Anaerobic metabolic evolution for homotypic L-valine fermentation

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Microbial Fermentation

Background:

• L-valine is a crucial essential amino acid for animal nutrition.
• Current production methods are limited, with no known organism capable of anaerobic D-glucose to L-valine conversion as the sole product.
• Efficient and sustainable L-valine production is highly desired.

Purpose of the Study:

• To engineer a microorganism for homotypic L-valine fermentation from D-glucose under anaerobic conditions.
• To enhance metabolic flux towards L-valine synthesis in Escherichia coli.
• To overcome growth limitations and improve production rates in engineered strains.

Main Methods:

• Metabolic engineering of Escherichia coli by blocking mixed-acid fermentation and L-alanine synthesis.
• Creation of an NADH driving force to favor L-valine production.
• Directed evolution for anaerobic growth rescue and strain improvement.
• Large-scale fermentation in a 320 m³ reactor.

Main Results:

• Engineered E. coli produced 83.6 g/L of L-valine from D-glucose within 60 hours.
• Achieved a high yield of 0.55 g/g glucose, representing 85% of the theoretical maximum.
• Demonstrated a >10-fold improvement in anaerobic growth and production rate through reverse engineering.

Conclusions:

• Successfully established a novel homotypic L-valine fermentation process using engineered E. coli.
• Identified key genetic and regulatory adaptations enabling enhanced anaerobic growth and L-valine production.
• L-valine synthesis acts as a primary NADH-consuming pathway, driving fitness in anoxic conditions.