OBTAINING HYBRIDS OF DISTILLERY YEASTS CHARACTERISED BY THE ABILITY OF FERMENTING STARCH

Joanna Kawa-Rygielska

ABSTRACT

The aim of the work was to obtain fusants of Saccharomyces and Schwanniomyces yeast strains. Biological material was composed of Schwanniomyces occidentalis ATCC 48086, Saccharomyces cerevisiae "malt", Saccharomyces diastaticus ATCC 13007. The use of natural markers, such as the growth of Schwanniomyces strain in medium with cyclohexymide and the growth of Saccharomyces yeast at 40⁰C helped select 26 fusion products. Genetic analysis of DNA using the PCR method showed the presence of three hybrids containing DNA of both parental strains.

Key words: yeast, protoplast fusion, DNA..

INTRODUCTION

The need to intensify the production of ethanol as an alternative source of energy leads to the necessity of lowering production costs so that the price of ethanol is more attractive than that of traditional fuels. Apart from new technological solutions proposed by numerous firms, scientists are looking for yeasts with improved biotechnological features that utilize a wider spectrum of carbohydrates, which are characterized by higher fermenting activity and better vitality. Saccharomyces cerevisiae, typical distillery strains, do not feature ability to utilize such complex carbohydrates as dextrin or starch. Due to that, fermentation of starch raw materials is preceded in classical technology of agricultural distilling by such expensive processes as evaporating and then liquefying and saccharifying of starch using commercial amylolytic preparations to the sugars undergoing fermentation release.

In order to improve the technological process, it is important to enrich the typical distilling yeasts with the ability to synthesize amylolytic enzymes, which would result in limiting or eliminating the necessity of using commercial enzymatic preparations and in lowering the costs of production.

Genetic methods are used in search for new, improved yeasts. The results of experiments presented in literature indicate the importance of protoplast fusion [7, 9, 12, 13, 16, 18, 25]. Chi et al. [3, 4] obtained hybrids with improved fermenting activity and the content up to 18.8% ethanol after fermentation of mash of raw and gelatinised corn starch.

Bearing the above in mind, the author of the present study used interspecific protoplast fusion to add the ability to synthesise amylolytic enzymes to improve the biochemical abilities of distilling yeasts. The starch fermenting strain Schwanniomyces occidentalis ATCC 48086 was used as a partner of Saccharomyces yeasts.

THE AIM OF THE RESEARCH

The aim of the study was to obtain interspecific hybrids of starch fermenting yeasts Schwanniomyces occidentalis ATCC 48086, and yeasts Saccharomyces diastaticus ATCC 13007 and Saccharomyces cerevisiae "malt".

The study comprised the following: choosing of markers for the selection of fusants, obtaining interspecific hybrids using protoplast fusion and confirmation of DNA recombination.

MATERIALS AND METHODS

Biological material was composed of distillery yeast Saccharomyces cervisiae "malt" from the International Department of Brewery and Distillery in Edinburgh, Saccharomyces diastaticus ATCC 13007 and Schwanniomyces occidentalis ATCC 48086.

Selection of markers differentiating parental strains was performed on solid media YPG [15] with cycloheximide (0.01 and 0.001%) and at the temperature 30°C and 40°C.

The yeast cells from the exponential phase were subjected to protoplasts formation with a lytic enzyme Novozym^TM 234 (Trichoderma harzianum).The rate of protoplasts formation [%] and regeneration of the protoplasts [%] were determined [7]. The fusion of protoplasts of yeasts was induced using a solution of polyethylene glycol (PEG 4000) in 10mM CaCl₂ [24, 28]. The hybrids obtained were transferred to YM slants and kept at 4°C. After 2, 4, 8, 12 weeks of storing, the stability of hybrids was examined by controlling their growth on selection medium containing starch as the sole carbon source.

