High intracellular trehalase activity prevents the storage of trehalose in the yeast Dekkera bruxellensis.

UNLABELLED: Dekkera bruxellensis hit the spotlight in the past decade mostly due to its rather high ability to adapt to several different fermentation processes. This yeast relies on different genetic and physiological aspects to achieve and preserve its high industrial fitness and some of these traits are shared with Saccharomyces cerevisiae. We have previously described that D. bruxellensis is unable to make use of accumulating trehalose as a strategy for cell adaptation and survival in the industrial scenario, as opposed to S. cerevisiae. Since trehalose is often involved in mechanisms related to cell protection, we aimed to investigate both cause and effect of the absence of this metabolite in the cell adaptive capacity in the industrial environment. Our results indicate that the major cause for the nonaccumulation of trehalose is the high constitutive activity of neutral trehalase. Therefore, the rate of trehalose degradation could be higher than its rate of synthesis, preventing accumulation. Altogether, our data elucidate the mechanisms involved in the lack of trehalose accumulation in D. bruxellensis as well as evaluates the implications of this feature. SIGNIFICANCE AND IMPACT OF THE STUDY: Dekkera bruxellensis can successfully take advantage of its peculiar physiological and genetic traits in order to adapt and survive in fermentation processes. So far, tolerance to stress has been credited to trehalose synthesis. The data presented in this work provided information on the underlying mechanism that prevents trehalose accumulation and corroborated the recent information that trehalose itself is not implicated in yeast stress tolerance. Second, it showed that D. bruxellensis responds differently to Saccharomyces cerevisiae to excess of sugar, which may explain its preference for respiration (oxidative metabolism) over fermentation (reductive metabolism) even at limited oxygen supply. These findings help to understand the drop on ethanol production in processes overtaken by this yeast.

Distribution of Dekkera bruxellensis in a sugarcane-based fuel ethanol fermentation plant.

UNLABELLED: We investigated the presence of the yeast Dekkera bruxellensis in samples collected at three points surrounding the industrial alcoholic fermentation plants of two distilleries where there are often cases of contamination caused by this yeast: this involved sugar cane wash water, feeding sugar cane juice and vinasse from the treatment pond. Total yeast was isolated in WLN medium with bromocresol green and cycloheximide and further selected on the basis of its ability to grow in synthetic medium containing nitrate. Following this, colonies were selected from the distribution on nitrate plates and identified by amplification with species-specific primers and DNA sequencing of the 26S-D1/D2 locus. The results showed that D. bruxellensis is introduced through the feeding substrate, which suggests that its cells originated with the harvested cane. Subsequently, its population circulates as a result of the reuse of water for washing the cane, in a continuous re-inoculation of the plant with yeasts. Furthermore, the yeast population is formed in the vinasse by the addition of wash water into the treatment ponds and then reintroduced to the culture fields by fertigation, so that the process can be renewed in the following season. It is now possible to adopt sanitation procedures that can prevent the entry of the contamination to the fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of the yeast Dekkera bruxellensis is sometimes attributed to a decline in the industrial productivity of ethanol since it has a more limited fermentation capacity than Saccharomyces cerevisiae. Although its adaptability to the industrial environment has been noted, so far, there has been no evidence to determine the source of this contamination. In this study, we provide evidence to show that D. bruxellensis comes from the fields together with the harvested cane and is then accumulated and recirculated. It might be possible to prevent the accumulation of this yeast by carrying out sanitation controls during the harvesting season.

Participation of CWI, HOG and Calcineurin pathways in the tolerance of Saccharomyces cerevisiae to low pH by inorganic acid.

AIMS: The present work aimed at identifying the metabolic response to acid stress and the mechanisms that lead to cell tolerance and adaptation. METHODS AND RESULTS: Two strategies were used: screening deletion mutants for cell growth at neutral and acid pH compared to wild type and measurement by qPCR of the expression of yeast genes involved in different pathways. CONCLUSIONS: The results complement our previous findings and showed that the Cell Wall Integrity pathway is the main mechanism for cell tolerance to acid pH, and this damage triggers the protein kinase C (PKC) pathway mainly via the Wsc1p membrane sensor. In addition, cell wall injury might mimic the effects of high osmotic shock and activates the High Osmolarity Glycerol pathway, which amplifies the signal in the upper part of PKC pathway and leads to the activation of Ca(2+) channels by SLT2 overexpression and this Ca(2+) influx further activates calcineurin. Together, these mechanisms induce the expression of genes involved in cell cycle regulation and cell wall regeneration. SIGNIFICANCE AND IMPACT OF THE STUDY: These interactions are responsible for long-term adaptation of yeast cells to the acidic environment, and the results could drive future work on the genetic modification of yeast strains for high tolerance to the stresses of the bioethanol fermentation process.

