Browsing by Author "Jansen, Trudy"
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- ItemExploring new Saccharomyces cerevisiae strains suitable for the production of cellulosic bioethanol(Stellenbosch : Stellenbosch University, 2019-03) Jansen, Trudy; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Natural Saccharomyces cerevisiae strains display an enhanced robustness that is associated with the environment they occupy. This robustness is expressed through complex biological networks and provides the organism with the ability to maintain cell viability during adverse environmental conditions. In this study, the natural diversity of S. cerevisiae was exploited to obtain strains with phenotypic characteristics beneficial for second-generation bioethanol production. Artificial hybridisation was employed to increase the genetic, and thus the phenotypic diversity of these strains, whereafter transcriptomics were utilised to elucidate the molecular mechanisms that allow adaptation to an increase in temperature and survival in an environment containing inhibitory compounds that are associated with the degradation of cellulosic feedstock. Our results indicated that there is an association between strain diversity, the environment and the geographic location, whilst, individual strains display phenotypes. The Cape West Coastal region was associated with inhibitor-resistant strains, whereas the Breede River valley was characterised by inhibitor-sensitive strains. Strains that displayed an increased fermentation capacity were associated with the Cape South Coast. Several strains with tolerant phenotypes i.e. the ability to grow and/or ferment under a range of environmental conditions, were identified, including a multi-tolerant strain, YI13, with growth tolerance against ethanol (15 % v/v), inhibitors (15 %) and increased temperature (45 °C). Two inhibitor tolerant (25 %) strains, HR4 and YI30, displayed improved fermentation capacity (0.22 and 0.35 g/L/h) during aerobic and anaerobic conditions, respectively. Artificial hybridisation generates genetic diversity that affects the phenotype of the organism and was applied to produce progeny strains. Several of these strains displayed inhibitor tolerance heterosis, whereas pH and salt tolerance decreased relative to the parental strains. In addition, unique phenotypes were generated, with strain HR4/YI30#6 displaying growth at 2 M NaCl and in 20 % ethanol. A single multi-tolerant strain, V3/YI30#6, with unique (2 M NaCl and 45 °C tolerance) and general (25 % inhibitor tolerance) traits was obtained. However, the fermentation capacity of this strain was decreased to a theoretical ethanol yield of 60 % compared to ~80 % for the parental strains. This indicates that although hybridisation produces heterosis and novel phenotypes, there is a limit to the degree of phenotypic diversity that can be obtained in a single strain. This may be due to the high energy demand (due to the increase in metabolic flux of certain biological processes) during the various stress responses, whilst maintaining cell viability. The molecular mechanisms for inhibitor and temperature tolerance of two natural strains were subsequently investigated. In addition to several biological processes, an upregulation of amino acid biosynthesis and ribosome biogenesis was observed in the temperature tolerant strain, YI13. This was possibly in response to irreversible protein damage inflicted by reactive oxygen species generated in response to an increase in temperature. The main contributor to inhibitor tolerance in strain YI30 was the activation of the oxidative stress response. This is probably due to the increased oxidoreductase activity required for the detoxification of the inhibitory compounds. Activation of the traditional heat shock response did not play a major role in combating temperature stress, however, an upregulation in this stress response was observed in reaction to inhibitor stress. This study indicates that the natural diversity of S. cerevisiae yields unique strains and that phenotype diversity can be enhanced through hybridisation. In addition, S. cerevisiae strains display similar mechanisms in response to environmental stress. However, the specific molecular mechanisms that allow robustness and the degree to which these stress responses are activated, are strain dependent. A single strain will not be able to display all the required characteristics for a particular process, therefore a compromise will have to be made where the characteristics of the host organism and the specific application are considered, including genetic engineering of yeast strains.