Biosynthesis of acetylacetone inspired by its biodegradation

Area of Science:

• Biotechnology
• Metabolic Engineering
• Enzyme Engineering

Background:

• Acetylacetone is a vital industrial chemical, but conventional petrochemical synthesis is inefficient and environmentally problematic.
• Existing production methods involve multiple steps, harsh conditions, low yields, and environmental concerns.
• There is a critical need for sustainable and cost-effective biosynthetic routes for acetylacetone.

Purpose of the Study:

• To establish an intuitive biosynthetic pathway for acetylacetone production.
• To engineer Escherichia coli (E. coli) for efficient acetylacetone biosynthesis.
• To improve acetylacetone yield through enzyme engineering.

Main Methods:

• Constructed a biosynthetic pathway by reversing the acetylacetone biodegradation route.
• Engineered E. coli by overexpressing the acetylacetone-cleaving enzyme (Dke1) from Acinetobacter johnsonii.
• Utilized sequence alignment and structure analysis for site-directed mutagenesis to enhance Dke1 activity.
• Performed fed-batch fermentation under anaerobic conditions for production optimization.

Main Results:

• Successfully produced acetylacetone using engineered E. coli.
• Site-directed mutagenesis of Dke1 led to a 3.6-fold increase in acetylacetone production capacity in a double-mutant (K15Q/A60D) strain.
• Achieved a final acetylacetone concentration of 556.3 ± 15.2 mg/L under optimized fermentation conditions.

Conclusions:

• Presented the first biosynthetic pathway for acetylacetone inspired by its biodegradation.
• Demonstrated the potential of engineered E. coli for efficient acetylacetone production.
• The developed method shows promise for the large-scale, sustainable manufacturing of acetylacetone.