L-malate production by metabolically engineered Escherichia coli

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biotechnology

Background:

• Previous work engineered Escherichia coli for succinate production.
• This study aimed to redirect carbon flux towards malate production in engineered E. coli.

Purpose of the Study:

• To engineer Escherichia coli strains for efficient malate production.
• To investigate and overcome metabolic challenges, such as D-lactate accumulation, during malate biosynthesis.

Main Methods:

• Genetic modification of E. coli, including gene deletions (fumarase isoenzymes, malic enzymes, pyruvate kinase).
• Metabolic flux analysis to understand carbon flow redirection.
• Two-stage fermentation process (aerobic growth, anaerobic production) for optimizing malate yield and productivity.

Main Results:

• A single mutation in fumarate reductase redirected carbon flow to malate, despite fumarase activity.
• Unexpected D-lactate accumulation occurred, influenced by pyruvate metabolism.
• The engineered strain XZ658 achieved 1.0 mol malate/mol glucose under anaerobic conditions.
• A two-stage process yielded 253 mM malate (34 g/L) in 72 hours with 1.42 mol/mol yield and 0.47 g/L/h productivity.

Conclusions:

• Metabolic engineering strategies for malate production in E. coli require careful consideration of competing pathways and by-product formation.
• A two-stage fermentation process significantly enhances malate yield and productivity, achieving levels competitive with existing biocatalysts.