Kluyveromyces marxianus developing ethanol tolerance during adaptive evolution with significant improvements of...

Area of Science:

• Biotechnology
• Microbial Physiology
• Synthetic Biology

Background:

• Kluyveromyces marxianus is a highly promising microorganism for bioethanol production due to its rapid growth, thermotolerance, and ability to utilize diverse agricultural residues.
• Current limitations in K. marxianus industrial application stem from its poor ethanol tolerance, necessitating strategies to enhance its resistance and understand the underlying mechanisms.

Purpose of the Study:

• To improve the ethanol tolerance of Kluyveromyces marxianus through adaptive laboratory evolution.
• To elucidate the molecular mechanisms, particularly transcriptional changes, responsible for enhanced ethanol resistance in K. marxianus.
• To assess the impact of improved ethanol tolerance on bioethanol production under industrial conditions.

Main Methods:

• Adaptive evolution of wild-type haploid K. marxianus FIM1 in a medium containing 6% (v/v) ethanol for 100 days.
• DNA analysis and RNA-sequencing (RNA-seq) to identify genetic and transcriptional changes in the evolved strain (KM-100d).
• Cell viability tests to confirm resistance to various stresses (ethanol, osmotic, oxidative, thermal).
• Measurement of ethanol production by the evolved strain compared to the wild-type.

Main Results:

• The evolved KM-100d population exhibited increased ethanol tolerance, rising from 6% to 10% (v/v).
• Enhanced ethanol tolerance was attributed to genome-wide transcriptional reprogramming rather than ploidy changes or significant mutations.
• Key upregulated pathways in KM-100d included ethanol consumption, membrane lipid biosynthesis, anti-osmotic pressure, anti-oxidative stress, and protein folding.
• KM-100d developed enhanced secretory pathway activity for osmotic stress resistance, distinct from glycerol production in Saccharomyces cerevisiae.
• The evolved strain demonstrated cross-tolerance to osmotic, oxidative, and thermic stresses and significantly improved ethanol production.

Conclusions:

• Ethanol-driven laboratory evolution effectively enhances K. marxianus ethanol tolerance via upregulation of multiple stress-resistance pathways.
• The improved strain exhibits increased ethanol yield under high-temperature and high-ethanol conditions, making it more suitable for industrial bioethanol production.
• Findings provide genetic insights for rational optimization of K. marxianus for bioethanol production and support a positive correlation between ethanol tolerance and production.