The volume of the parental and hybrid cells was measured by microscope and calculated according to Sakai et al. [21]. Isolation of DNA from the cells of parental and hybrid strains was performed using the Rose et al. method [20] in Skała modification [23]. Amplification of DNA was performed using microsatellite primer (GTG)₅ GTG GTG GTG GTG GTG [19]. Polymerase chain reaction (PCR) was conducted using thermostable Taq DNA of polymerase from QIAGEN Company and in thermocycler DNA Peltier Engine PTC – 200 Peltier Thermal Cycler - MJ Research. The products of PRC reaction were analyzed by electrophoresis. The mass of DNA fragments was determined in the presence of the standard of mass (100 bp DNA Ladder Plus, Promega) on 0.8 % agarose gel with ethidium bromide.

RESULTS

The parental strains used in the process of obtaining protoplasts and their fusion should be characterised by properties allowing to make a distinction between the products of fusion and the cells of parental strains. There were analysed differences between natural features of the strains. The results are presented in Table 1.

Schwanniomyces occidentalis ATCC 48086 was the only strain which did not grow at 40°C and was resistant to the cycloheximide concentrations used in the research. The Saccharomyces strains did not show ability of growing in the presence of cycloheximide, even when its concentration was 0.001%. Based on the above observations, tolerance to cycloheximide (0.001%) and the ability to grow at 40°C were selected as markers allowing to make a distinction between the hybrids and initial strains in selection regeneration medium.

The first stage of the research was obtaining the protoplasts of parental strains. The rate of protoplast formation of yeast cells at set conditions exceeded 90% (Table 2). The rate of protoplasts regeneration was also high and reached over 50%. The best ability to rebuild cell wall was observed in Saccharomyces cerevisiae "malt whiskey".

26 hybrids were obtained as a result of protoplast fusion of Schwanniomyces occidentalis and Saccharomyces. The frequency of fusion ranged from 7.8 x 10^-6 to 6.4 x 10^-5 (Table 3).

One of the crucial factors deciding about the usefulness of the fusion products is their stability during storing. The hybrids ability to grow in selection conditions within 12 weeks of storing was assessed. Starch was used as a source of carbon to eliminate the hybrids with no ability to assimilate that source of carbon. Table 4 presents the number of fusants growing in the conditions described above. 52.6 % of hybrids S and 42.6% of hybrids M showed growing ability after 12 weeks of storing. The hybrids which were stable after 12 weeks of storing were used in the further research.

In the next stage of the selection the volume of the fusant cells and the parental strains were compared. No significant differences were observed in the volume of the Saccharomyces yeasts (7.0 – 7.6) x 10^-7 mm³. The cells of all Saccharomyces strains were larger than those of the partner strain Schwanniomyces (5.2 x 10^-7 mm³), Table 5.

From among the hybrids of S group (Saccharomyces diastaticus ATCC 13007 x Schwanniomyces occidentalis ATCC 48086), strains S1, S2, S3, S4, S6 and S8 distinguished larger volume of the cells. In M family (Saccharomyces cerevisiae "malt" x Schwanniomyces occidentalis ATCC 48086), the M1 hybrid was worth noting. The other hybrids had their volume of the cells similar to one of the fusion partners.

The fusants S1, S2, S3, S4, S6, S8 and M1, whose cell volume was significantly larger than that of parental strains, were selected to be used in the next stage.

It is at this stage that authors of some studies decided which hybrids to consider fusants. However, as indicated in the present research, genomic DNA sequence was necessary to confirm effectiveness of fusion.

