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

Journal Article · · ACS Catalysis
 [1];  [2];  [3];  [4];  [2];  [2];  [4];  [2];  [2];  [3];  [2];  [4]
  1. Pennsylvania State Univ., University Park, PA (United States). Dept. of Chemical Engineering; Department of Chemical Engineering, Pennsylvania State University
  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
  4. Pennsylvania State Univ., University Park, PA (United States). Dept. of Chemical Engineering
+ Show Author Affiliations
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 enhanced 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.
Research Organization:
Pennsylvania State Univ., University Park, PA (United States)
Sponsoring Organization:
National Institutes of Health (NIH); USDOE Advanced Research Projects Agency - Energy (ARPA-E)
Grant/Contract Number:
AR0000432
OSTI ID:
1408279
Journal Information:
ACS Catalysis, Journal Name: ACS Catalysis Journal Issue: 6 Vol. 7; ISSN 2155-5435
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
English

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Cited By (11)

PoreDesigner for tuning solute selectivity in a robust and highly permeable outer membrane pore journal September 2018
From directed evolution to computational enzyme engineering—A review journal September 2019
Fatty Acid Biosynthesis: Chain‐Length Regulation and Control journal June 2019
Microbial synthesis of medium-chain chemicals from renewables journal December 2017
Computational redesign of penicillin acylase for cephradine synthesis with high kinetic selectivity journal January 2018
Using molecular dynamics simulations to evaluate active designs of cephradine hydrolase by molecular mechanics/Poisson–Boltzmann surface area and molecular mechanics/generalized Born surface area methods journal January 2019
Advanced strategy for metabolite exploration in filamentous fungi journal January 2020
Structure-guided protein engineering increases enzymatic activities of the SGNH family esterases journal June 2020
Recent advances in high-throughput mass spectrometry that accelerates enzyme engineering for biofuel research journal February 2020
Carboxylic Ester Hydrolases in Bacteria: Active Site, Structure, Function and Application journal November 2019
Optimization of Diverse Carbon Sources and Cultivation Conditions for Enhanced Growth and Lipid and Medium-Chain Fatty Acid (MCFA) Production by Mucor circinelloides journal April 2019

Figures / Tables (33)

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Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
Dodecanoic acid
IPRO
fatty acid
molecular dynamics
octanoic acid
redesign
thioesterase
‘TesA