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. 2022 Jan 11:9:799756.
doi: 10.3389/fbioe.2021.799756. eCollection 2021.

Meiosis-Based Laboratory Evolution of the Thermal Tolerance in Kluyveromyces marxianus

Li Wu  1   2 , Yilin Lyu  1   2 , Pingping Wu  1   2 , Tongyu Luo  1   2 , Junyuan Zeng  1   2 , Tianfang Shi  1   2 , Jungang Zhou  1   2 , Yao Yu  1   2 , Hong Lu  1   2   3
Affiliations

Meiosis-Based Laboratory Evolution of the Thermal Tolerance in Kluyveromyces marxianus

Li Wu et al. Front Bioeng Biotechnol. .

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.

Keywords: Kluyveromyces marxianus; PDE2; PSR1; iterative cycle; laboratory evolution; meiosis; thermal tolerance.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The meiosis-based evolution of the thermotolerance in K. marxianus. (A) Switches of mating type in K. marxianus. (B) Constructions of stable MATa and MATα cells. (C) Constructions of diploid, triploid and tetraploid cells. (D) Spot assay of FIM1, KM-Diploid, KM-Triploid and KM-Tetraploid strains at different temperatures. (E) Spot assay of spores generated by KM-Diploid, KM-Triploid and KM-Tetraploid at different temperatures. (F) Spot assay of spores generated by 2–2 and 3–2 at different temperatures. (G, H) Survival rates of spores generated in the first (G) and second (H) rounds of the screen at different temperatures.
FIGURE 2
FIGURE 2
Comparison of the thermotolerant spores generated by diploid and triploid strains. (A, B) Spot assays of the thermotolerant spores generated by diploid strains (A) and triploid strains (B). (C, D) The growth curves of the thermotolerant spores generated by diploid (C) and triploid strains (D) at 47°C. Values here and below represented mean ± SD (n = 3). (E) The maximum growth rate of representative spores generated by diploid and triploid strains at 47°C. (F) OD600 of representative spores at 72 h. The significant difference was measured by a Student’s t-test. *p < 0.05, **p < 0.01.
FIGURE 3
FIGURE 3
Spot assays of FIM1 carrying the deletion of PSR1, PDE2 or a combination of both deletions. FIM1, Dip2-8 and Tri2-8 were spotted as controls.
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