Mutants with Enhanced Multi-Stress Tolerance of Kluyveromyces marxianus and Their Ability for Ethanol Fermentation

Kluyveromyces marxianus is an attractive thermotolerant yeast species for ethanol production because of its ability to utilize various carbon sources as a fermentation substrate. The use of thermotolerant microorganisms enables the performance of high-temperature ethanol fermentation, which has several advantages, including the reduction of cooling costs and minimization of contamination risks. To improve K. marxianus for ethanol fermentation under stress conditions, two strains, DMKU 3-1042 and DMKU 3-118, were adapted for heat resistance and resistance to toxic substances in pulp wastewater from a paper mill, respectively, resulting in the generation of KMR1042 and KMR118, respectively. Both adapted mutants exhibited clumpy clusters of cells as pseudo-hyphae and altered colony morphology, and their sedimentation speeds were much faster than those of the corresponding parent strains. The two mutants showed stronger tolerance to various stresses and higher performance for ethanol production than those of the corresponding parent strains at high temperatures or in the presence of toxic substances. Genome sequencing analysis revealed that both mutants had disruption of the same gene, SWI5, despite adaptation under different stress conditions, suggesting that the formation of pseudo-hyphae is a common strategy of K. marxianus for coping with stresses.