Ключові слова: резистентність до високої температури, толерантність до етанолу, паливний етанол, ксилоза, Hansenula polymorpha, Dekkera bruxellensis.

АННОТАЦИЯ

Ищук Е.П. Молекулярные механизмы резистентности к высоким температурам и этиловому спирту у неконвенционных дрожжей. - Рукопись.

Диссертация на соискание ученой степени кандидата биологических наук по специальности 03.00.07 - микробиология. - Институт биологии клетки НАН Украины, Львов, 2010.

Диссертация посвящена идентификации генов, ответственных за резистентность к высоким температурам и этиловому спирту у неконвенционных дрожжей Hansenula polymorpha и Dekkera bruxellensis. Показано роль кислой трегалазы (Ath1p) и белков теплового шока (Hsp104p, Hsp16p) в термотолерантности H. polymorpha. Идентифицирован новый ген H. polymorpha (ETT1), ответственный за резистентность к этанолу и высоким температурам. Трансформанты, которые надэксрессируют ETT1, характеризируется в 3-10 раз повышенной резистентностью к этанолу, денатурирующему агенту (AZC) и тепловому шоку. Показано позитивное влияние гетерологической экспрессии гена MPR1 S. cerevisiae на этанол-резистентность H. polymorpha. Трансформант, содержащий 3 копии гена MPR1, характеризируется в 10 раз повышенной этанол-резистентностью. Надэкспрессия гена PDC1, кодирующего пируватдекарбоксилазу, у H. polymorpha может использоваться для конструирования улучшенных продуцентов этанолу при повышенных температурах, поскольку PDC1-трансформанты продуцируют в 3 раза больше этанола. Разработаны методы генной инженерии для D. bruxellensis: систему трансформации на основе векторов, которые содержат ауксотрофные (URA3 и URA5) и доминантный (APH) маркеры, репортерную систему на основе гена LAC4 Kluyveromyces lactis. Клонировано и проведено анализ центромерных последовательностей D. bruxellensis.

Ключевые слова: высокотемпературная резистентность, этанол-резистентность, топливный этанол, ксилоза, Hansenula polymorpha, Dekkera bruxellensis.

SUMMARY

Ishchuk O.P. Molecular mechanisms of resistance to high-temperatures and ethanol in non-conventional yeasts. - Manuscript.

Thesis for a Doctor of Philosophy (PhD) degree in Biology, speciality 03.00.07 - Microbiology. - Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, 2010.

The dissertation is devoted to the identification of genes involved in the resistance to high-temperature and ethanol in non-conventional yeasts Hansenula polymorpha and Dekkera bruxellensis.

It was reported previously that trehalose is important for H. polymorpha thermotolerance. The loss of trehalose-6-phosphate synthase activity (enzyme of trehalose biosynthesis) makes H. polymorpha incapable of trehalose synthesis and sensitive to heat-shock. However, the effect of increased trehalose level on H. polymorpha thermotolerance has not been studied yet. To evaluate the role of the ATH1 gene (coding for acid trehalase) in H. polymorpha trehalose mobilization and thermotolerance we constructed Дath1 mutant of this yeast. It was shown, that Дath1 mutant has 2-fold elevated level of intracellular trehalose. Along with an increase in trehalose content, the Дath1 strain has 2-fold increased resistance to heat-shock. In addition, the deletion of ATH1 gene results in improved high-temperature xylose fermentation. Дath1 mutant produces 6-fold higher amount of ethanol during fermentation at 50 °C.

Heat-shock proteins are referred as major stress protectors of proteins and membranes. In the current study, we constructed the H. polymorpha strains carrying the expression plasmids of heat-shock proteins. The selected genes of H. polymorpha were HSP16 and HSP104 that encode a small heat-shock protein and an ATP-dependent molecular chaperone correspondingly. Transformants carrying heat-shock proteins expression plasmids showed high constitutive level of Hsp16p and Hsp104p expression that resulted in 2-10-fold increased resistance to heat-shock and 3-6-fold higher ethanol production during xylose fermentation at 50 °C.

