Optimization of an acetate reduction pathway for producing cellulosic ethanol by engineered yeast

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Xylose fermentation by engineered Saccharomyces cerevisiae using xylose reductase (XR) and xylitol dehydrogenase (XDH) is limited by redox imbalance, particularly under anaerobic conditions.
• The cofactor difference between XR (NADPH-dependent) and XDH (NAD+-dependent) creates a metabolic bottleneck.
• Acetic acid in cellulosic hydrolysates inhibits yeast fermentation and reduces ethanol yield.

Purpose of the Study:

• To enhance ethanol yield from xylose fermentation by optimizing an NADH-dependent acetate reduction pathway coupled with xylose metabolism.
• To achieve simultaneous co-utilization of xylose and acetate for improved fermentation efficiency and in situ detoxification of acetic acid.
• To investigate the impact of acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) activities on acetate consumption and ethanol production.

Main Methods:

• Engineered Saccharomyces cerevisiae strains with varying levels of AADH and ACS activities were constructed.
• Fermentation experiments were conducted using mixtures of glucose, xylose, and acetate, as well as cellulosic hydrolysate.
• Environmental (glucose-pulse feeding) and genetic (promoter truncation, gene deletion) perturbations were employed to improve acetate consumption.

Main Results:

• The highest ethanol yield from xylose (0.463 g ethanol/g xylose) was achieved through optimization of the acetate reduction pathway.
• Engineered strains with higher AADH and ACS activities demonstrated increased acetate consumption and ethanol production.
• The optimized yeast strain (SR8A6S3) produced 18.4% more ethanol and 41.3% less glycerol and xylitol when using cellulosic hydrolysate, with significant acetate consumption.
• Environmental and genetic perturbations did not further improve acetate consumption, suggesting enzyme activity as the limiting factor.

Conclusions:

• Coupling an NADH-dependent acetate reduction pathway with xylose metabolism effectively addresses the redox imbalance in engineered yeast.
• Optimizing AADH and ACS activities is crucial for enhancing acetate reduction during xylose fermentation.
• This engineered metabolic strategy enables efficient xylose fermentation, in situ detoxification of acetic acid, and increased ethanol productivity in cellulosic hydrolysates.