Genetic and genomic characterization of thermotolerance in Kluyveromyces marxianus

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Montini, Noemi
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University College Cork
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This thesis was carried out as part of the Horizon 2020 project CHASSY (, funded by the European Union. The CHASSY project was a multi-partner research collaboration focused on unlocking the potential of yeast as a microbial cell factory. The research scope of CHASSY included investigating the basis of stress adaptation response in various yeast species, including K. marxianus, in order to engineer robustness in industrial strains. In fact, microbial cell factories face suboptimal growth conditions during the bioprocesses that cause stress on the cell and hinder productivity. Stress responses and adaptation mechanisms are a complex phenomenon and involve numerous genes and processes in yeast. Therefore, a better understanding of these is needed in order to design more robust yeast strains and broaden their application in industrial biotechnology. In this context, K. marxianus possesses valuable physiological traits: the yeast is thermotolerant, has a rapid growth rate and is able to utilise a wide range of substrates. Particularly promising for biotechnological applications, is its innate thermotolerance, with certain strains being able to grow up to 45 °C. Since thermotolerance is desirable from an industrial standpoint due to reduced cooling cost and contamination risk, K. marxianus thermotolerance mechanisms were investigated in this study. However, since a wide physiological variability is known between K. marxianus strains, and the physiological studies available focus on different strains and conditions, a systematic physiological comparison was needed in order to efficiently compare the data. Therefore, batch cultivations in bioreactors were carried out with three strains: the haploids CBS 6556 and NBRC 1777 and the diploid CBS 397 in Chapter III. Growth characteristics of each strain were compared under two growth temperatures (30 °C and 40 °C) in order to investigate the physiological response of different K. marxianus strains to elevated temperature. NBRC 1777 was the fastest growing strain at 30 °C among the three compared, while both CBS 6556 and CBS 397 showed an almost doubled growth rate at 40 °C compared to 30 °C, indicating that higher growth temperatures are preferred by some K. marxianus strains. NBRC 1777 instead showed decreased biomass yield on substrate at 40°C and higher oxygen uptake rate, in addition to slower growth, suggesting that these parameters are linked to growth under high temperature. Subsequently, K. marxianus’ thermotolerance response was further investigated under long-term exposure to stress, since adaptation mechanisms are better observed under these premises. Specifically, the haploid strain CBS 6556 was subjected to chemostat cultivation under three industrially relevant stresses, namely: high temperature, low pH and high osmolarity, which allowed the simulation of long term stress. The analysis of the transcriptional changes in response to the three stresses revealed that, while some biological processes are involved in the response to all tested stresses, there are no genes upregulated under all three stress condition, which suggests a stress-specific response. The physiological data collected under high temperature stress revealed that the sugar uptake rate is higher in CBS 6556 under high temperature stress, likely to fulfil a higher energy demand to cope with the condition. In addition, and contrary to what might be expected, analysis of the transcriptional responses showed that the genes involved in the respiratory activity, also involved in energy generation, were downregulated. This strategy could be used by K. marxianus to control reactive oxygen species (ROS) accumulation, a phenomenon naturally occurring under high temperature stress, and contributing therefore to its innate thermotolerance. A collaborative study within CHASSY analysing K. marxianus and other yeast species’ transcriptomic data, found that evolutionarily young genes are proportionally more responsive, in yeast, to growth under stressful conditions than core genes shared by many species. In the study, it was therefore postulated that such genes could be important for long-term adaptation to stress. This hypothesis was tested in K. marxianus in regards to thermotolerance in Chapter IV by identifying and studying species-specific genes that showed upregulation during high temperature growth. Twelve such genes were identified and eleven were successfully inactivated using CRISPR-mediated mutagenesis. One gene, KLMX_70384, was shown to be required for competitive growth at high temperature. KLMX_70384 was predicted to encode an 83AA peptide and RNA-Seq and Ribo-Seq were used to confirm transcription and translation of the gene. Although the precise function of KLMX_70384 remains unknown, the generic structure and short sequence suggest RNA-binding activity. Since KLMX_70384 is among the genes that arose de novo in K. marxianus post the speciation event that separated K. marxianus and K. lactis, this confirmed the hypothesis that evolutionary young genes contribute to the unique traits of K. marxianus’, such as thermotolerance. In yeast, high temperatures trigger ROS accumulation, therefore the thermal stress response is tightly connected to the oxidative stress response. This is suggested by evidences of a crosstalk between signalling mechanisms for the two stresses. In Chapter V, a physiological and genomic comparison was carried out between two K. marxianus strains: ATCC 26548 and UCC01, the latter was derived from ATCC 26548 but displays a thermosensitive phenotype. The comparison of the two genome sequences showed a high similarity between the two strains, with only 1012 variants between the two genomes, suggesting that UCC01 spontaneously accumulated mutations which resulted in thermosensitivity. A physiological comparison via bioreactor cultivation under high temperature and different aeration levels (0.5 and 2 L/min), revealed that UCC01’s thermosensitive phenotype is linked to oxygen availability. An accumulation of ROS was measured at 45 °C in UCC01 under 2L/min aeration and, accordingly, single nucleotide polymorphisms (SNPs) analysis found five SNPs-containing genes of which four (CYC8, SWI1, BNR1 and YAK1) with functions related to oxidative and general stress response, and one unannotated gene KLMX_30726. Although individual deletion of these genes in ATCC 26548 did not reproduce a thermosensitive phenotype, deletion of CYC8 and YAK1 resulted in an oxidative stress-sensitive phenotype, confirming the existence of a crosstalk between oxidative and thermal stress responses in K. marxianus, possibly linked to its innate thermotolerance. Finally, trehalose metabolism was investigated in K. marxianus in Chapter VI, since trehalose is considered a thermoprotectant allowing yeast survival to heat stress. Recent studies also demonstrated a more complex role of trehalose, such as regulating central carbon metabolism and cell homeostasis. Knock-out of different enzymatic subunits of the trehalose synthase complex is known to generate diverse phenotypes in various yeast species. In addition, several genes of trehalose metabolism were found upregulated under long term temperature stress by differential expression (DE) analysis in K. marxianus. Here, we investigated the individual roles of TPS1 and TPS2, enzymatic subunits of the trehalose synthase complex, on K. marxianus thermotolerance by construction and analysis of knock-out mutants. The deletion of TPS1 was not found to be lethal in K. marxianus, unlike in S. cerevisiae, but yielded a thermosensitive phenotype. Moreover, deletion of TPS2 yielded a thermosensitive phenotype exclusively on galactose. The thermosensitive phenotype was found to be linked to the accumulation of trehalose-6- phosphate (T6P) under high temperature, which deregulates the galactose metabolism genes. In accordance, GAL1 and GAL7 genes were found to be upregulated, and PGM2 strongly upregulated in the Δtps2 mutant under high temperature on galactose. The study shows evidence of T6P playing a role in regulating central carbon metabolism in K. marxianus; it also reveals a previously unknown layer of metabolite repression acting on galactose genes in this yeast, linked to thermotolerance. The results presented in this thesis contribute to better the current understanding of the many mechanisms involved K. marxianus’ thermotolerance, providing a knowledge resource to further develop this yeast for cell factory applications.
Kluyveromyces marxianus , Thermotolerance , Yeast , Microbial cell factory
Montini, N. 2022. Genetic and genomic characterization of thermotolerance in Kluyveromyces marxianus. PhD Thesis, University College Cork.