Structural Insights\ninto Novel Thiolases for Enhancing\nthe Production of Tailored Biochemicals

Thiolase is essential in the first step of synthesizing\nmany value-added\nbiochemicals. However, only a few feasible THLs have been reported,\nand a limited understanding of these enzymes has restricted their\nbroader application in valuable production processes. In this study,\nbiochemical and structural analysis identified the specific properties\nof THLs belonging to three groups for synthesizing medium- to long-chain\nfatty acids, polyketide, and dicarboxylic acid, represented by adipic\nacid. We further demonstrated that hydrolysis activity, considered\na rate-limiting step in the biosynthetic process, can be alleviated\nthrough flux balance and site-directed mutagenesis. Notably, we discovered\ntwo THLs with strong biosynthetic abilities and low hydrolysis: <i>Pa</i>THL4, effective for medium- to long-chain fatty acids\nand polyketides, and <i>Pa</i>THL7, tailored for adipic\nacid production. This study provides valuable insights into tailored\nTHLs for specific biochemical production.