Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

Area of Science:

• Biotechnology
• Synthetic Biology
• Microbial Engineering

Background:

• Efficient fermentation of cellulosic sugars is crucial for biofuels and chemicals.
• Glucose repression limits Saccharomyces cerevisiae's fermentation of cellulosic sugar mixtures.
• Engineered yeast with cellobiose transporter (cdt-1) and intracellular β-glucosidase (gh1-1) can coferment sugars, alleviating glucose repression.

Purpose of the Study:

• To enhance cellobiose fermentation rates in engineered yeast.
• To identify the mechanisms behind improved cellobiose fermentation through laboratory evolution.
• To explore gene amplification as a metabolic engineering strategy.

Main Methods:

• Laboratory evolution of engineered yeast strains.
• Serial subculturing on cellobiose media.
• Genome sequencing to identify genetic modifications.
• Quantitative analysis of gene copy numbers.

Main Results:

• Evolved yeast strains exhibited a 15-fold increase in cellobiose fermentation rate.
• Massive amplification of cdt-1 (9 copies) and gh1-1 (23 copies) genes was observed in evolved strains.
• A positive correlation was found between gene copy numbers and cellobiose fermentation rates.

Conclusions:

• The cellobiose assimilation pathway (transport and hydrolysis) is a rate-limiting step for efficient fermentation.
• On-demand gene amplification is a feasible and effective strategy for yeast metabolic engineering.
• This approach can optimize heterologous metabolic pathways and improve biofuel production.