The insertional mutagenesis was used to identify genes involved in H. polymorpha ethanol resistance. One insertional mutant, highly sensitive to ethanol, was selected. In this mutant the insertional cassette disrupted the ORF448 (herein designated ETT1) sharing homology with the S. cerevisiae MPE1 gene, which was reported to be an essential gene involved in RNA 3'-end processing. In contrary to S. cerevisiae MPE1, H. polymorpha ETT1 is not an essential gene. H. polymorpha ett1 mutant is viable but sensitive to different stress factors, particularly to ethanol, high temperature, has decreased capacity of xylose fermentation. To study the effect of ETT1 expression on H. polymorpha stress tolerance, the recombinant strain carrying 6-7 copies of the ETT1 gene was constructed. This transformant was shown to have increased resistance to ethanol, high temperature and protein misfolding reagent (AZC). The S. cerevisiae MPE1 gene does not complement the ett1 mutation of H. polymorpha. However, it was partially complemented by MPE1 gene of other xylose utilizing yeast, Pichia stipitis. To investigate the Ett1p molecular function, it was overexpressed and purified. It was shown that Ett1p does not participate in pre-mRNA cleavage. The alignment of amino acid sequences of DWNN domains of Mpe1p S. cerevisiae, P. stipitis and Ett1p of H. polymorpha with ubiquitin revealed the difference in the conserved residues. Despite that Mpe1p is evolutionary conserved, this protein has distinct functions in H. polymorpha and P. stipitis. Ett1p of H. polymorpha is likely involved in the ubiquitination that plays an important role in controlling a number of cellular processes, among them the degradation of abnormal proteins generated during stress exposure.

The MPR1 gene of S. cerevisiae encoding acetyltransferase was recently reported to confer ethanol tolerance in S. cerevisiae. We studied the effect of heterologous expression of the S. cerevisiae MPR1 gene on H. polymorpha ethanol resistance. MPR1 gene of S. cerevisiae proved to be efficiently expressed in H. polymorpha. It was shown that MPR1 expression confers tolerance to ethanol and AZC in H. polymorpha and can be used in the construction of improved ethanol producers of this yeast species.

One of the approaches for high-temperature fermentation improvements in H. polymorpha involved the overexpression of the PDC1 gene coding for pyruvate decarboxylase. Pyruvate decarboxylase is a key enzyme in alcoholic fermentation and the increase of its activity may be important for pyruvate distribution between pyruvate dehydrogenase and pyruvate decarboxylase, and therefore improve the ethanol yield. To test this hypothesis native and heterologous (Kluyveromyces lactis) PDC1 genes were overexpressed in H. polymorpha. The PDC1 overexpression resulted in an increased pyruvate decarboxylase activity and higher ethanol production from glucose and xylose. Multi-copy integration of PDC1 in the genome resulted in 20-fold increase in pyruvate decarboxylase activity and 3-fold improved ethanol yield during high-temperature xylose fermentation. Thus the overexpression of PDC1 can be used in the construction of improved H. polymorpha ethanol producers from lignocellulose at high-temperature.

As methods of molecular genetics are not reported for D. bruxellensis, we aimed at the development of corresponding methods to be able to clone genes of ethanol tolerance and construct improved ethanol producers of this yeast. The transformation system was developed based on the integrative vectors with auxotrophic (URA3 and URA5) and dominant (APH) selective markers. To develop replicative plasmids the centromeric regions of D. bruxellensis were also cloned. Cloned centromeres differ in their sequences and contain motifs similar to S. cerevisiae CDEI- and CDEIII-elements. To study the promoter activities in D. bruxellensis, the reporter system was developed based on K. lactis LAC4 gene using similar approaches to those developed previously by us for Candida famata.

Key words: high-temperature tolerance, ethanol resistance, fuel ethanol, xylose, Hansenula polymorpha, Dekkera bruxellensis.

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