After the analysis of the distribution of DNA fragments of strains Schwanniomyces occidentalis ATCC 48086, Saccharomyces diastaticus ATCC 13007 and their hybrids S1, S3, S4, S6 and S8 (Fig. 1), only two strains - S2 and S3 could be considered fusants. The hybrid S2 contained two DNA fragments (over 1500 bp and 675 bp), which were also present in the parental strain Saccharomyces diastaticus ATCC 13007, and two other fragments (1300 bp and 600 bp) typical of the other parent Schwanniomyces occidentalis ATCC 48086. Unlike the fusant S2, the fusant S3 contained only one region typical of each parental strain (675 bp) typical of Saccharomyces diastaticus ATCC 13007 and (1300 bp) typical of Schwanniomyces occidentalis ATCC 48086. Moreover, in both hybrids, the presence of one DNA fragment (400 bp) from both parental strains was observed. It would require further analysis to definitely say from which parental strai n this fragment is derived. The remaining products of the fusion of Saccharomyces diastaticus ATCC 13007 and Schwanniomyces occidentalis ATCC 48086 contained DNA fragments identical with one of the fusion partners – S8 and S6 with Schwanniomyces, whereas S1 and S4 with Saccharomyces (Fig. 1). The M1 fusant contained two DNA fragments (1300 bp and 600 bp) typical of Schwanniomyces and one (675 bp) typical of Saccharomyces “malt” (Fig. 2).

The ability of the obtained interspecific hybrids to synthesise amylolytic enzymes and actively ferment media containing compound carbohydrates was examined at further stages (study in preparation).

DISCUSSION

Yeasts with a rich amylolytic complex may find numerous applications [2]. Many of them cannot be used in distilling of alcohol as they are characterised by low ethanol tolerance and sensitivity to the products of starch hydrolysis [27]. The yeasts Schwanniomyces, with the ability to produce amylolytic enzymes and to fully hydrolyse and ferment starch, are especially interesting [1, 6, 8, 22]. The strain Schwanniomyces occidentalis ATCC 48086 was used in the research to improve the fermenting ability of distilling yeasts Saccharomyces characterised by good fermentation dynamics and high degree of attenuation.

Saccharomyces cerevisiae "malt", characterised by an ability to use such carbohydrates as maltotriose and maltotetrose, is a strain used in the production of Scotch whisky. The strain Saccharomyces diastaticus [15] is able to hydrolyse dextrin to glucose and contains active glucoamylase [10]. Its big advantage is high tolerance to ethanol [11].

The parental strains used in the study were easily formed into protoplasts. The use of Novozym 234 enzyme resulted in obtaining over 90% of protoplasts of all strains used in the research. The frequency of fusion was at the level from 6.4 x 10^-5 to 7.9 x 10^-6 and it was higher than the frequency indicated in literature. The frequency of fusion between the auxotrophic strain Saccharomyces cerevisiae and Schwanniomyces castelli was 5.3 x 10^-7 [26].

The fusants obtained in the study were genetically stable despite a big taxonomical difference. This may have resulted from the selection of markers which represented the natural biochemical and physiological qualities of the strains of both types.

The genetic DNA examination of the interspecific hybrids performed with the use of PCR method made it possible to identify and differentiation of the strains within the kind and species and proper selection of primers was of key importance. Couto et al. [5] indicated special usefulness of a microsatellite primer (GTG)₅ which made it possible to differentiate the strains interspecifically. Other researchers were successfully using microsatellite primers for detecting the species within Saccharomyces and Debaryomyces [9]. In the present research, the use of PCR method and a microsatellite primer (GTG)₅ enabled differentiation of the fusion partners within Schwanniomyces and Saccharomyces and confirm the presence of 3 interspecific hybrids of the strains: S2, S3 and M1.

CONCLUSIONS

1. The use of protoplast fusion and such natural markers as tolerance to cycloheximide (0.001%) in case of Schwanniomyces occidentalis ATCC 48086 and the ability of Saccharomyces yeasts to grow at 40°C resulted in obtaining 26 interspecific hybrids of which 16 were stable after 12 weeks of storing.

2. The analysis of DNA homology of the hybrids used in the study performed using a method of chain reaction of polymerase and a microsatellite primer (GTG)₅ confirmed interchange of genetic material in 3 hybrids: S2, S3 and M1.

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