Abstract
Kluyveromyces marxianus is the fastest-growing eukaryote and a promising host for producing bioethanol and heterologous proteins. To perform a laboratory evolution of thermal tolerance in K. marxianus, diploid, triploid and tetraploid strains were constructed, respectively. Considering the genetic diversity caused by genetic recombination in meiosis, we established an iterative cycle of “diploid/polyploid - meiosis - selection of spores at high temperature” to screen thermotolerant strains. Results showed that the evolution of thermal tolerance in diploid strain was more efficient than that in triploid and tetraploid strains. The thermal tolerance of the progenies of diploid and triploid strains after a two-round screen was significantly improved than that after a one-round screen, while the thermal tolerance of the progenies after the one-round screen was better than that of the initial strain. After a two-round screen, the maximum tolerable temperature of Dip2-8, a progeny of diploid strain, was 3°C higher than that of the original strain. Whole-genome sequencing revealed nonsense mutations of PSR1 and PDE2 in the thermotolerant progenies. Deletion of either PSR1 or PDE2 in the original strain improved thermotolerance and two deletions displayed additive effects, suggesting PSR1 and PDE2 negatively regulated the thermotolerance of K. marxianus in parallel pathways. Therefore, the iterative cycle of “meiosis - spore screening” developed in this study provides an efficient way to perform the laboratory evolution of heat resistance in yeast.
Highlights
The mutation is the major driving force of the adaptive evolution, leading to traits in coping with various environmental stress, including heat, salinity, alkalinity, acidity and so on (Wright, 2004)
The growth of FIM1-psr1Δpde2Δ was better than that of FIM1-psr1Δ and FIM1pde2Δ at 45, 46, and 47°C (Figure 3). These results suggested that PSR1 and PDE2 were key genes that negatively regulated the thermotolerance in K. marxianus and that two genes functioned in parallel pathways
Based on the genetic diversity produced by the recombination of meiosis, we established an iterative cycle of “diploid/polyploid - meiosis - selection of spores at high temperature” to improve the thermal tolerance of K. marxianus
Summary
The mutation is the major driving force of the adaptive evolution, leading to traits in coping with various environmental stress, including heat, salinity, alkalinity, acidity and so on (Wright, 2004). Called thermal tolerance, is well-recognized as a quantitative trait controlled by a range of genes and pathways (Gao et al, 2016). Mutations of Rho1-Pkc pathway increase the thermal tolerance by affecting the integrity of the cell wall (Huang et al, 2018). Glutathione directly reduces the hydroxyl radical to form H2O, thereby removing oxygen free radicals in cells, to increase the heat resistance of cells (Grant et al, 1996). Trehalose mainly protects cells from high-temperature damage by stabilizing the cell membrane structure and maintaining the conformation of intracellular proteins (Martinez-Esparza et al, 2011)
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