and 34 strains to group 3. The latter included 23 S. bayanus var. uvarum strains from grapes and various wines from France, Spain, Hungary and Slovakia, plus 5 natural isolates (Table 1 and Fig. 1, lanes 2, 3, 6, 8–10). Halo diameter sizes from 15 to 20 mm were observed in five out of ten studied S. eubayanus strains and in the S. bayanus var. bayanus NBRC 1948 strain (18.7 mm) (Table 1 and Fig. 1, lane 12).

Fig. 1.
figure 1

Screening of Saccharomyces bayanus strains grown on a medium containing polygalacturonic acid for the presence of pectinolytic activity: reference strain S. cerevisiae VKPM Y-718 (1); S. bayanus var. uvarum М489 (2); VKM Y-361 (3); SCU 197 (4); M472 (5); CBS 395 (6); CBS 431 (7); PYCC 6867 (8); PYCC 6869 (9); PYCC 7083 (10); S. bayanus var. bayanus CBS 378 (11); NBRC 1948 (12); S. eubayanus CBS 12357 (13); PYCC 7086 (14); and yHKS 212 (15). Dashed circles outline polygalacturonic acid hydrolysis zones around yeast colonies.

Full size image

The highest pectinolytic activity was found in the following five strains: M472 (20.7 mm), CBS 8711 (20.1 mm), CBS 431 (23.7 mm), NCAIM Y-00676 (23.8 mm) and CBS 378 (23.7 mm) (Fig. 1). The first four strains belong to S. bayanus var. uvarum, and the fifth one, to S. bayanus var. bayanus (Table 1). The strains CBS 431 (fermenting pear juice), NCAIM Y-00676 (alcoholic drink) and CBS 378 (beer) were comparable in efficiency of polygalacturonic acid hydrolysis with the control strain S. cerevisiae VKPM Y-718 (27.0 mm). The latter is a tetraploid strain obtained by treating the wine diploid strain Kokur-3 with a colchicine solution [44]. It should be noted that all the studied strains of S. bayanus var. bayanus, S. bayanus var. uvarum and S. eubayanus are diploid and are represented by fertile monospore cultures. Previously, we found that the type culture of the S. pastorianus CBS 1538 hybrid yeast is also diploid [21].

Chromosomal Mapping of PGU Genes in the S. bayanus Genome

The S. cerevisiae yeast has only one pectinase gene, PGU1, located on the X chromosome [11, 25, 26]. We have previously shown that S. bayanus var. uvarum yeasts have three PGU genes with different chromosomal location: PGU1b, chromosome X; PGU2b, chromosome I and PGU3b, chromosome XIV [18, 47, 48]. It was established that the PGU1b gene nucleotide sequence has 86–87% similarity to the PGU2b and PGU3b genes, while the last two genes are 96% identical to each other.

In this work, we determined the number of the PGU genes in the strains of S. bayanus var. bayanus (CBS 380, CBS 378, CBS 425, CBS 424, and NBRC 1948) and S. eubayanus (CBS 12357, PYCC 7085, PYCC 7084, PYCC 7086, PYCC 7087, PYCC 7088, PYCC 7089, yHKS210, yHKS211, and yHKS212). Using the three primer pairs described in the MATERIALS AND METHODS section, PCR fragments corresponding to the PGU1b, PGU2b and PGU3b genes of the studied strains were amplified. Southern hybridization confirmed the presence of these PGU genes in the genomes of the S. bayanus var. bayanus and S. eubayanus yeasts. According to PCR analysis and Southern hybridization, the type culture of the hybrid S. pastorianus CBS 1538 yeast also has the three pectinase genes. On the other hand, the CBS 1538 genome was shown not to contain the PGU1 gene characteristic of S. cerevisiae yeasts.

Thus, S. bayanus var. bayanus, S. bayanus var. uvarum and S. eubayanus have the PGU1b PGU2b PGU3b genotype regardless of the source and region of their isolation. The only exception is the French wine strain S. bayanus var. uvarum PJS1.94, which possesses only two pectinase genes, PGU1b (chromosome X) and PGU3b (chromosome XIV) [48]. Interestingly, this is the only one of the 87 studied strains that is unable to hydrolyze polygalacturonic acid (Table 1).

