Elucidation of weak organic acid resistance mechanisms in non-Saccharomyces yeast: a case study of Zygosaccharomyces parabailii and Kluyveromyces marxianus

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Kuanyshev, Nurzhan
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University College Cork
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The efficient implementation of biorefineries to produce bio-based chemicals and fuels requires sustainable source of feedstock and robust microbial factories. Among others, lignocellulose and whey, which are residual wastes deriving from wood/agriculture and dairy industries, represent cheap, sugar-enriched feedstocks. The conversion of lignocellulose and whey into the desired products using microbial cell factories is a promising option to replace the fossil based petrochemical refinery. Different bacteria, algae and yeasts are currently used as microbial hosts, and their number is predicted to increase over next years. Minimum nutritional requirements and robustness have made yeasts a class of microbial hosts widely employed in industrial biotechnology, exploiting their natural abilities as well as genetically acquired pathways for production of natural and recombinant products, including bulk chemicals such as organic acids. However, efficient and economically viable production of organic acids has to face problems related to low productivity/titer and toxicity of the final product. Therefore, the exploration of yeast biodiversity to exploit unique native features and the understanding of mechanisms to endure harsh conditions are essential to develop ultraefficient and robust industrial yeast with novel properties. The aim of the research thesis is to evaluate the mechanism of weak acid stress response in the non-Saccharomyces yeasts Zygosaccharomyces parabailii and Kluyveromyces marxianus. To better understand the weak acid stress response of Z. parabailii, we summarized recent finding on the species. Knowing the relevant scientific reports, the next study was focused on the effect of lactic acid stress on Z. parabailii. This organic acid can be used as monomer for the production of biodegradable bioplastic polymers, such as poly lactic acid (PLA). The study revealed that cells are able to tolerate 40g/l of lactic acid without inducing a lag phase of growth and exhibit a negligible percentage of dead cells. More importantly, during lactic acid exposure, we observed structural modifications at the level of cell wall and membrane. These findings confirmed the peculiar ability of Z. parabailii to adapt to weak organic acids via remodeling of cellular components. The lack of a complete genome assembly and annotation encouraged us to perform a genome sequencing and genome study of our Z. parabailii strain. The results revealed that Z. parabailii is undergoing fertility restoration after interspecies hybridization event, which may shed a light to the process of whole genome duplication. The availability of Z. parabailii complete genome information allowed us to perform the first RNA-sequencing analysis on the species exposed to lactic acid stress. The results showed upregulation of mitochondrial and oxidative stress genes, and downregulation of a subset of cell wall genes, in addition to other specific regulation related to redox balance and ion homeostasis. Remarkably, several differentially regulated genes differ significantly from the S. cerevisiae counterpart or, in some cases, even seem not to have a homologue. Increased interest of K. marxianus application in industrial biotechnology led us to study its multidrug resistance transporters during acetic and lactic acid stress, the first being a contaminant related to the use of lignocellulose as feedstocks, while the second as final product of interest, as mentioned above. The results showed a strain-specific response to weak organic acid stress, and a possible involvement of KmPDR12 in acetic and lactic acid stress resistance, opening potential for future discoveries and novel studies. Overall, this work contributes to the vast array of studies that are shedding light on yeasts biodiversity, both as a way for understanding their natural potential and as an instrument for tailoring novel cell factories.
Zygosaccharomyces parabailii , Kluyveromyces marxianus , FTIR , RNA-seq , PDR12 , Biorefinery , Acetic acid , Lactic acid , Hybrid , Fertility restoration
Kuanyshev, N. 2017. Elucidation of weak organic acid resistance mechanisms in non-Saccharomyces yeast: a case study of Zygosaccharomyces parabailii and Kluyveromyces marxianus. PhD Thesis, University College Cork.
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