Structural Insights into Novel Thiolases for Enhancing the Production of Tailored Biochemicals

Thiolase is essential in the first step of synthesizing many value-added biochemicals. However, only a few feasible THLs have been reported, and a limited understanding of these enzymes has restricted their broader application in valuable production processes. In this study, biochemical and structural analysis identified the specific properties of THLs belonging to three groups for synthesizing medium- to long-chain fatty acids, polyketide, and dicarboxylic acid, represented by adipic acid. We further demonstrated that hydrolysis activity, considered a rate-limiting step in the biosynthetic process, can be alleviated through flux balance and site-directed mutagenesis. Notably, we discovered two THLs with strong biosynthetic abilities and low hydrolysis: PaTHL4, effective for medium- to long-chain fatty acids and polyketides, and PaTHL7, tailored for adipic acid production. This study provides valuable insights into tailored THLs for specific biochemical production.