Phylogenetic Analysis of S. bayanus PGU1b Genes and their Encoded Amino-Acid Sequences

We have sequenced the PGU1b genes from 18 S. bayanus strains of different origins with different pectinolytic activity: S. bayanus var. uvarum (SC4, SRC258, CBS 8711, TBVIC2.95, PJS2.95, CECT 10560, PYCC 6330, PYCC 7082 and PYCC 7083), S. bayanus var. bayanus (CBS 380, CBS 378 and NBRC 1948) and S. eubayanus (PYCC 7084, PYCC 7085, PYCC 7086, PYCC 7087, PYCC 7088 and yHKS 210). The PGU1b gene of the type S. pastorianus CBS 1538 strain was also sequenced. The nucleotide sequences of 14 S. bayanus var. uvarum strains (CBS 7001, CBS 395, CBS 377, M300, VKM Y-361, VKM Y-1140, NCAIM Y.00677, T5/6, T13/30, PJS1.94, UWO (PS) 99-808.3, CCY21-31-12, 136.01 and 148.01) and of the type S. eubayanus CBS 12357 were taken from [18, 48] and the GenBank database. The resulting sequences contained 1077 nucleotides, which covers the main part of the PGU1b gene-encoding region.

Comparative analysis of the obtained nucleotide sequences revealed two groups of S. bayanus var. uvarum strains, which differ by 18–20 nucleotide substitutions. The first group includes 16 strains differing in the diameter of the polygalacturonic acid lysis zone, i.e., in endo-polygalacturonase activity. The nucleotide sequences in the following strains were shown to be identical and differ by one substitution from the PGU1b sequence of strains CBS 395 (18.0 mm) and SC4 (12.7 mm): PJS1.94 (0 mm), UWO(PS) 99–808.3 (10.7 mm), PJS2.95 (11.0 mm), M300 (11.2 mm), CCY21-31-12 (11.5 mm), PYCC 6330 (11.7 mm), PYCC 7082 (14.5 mm). CBS 377 (15.2 mm), TBVIc2.95 (15.3 mm), CBS 7001 (15.5 mm), VKM Y-1146 (17.2 mm), T5/6 (17.0 mm), VKM Y-361 (17.7 mm) and T13/30 (18.8 mm). The second group contains nine strains: 148.01 (13.9 mm), 136.0 (14.7 mm), VKM Y-508 (15.2 mm), PYCC 7083 (15.6 mm), M488 (16.4 mm), CECT 10560 (19.2 mm), SRC258 (18.0 mm), NCAIM Y.00677 (18.9 mm) and CBS 8711 (20.1 mm). The PGU1b gene of the latter strain contained two substitutions compared to the rest of the members of this group. It should be noted that most of the nucleotide substitutions were synonymous and did not affect the protein structure. Pectinases of the S. bayanus var. uvarum strains from both groups only differed in two amino-acid substitutions.

The S. bayanus var. bayanus CBS 380 (14.1 mm) and CBS 378 (23.7 мм) strains have identical nucleotide sequences of the PGU1b gene, which differs by one position (C–T) from that of the NBRC 1948 strain (18.7 mm), and by three positions (A–G and C–T transitions, and T–A transversion) from the gene sequence of the hybrid S. pastorianus CBS 1538 yeast (13.2 mm). The amino-acid sequences of pectinases from S. bayanus var. bayanus and the S. pastorianus CBS 1538 hybrid yeast were identical. Interestingly, the nucleotide sequence of the PGU1b gene of the CBS 1538 strain is similar to that of other yeasts deposited in GenBank, including both old collection strains CBS 1486, CBS 1503, CBS 1513 and the modern bottom fermenting brewer’s yeast strains W34/70 and Weihenstephan 34/70. It is important to note that the S. cerevisiae-type pectinase genes were not observed in the genome of all the listed S. pastorianus strains. The differences between the pectinase genes of the S. eubayanus strains range from zero to five nucleotides; in S. eubayanus, four to seven substitutions were observed compared to S. pastorianus and seven to ten substitutions compared to S. bayanus var. bayanus. The PGU1b nucleotide sequences of S. bayanus var. uvarum and S. bayanus var. bayanus/S. eubayanus differ by more than 80 positions, while the pectinase gene sequences from S. bayanus and other seven Saccharomyces species (S. arboricola CBS 10644, S. cariocanus UFRJ50816, S. jurei NCYC 3947, S. cerevisiae CBS 1171, S. kudriavzevii NBRC 1802, S. mikatae NBRC 1815 and S. paradoxus CBS 432) differ by more than 190 nucleotides.

