skip to main content


Title: Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources to a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis, in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e., binding selectivity). We demonstrate this approach by identifying medium-chain (C8–C12) acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, which are products of thioesterase-catalyzed hydrolysis, are limited in natural abundance, compared to long-chain fatty acids; the limited supply leads to high costs of C6–C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding of the highly active ‘TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12 or C8 specificity, while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified 3 variants with enhanced production of dodecanoic acid (C12) and 27 variants with enhancedmore » production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity. Finally, we expect the design rules that we uncovered and the thioesterase variants that we identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain-length oleochemicals.« less
 [1] ;  [2] ;  [3] ;  [1] ;  [2] ;  [2] ;  [1] ;  [2] ;  [2] ;  [3] ;  [2] ; ORCiD logo [1]
  1. Pennsylvania State Univ., University Park, PA (United States). Dept. of Chemical Engineering
  2. Univ. of Wisconsin, Madison, WI (United States). Dept. of Chemical and Biological Engineering
  3. Univ. of Wisconsin, Madison, WI (United States). Dept. of Biochemistry
Publication Date:
Grant/Contract Number:
AR0000432; GM115921; GM008505
Accepted Manuscript
Journal Name:
ACS Catalysis
Additional Journal Information:
Journal Volume: 7; Journal Issue: 6; Journal ID: ISSN 2155-5435
American Chemical Society (ACS)
Research Org:
Pennsylvania State Univ., University Park, PA (United States)
Sponsoring Org:
USDOE Advanced Research Projects Agency - Energy (ARPA-E); National Institutes of Health (NIH)
Country of Publication:
United States
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Dodecanoic acid; fatty acid; IPRO; molecular dynamics; octanoic acid; redesign; thioesterase; ‘TesA
OSTI Identifier: