Browsing by Subject "Zygosaccharomyces"

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  • Gonzalez Ramos, Victor Manuel (Helsingin yliopisto, 2020)
    Yeasts are a major spoilage threat in carbonated and fermented beverages, causing considerable economic losses for the manufacturers. Dekkera bruxellensis and Zygosaccharomyces bailii are the two most common spoilage yeast in beverages due to their high tolerance towards beverage-related stress factors. For industry, early and reliable detection of contamination is necessary to minimize spoilage potential and maintain product quality. Cultivation on selective/differential media remains the main method for detection of these organisms, with incubation times from 3 to 15 days. Beverage-related stresses may generate sub-population of injured yeast cells and further delay or even prevent the detection in regular media. PCR, flow cytometry and other alternative detection methods also rely on enrichment cultivation to achieve the required sensitivity for the industry. Therefore, reduced incubation time of sample enrichment and improved detection of injured cells is crucial for a more rapid and reliable detection method. Modification of specific compounds in the culture medium composition has been reported to improve recovery of bacteria after stress. As analogue studies have not been performed on spoilage yeast, modification of the culture medium composition offers a possibility to improve the growth of injured and healthy yeast cells. The aim of this study is to reduce cultivation time required for detection of healthy and injured Dekkera bruxellensis and Zygosaccharomyces bailii cells. Initially, conditions for inducing organic acid and heat injury in D. bruxellensis, D. anomala and Z. bailii cells were studied in an artificial beverage containing basic components of soft drinks. Selective and non-selective plate cultivation and fluorescent viability stains were used to assess the level of injury. The organic acid treatments resulted in inconsistent injury of spoilage yeasts, and thus, recovery from organic acid injury could not be screened. The heat treatments resulted in consistent 1-3 log reduction of viable cell counts. Altogether, 46 potential injury-relieving or growth-enhancing supplements were screened for their effects on the growth rate and lag time of heat-treated and untreated cells in non-selective YM broth using high-throughput automated turbidometry. During individual screening, the growth of Z. bailii strains was significantly improved (p<0.05) only by supplementation with three ion sources: calcium chloride, potassium chloride, and magnesium sulphate. Synergistic effects of the three ion sources was optimized for D. bruxellensis and Z. bailii individually using surface response analysis. Optimized D. bruxellensis YM medium showed no consistent impact on healthy or heat-treated D. bruxellensis strains. On the other hand, two out of the three Z. bailii strains showed significant lag time reduction of 63-66% in untreated cells and 34% in heat-treated cells when incubated in optimized Z. bailii YM medium. The lack of differentiation between improvement of growth of untreated and heat-treated cells point to a generalized ionic deficiency in YM medium. In conclusion, the optimized Z. bailii YM medium is a promising candidate for reducing the detection time of the common spoilage yeast, but it would still require validation with additional Z. bailii strains and quality control samples. It would be also interesting to study the benefits of the medium for cultivation of other spoilage yeasts and in the presence of Z. bailii selective compounds. The information about the importance of various salts for growth of Z. bailii may also prove useful in biotechnological applications of this yeast.
  • Salumäe, Astrid (Helsingin yliopisto, 2020)
    In biotechnological protein production and metabolic engineering, regulating the expression of genes is essential. For this, expression systems composed of promoters, terminators and transcription factors are essential. So far, majority of these systems use native promoters and transcription factors. That however rises two problems: 1) these systems usually work in only a set of closely related species, 2) native regulatory components can cause unintended expression levels due to the complexity of cellular regulation. Recently, a synthetic expression system (SES) was established for a wide range of fungal species. The transcription factor used in this system comprises an activation domain that originates from a virus. However, in the field of biotechnology and especially food industry, viral DNA constructs are not favorable because of customer concerns. In this paper, plant-derived activation domains were screened in Trichoderma reesei and Pichia pastoris using mCherry as a target gene for measuring the expression levels. The best expression systems were also tested for protein production in T. reesei and P. pastoris. We tested the production of two different proteins – a bacterial xylanase and a phytase. Two of the novel activation domains provided similar expression levels to the viral activation domain in both fungi. In addition, we developed optimized expression systems for an unconventional yeast from Zygosaccharomyces spp. using the novel transcription factors. The best SES version was used for secretion signal sequence screening for xylanase protein production. To further improve the use of T. reesei as a production host, the CRISPR-Cas9 system with the Cas9 D10A nickase version was tested for transformation of T. reesei. Here, we demonstrated the genomic integration and expression of Cas9 D10A nickase in T. reesei using the SES system with the novel plant-derived activation domain. Furthermore, we successfully transformed the T. reesei Cas9 D10A nickase expressing strain using only guide-RNAs and a donor DNA.