The resulting PGU gene nucleotide sequences served as the basis for determining the corresponding protein sequences, which in turn, were used to construct a phylogenetic tree (Fig. 2).

Fig. 2.
figure 2

Phylogenetic analysis of endo-polygalacturonase amino-acid sequences of Saccharomyces bayanus and other species of the Saccharomyces genus. Endo-polygalacturonase of Kluyveromyces marxianus (Epg1) was used as an outgroup. Bootstrap support values higher than 70% are represented. The scale bar corresponds to 50 amino-acid substitutions per 1000 amino-acid positions. The numbers in brackets indicate groups of strains with identical amino-acid sequences: (1) M300, VKM Y-1146, T5/6, T13/30, UWO (PS)99-808, PYCC 6330, PYCC 7082, CCY21-31-12, CBS 377, PJS1.94, PJS2.95, TBVIc 2.95, VKM Y-361, SC4; (2) 136.01, 148.01.3, NCAIM Y.00677, VKM Y-508, CBS 8711, CECT 10560, SRC258, PYCC7083; (3) NBRC 1948; (4) yHKS210, PYCC 7085, PYCC 7086, PYCC 7087, PYCC 7088.

Full size image

Kluyveromyces marxianus Epg1 was used as an outgroup. There are two main clusters on the phylogenetic tree. The first includes with a 97% bootstrap support pectinases from S. bayanus var. uvarum, S. bayanus var. bayanus, S. eubayanus and the hybrid S. pastorianus brewer’s yeast, which are 94–99% identical. The highest similarity (99–100%) is characteristic of Pgu1 from the last three taxa.

The second cluster contains the remaining seven Saccharomyces species. Two subclusters within it differ from the rest. The first subcluster is composed by the S. cerevisiae, S. paradoxus and S. cariocanus strains, the Pgu1 of which are 97–98% similar. The second subcluster includes S. mikatae and S. jurei, which have endo-polygalacturonases identical by 96%. S. kudriavzevii pectinase Pgu1k adjoins this subcluster; it has 88–92% similarity with the others. S. arboricola Pgu1a protein also adjoints the second cluster; its identity with the corresponding S. cerevisiae, S. cariocanus, S. jurei, S. kudriavzevii, S. mikatae and S. paradoxus proteins is 87–88%. The similarity between Pgu1 of S. bayanus and the remaining Saccharomyces species is 86–88%.

CONCLUSIONS

Thus, based on a phylogenetic analysis, it can be concluded that the Saccharomyces yeast PGU genes are species-specific. As an example, the PGU genes of the hybrid brewer’s yeast S. pastorianus are likely to originate from the cold-tolerant S. bayanus and not from S. cerevisiae. This is in good agreement with the fact that S. bayanus is common in breweries, while the S. cerevisiae beer yeast does not have pectinolytic activity. The type culture S. cerevisiae CBS 1171 isolated from the brewing process is unable to degrade pectin (Table 1). All S. bayanus strains studied in this work have rather high pectinolytic activity, which is likely to be a feature of this species. Regardless of where and from what source they were isolated, the studied strains of S. bayanus var. uvarum, S. bayanus var. bayanus and S. eubayanus have the PGU1b PGU2b PGU3b genotype. The only exception is the French wine strain S. bayanus var. uvarum PJS1.94 with its PGU1b PGU3b genotype, which is unable to decompose polygalacturonic acid.

Strains M472, CBS 8711, CBS 431, NCAIM Y-00676, and CBS 378, which secrete active endo-polygalacturonase, are of interest for further molecular genetic research and selection, including those aimed at creating new wine strains.