Multiple Lignocellulosic Inhibitor-Tolerant Saccharomyces cerevisiae Strains Developed by Evolutionary Engineering...

Area of Science:

• Biotechnology
• Microbial Engineering
• Synthetic Biology

Background:

• Saccharomyces cerevisiae is a key organism for industrial fermentation.
• Lignocellulosic biomass presents challenges due to inhibitory compounds like furfural, acetic acid, and vanillin.
• Enhancing yeast tolerance to these inhibitors is crucial for efficient biofuel production.

Purpose of the Study:

• To develop Saccharomyces cerevisiae strains with improved tolerance to lignocellulosic inhibitors.
• To investigate the mechanisms underlying enhanced stress resistance.
• To improve ethanol conversion rates from lignocellulosic feedstocks.

Main Methods:

• Continuous culture of Saccharomyces cerevisiae CEN.PK113-7D for 80 days with lignocellulosic inhibitors.
• CRISPR/Cas9 gene editing to create a double mutant (Rad18 and Gcn1).
• Analysis of stress tolerance, ethanol conversion rates, and underlying molecular mechanisms (enzyme activity, metabolite levels).

Main Results:

• An evolved strain (CEN.PK113-AL80-4) showed a 12-hour reduction in lag phase and a 17% increase in ethanol conversion rate.
• A double mutant strain (RG) exhibited significant ethanol production (5.88 ± 0.28 g/L) under stress, while the original strain could not grow.
• Mechanisms involved increased catalase and superoxide dismutase activity, higher intracellular glycerol, and strengthened carbon metabolism and oxidative stress responses.

Conclusions:

• Engineered Saccharomyces cerevisiae strains demonstrate robust tolerance to lignocellulosic inhibitors.
• Rad18 and Gcn1 genes play critical roles in enhancing stress tolerance and metabolic efficiency.
• This research provides a foundation for developing superior yeast strains for lignocellulosic biomass valorization.