Rational design of thiolase substrate specificity for metabolic engineering applications

Area of Science:

• Biocatalysis and Metabolic Engineering
• Enzyme Engineering
• Synthetic Biology

Background:

• Metabolic engineering requires highly active and specific enzymes for commercial viability.
• The 3-hydroxyacid (3HA) pathway synthesizes valuable compounds but suffers from byproduct formation, reducing yields.
• Thiolase enzymes initiate the 3HA pathway, and their substrate specificity dictates product chain length.

Purpose of the Study:

• To rationally engineer thiolase substrate specificity to enhance the production of longer-chain products in the 3HA pathway.
• To increase the ratio of C6/C4 products (3-hydroxy-hexanoic acid/3-hydroxybutyric acid) synthesized via the 3HA pathway.

Main Methods:

• Employed a model-guided, rational design approach focusing on ordered substrate binding.
• Applied this design strategy to two specific biosynthetic thiolases.
• Screened and identified thiolase mutants with altered substrate specificity.

Main Results:

• Identified specific thiolase mutants that significantly enhance C6/C4 product selectivity.
• Achieved nearly 10-fold increases in the ratio of C6/C4 products formed.
• Demonstrated the effectiveness of rational design in altering enzyme specificity for metabolic engineering.

Conclusions:

• Rational engineering of thiolase substrate specificity can overcome byproduct formation in the 3HA pathway.
• The developed strategy can improve yields of desired longer-chain products, reducing downstream costs.
• Findings contribute to understanding enzyme sequence-structure-function relationships and can be applied to other chain-elongation pathways.