Detection and identification of wild yeast contaminants of the industrial fuel ethanol fermentation process.

Monitoring for wild yeast contaminants is an essential component of the management of the industrial fuel ethanol manufacturing process. Here we describe the isolation and molecular identification of 24 yeast species present in bioethanol distilleries in northeast Brazil that use sugar cane juice or cane molasses as feeding substrate. Most of the yeast species could be identified readily from their unique amplification-specific polymerase chain reaction (PCR) fingerprint. Yeast of the species Dekkera bruxellensis, Candida tropicalis, Pichia galeiformis, as well as a species of Candida that belongs to the C. intermedia clade, were found to be involved in acute contamination episodes; the remaining 20 species were classified as adventitious. Additional physiologic data confirmed that the presence of these major contaminants cause decreased bioethanol yield. We conclude that PCR fingerprinting can be used in an industrial setting to monitor yeast population dynamics to early identify the presence of the most important contaminant yeasts.

Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation.

AIMS: To identify and characterize the main contaminant yeast species detected in fuel-ethanol production plants in Northeast region of Brazil by using molecular methods. METHODS AND RESULTS: Total DNA from yeast colonies isolated from the fermentation must of industrial alcohol plants was submitted to PCR fingerprinting, D1/D2 28S rDNA sequencing and species-specific PCR analysis. The most frequent non-Saccharomyces cerevisiae isolates were identified as belonging to the species Dekkera bruxellensis, and several genetic strains could be discriminated among the isolates. The yeast population dynamics was followed on a daily basis during a whole crop harvesting period in a particular industry, showing the potential of D. bruxellensis to grow faster than S. cerevisiae in industrial conditions, causing recurrent and severe contamination episodes. CONCLUSIONS: The results showed that D. bruxellensis is one of the most important contaminant yeasts in distilleries producing fuel-ethanol from crude sugar cane juice, specially in continuous fermentation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Severe contamination of the industrial fermentation process by Dekkera yeasts has a negative impact on ethanol yield and productivity. Therefore, early detection of D. bruxellensis in industrial musts may avoid operational problems in alcohol-producing plants.

Contaminant yeast detection in industrial ethanol fermentation must by rDNA-PCR.

AIMS: The present work focuses on the possibility to use conserved primers that amplify yeast ITS1-5.8S-ITS2 ribosomal DNA locus (rDNA) to detect the presence of non-Saccharomyces cerevisiae yeast in fermentation must of bioethanol fermentation process. METHODS AND RESULTS: Total DNA was extracted from pure or mixed yeast cultures containing different cell concentrations and different contaminant/fermenting yeast concentrations and submitted to PCR. Upon improvement of detection limits and DNA extraction protocol, must samples of distillery were checked for the presence of contaminant yeast. Contaminant rDNA bands were detected only in industrial samples during contamination episodes, but not in noncontaminated must. CONCLUSIONS: The method described here could detect the presence of contaminant yeast from industrial must in eight hours after sampling. SIGNIFICANCE AND IMPACT OF THE STUDY: The improved procedure may help to avoid severe contamination episodes at fermentation industries by decreasing the detection time from 5 days to 8 h and possible quantification of contaminant yeasts that can impose economical loss to the process.

Cloning and characterization of SmZF1, a gene encoding a Schistosoma mansoni zinc finger protein.

The zinc finger motifs (Cys2His2) are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1.

Further characterization of the yeast pso4-1 mutant: interaction with rad51 and rad52 mutants after photoinduced psoralen lesions.

The pso4-1 mutant was characterized as deficient in some types of recombination, including gene conversion, crossing over, and intrachromosomal recombination. The mode of interaction between pso4-1 and rad51 and between pso4-1 and rad52 mutants indicated that the PSO4 gene belongs to the RAD52 epistasis group for strand-break repair. Moreover, the presence of the pso4-1 mutation decreased 8-MOP-photoinduced mutagenesis of the rad51 and rad52 mutants. Complementation tests using heterozygous diploid strains showed that the pso4 protein might interact with the rad52 protein during repair of 8-mop photolesions. The pso4-1 mutant, even though defective in inter- and intra-chromosomal recombination, conserves the ability for plasmid integration of circular and linear plasmid DNA. On the other hand, similar to the rad51 mutant, pso4-1 was able to incise but did not restore high-molecular-weight DNA during the repair of cross links induced by 8-MOP plus UVA. These results, together with those of previous reports, indicate that the PSO4 gene belongs to the RAD52 DNA repair group and its product participates in the DNA rejoining step of the repair of cross-link lesions, which are crucial for induced mutagenesis and recombinogenesis.