Precursor balancing by reprogramming pyruvate synthesis for high-yield l-isoleucine production in engineered...

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biochemical Production

Background:

• l-isoleucine (l-Ile) is an essential branched-chain amino acid vital for food, feed, and pharmaceutical applications.
• Current microbial fermentation yields for l-Ile are suboptimal, limiting its industrial-scale production.
• Efficient synthesis requires precise control over precursor molecules, 2-oxobutyrate and pyruvate.

Purpose of the Study:

• To enhance l-isoleucine (l-Ile) yield from glucose through metabolic engineering.
• To optimize the balance between 2-oxobutyrate and pyruvate precursors for increased l-Ile production.
• To develop a robust microbial platform for efficient and cost-effective l-Ile synthesis.

Main Methods:

• Constructed a plasmid-free l-threonine (l-Thr) synthesis module to boost 2-oxobutyrate supply.
• Optimized the l-Ile synthesis pathway by modifying transport systems and enzyme expression.
• Engineered precursor balancing by deleting pykFA and utilizing the glucose PTS transport system to partition phosphoenolpyruvate.
• Attenuated TCA cycle flux and introduced leucine dehydrogenase to minimize losses and address cofactor imbalances.

Main Results:

• Achieved a high l-isoleucine yield of 0.67 mol/mol glucose and a productivity of 1.72 g/L/h.
• Significantly reduced the byproduct l-valine (l-Val) by balancing the 2-oxobutyrate/pyruvate ratio.
• Demonstrated a viable strategy for facilitating downstream purification of l-isoleucine.

Conclusions:

• The integrated metabolic engineering strategies successfully enhanced l-isoleucine production.
• Balancing precursor ratios is critical for high-yield microbial synthesis of l-Ile.
• The developed engineered strain serves as a promising cell factory for l-isoleucine and derivative production.