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Title: Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression

Abstract

Medium-chain fatty acids are commodity chemicals. Increasing and modifying the activity of thioesterases (TEs) on medium-chain fatty acyl-acyl carrier protein (acyl-ACP) esters may enable a high-yield microbial production of these molecules. The plant Cuphea palustris harbors two distinct TEs: C. palustris FatB1 ( CpFatB1) (C 8 specificity, lower activity) and Cp FatB2 (C 14 specificity, higher activity) with 78% sequence identity. We combined structural features from these two enzymes to create several chimeric TEs, some of which showed nonnatural fatty acid production as measured by an enzymatic assay and gas chromatography-mass spectrometry (GC-MS). Substantially, chimera 4 exhibited an increased C 8 fatty acid production in correlation with improved microbial expression. This chimera led us to identify CpFatB2-specific amino acids between positions 219 and 272 that lead to higher protein levels. Chimera 7 produced a broad range of fatty acids and appeared to combine a fatty acid binding pocket with long-chain specificity and an ACP interaction site that may activate fatty acid extrusion. Using homology modeling and in silico docking with ACP, we identified a “positive patch” within amino acids 162 to 218, which may direct the ACP interaction and regulate access to short-chain fatty acids. On the basis of thismore » modeling, we transplanted putative ACP interaction sequences from CpFatB1 into CpFatB2 and created a chimeric thioesterase that produced medium-chain as well as long-chain fatty acids. Hence, the engineering of chimeric enzymes and characterizing their microbial activity and chain-length specificity suggested mechanistic insights into TE functions and also generated thioesterases with potentially useful properties. These observations may inform a rational engineering of TEs to allow alkyl chain length control. IMPORTANCE: Medium-chain fatty acids are important commodity chemicals. These molecules are used as plastic precursors and in shampoos and other detergents and could be used as biofuel precursors if production economics were favorable. Hydrocarbon-based liquid fuels must be optimized to have a desired boiling point, low freezing point, low viscosity, and other physical characteristics. Furthermore, the solubility and harshness of detergents and the flexibility of plastic polymers can be modulated. The length and distribution of the carbon chains in the hydrophobic tails determine these properties. The biological synthesis of cell membranes and fatty acids produces chains of primarily 16 to 18 carbons, which give rise to current biofuels. The ultimate goal of the work presented here is to engineer metabolic pathways to produce designer molecules with the correct number of carbons in a chain, so that such molecules could be used directly as specialty commodity chemicals or as fuels after minimal processing.« less

Authors:
ORCiD logo [1];  [1];  [2];  [2];  [2];  [1];  [2];  [3]
  1. Harvard Medical School, Boston, MA (United States). Wyss Institute for Biologically Inspired Engineering and Dept. of Systems Biology
  2. Harvard Medical School, Boston, MA (United States). Wyss Institute for Biologically Inspired Engineering
  3. Goethe Univ., Frankfurt (Germany)
Publication Date:
Research Org.:
Harvard College, Boston, MA (United States)
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
OSTI Identifier:
1503630
Grant/Contract Number:  
AR0000079
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 84; Journal Issue: 10; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; protein engineering; chimeric enzymes; octanoic acid

Citation Formats

Ziesack, Marika, Rollins, Nathan, Shah, Aashna, Dusel, Brendon, Webster, Gordon, Silver, Pamela A., Way, Jeffrey C., and Müller, Volker. Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression. United States: N. p., 2018. Web. doi:10.1128/aem.02868-17.
Ziesack, Marika, Rollins, Nathan, Shah, Aashna, Dusel, Brendon, Webster, Gordon, Silver, Pamela A., Way, Jeffrey C., & Müller, Volker. Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression. United States. doi:10.1128/aem.02868-17.
Ziesack, Marika, Rollins, Nathan, Shah, Aashna, Dusel, Brendon, Webster, Gordon, Silver, Pamela A., Way, Jeffrey C., and Müller, Volker. Fri . "Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression". United States. doi:10.1128/aem.02868-17. https://www.osti.gov/servlets/purl/1503630.
@article{osti_1503630,
title = {Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression},
author = {Ziesack, Marika and Rollins, Nathan and Shah, Aashna and Dusel, Brendon and Webster, Gordon and Silver, Pamela A. and Way, Jeffrey C. and Müller, Volker},
abstractNote = {Medium-chain fatty acids are commodity chemicals. Increasing and modifying the activity of thioesterases (TEs) on medium-chain fatty acyl-acyl carrier protein (acyl-ACP) esters may enable a high-yield microbial production of these molecules. The plant Cuphea palustris harbors two distinct TEs: C. palustris FatB1 (CpFatB1) (C8 specificity, lower activity) and Cp FatB2 (C14 specificity, higher activity) with 78% sequence identity. We combined structural features from these two enzymes to create several chimeric TEs, some of which showed nonnatural fatty acid production as measured by an enzymatic assay and gas chromatography-mass spectrometry (GC-MS). Substantially, chimera 4 exhibited an increased C8 fatty acid production in correlation with improved microbial expression. This chimera led us to identify CpFatB2-specific amino acids between positions 219 and 272 that lead to higher protein levels. Chimera 7 produced a broad range of fatty acids and appeared to combine a fatty acid binding pocket with long-chain specificity and an ACP interaction site that may activate fatty acid extrusion. Using homology modeling and in silico docking with ACP, we identified a “positive patch” within amino acids 162 to 218, which may direct the ACP interaction and regulate access to short-chain fatty acids. On the basis of this modeling, we transplanted putative ACP interaction sequences from CpFatB1 into CpFatB2 and created a chimeric thioesterase that produced medium-chain as well as long-chain fatty acids. Hence, the engineering of chimeric enzymes and characterizing their microbial activity and chain-length specificity suggested mechanistic insights into TE functions and also generated thioesterases with potentially useful properties. These observations may inform a rational engineering of TEs to allow alkyl chain length control. IMPORTANCE: Medium-chain fatty acids are important commodity chemicals. These molecules are used as plastic precursors and in shampoos and other detergents and could be used as biofuel precursors if production economics were favorable. Hydrocarbon-based liquid fuels must be optimized to have a desired boiling point, low freezing point, low viscosity, and other physical characteristics. Furthermore, the solubility and harshness of detergents and the flexibility of plastic polymers can be modulated. The length and distribution of the carbon chains in the hydrophobic tails determine these properties. The biological synthesis of cell membranes and fatty acids produces chains of primarily 16 to 18 carbons, which give rise to current biofuels. The ultimate goal of the work presented here is to engineer metabolic pathways to produce designer molecules with the correct number of carbons in a chain, so that such molecules could be used directly as specialty commodity chemicals or as fuels after minimal processing.},
doi = {10.1128/aem.02868-17},
journal = {Applied and Environmental Microbiology},
issn = {0099-2240},
number = 10,
volume = 84,
place = {United States},
year = {2018},
month = {3}
}

Journal Article:
Free Publicly Available Full Text
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Cited by: 1 work
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Figures / Tables:

FIG 1 FIG 1: FatB-type thioesterase (TE) biochemistry. (a) Synthetic biochemical pathway integrating plant-derived TEs that have different chain length specificities with bacterial metabolism. During E. coli fatty acid biosynthesis, the hydrocarbon chains are tethered to an acyl carrier protein (ACP). Fab enzymes elongate the fatty acid chain, through sequential malonyl-ACP condensation,more » reduction, dehydration, and further reduction. Heterologous TEs (CpFatB1 and CpFatB2) interrupt this elongation cycle by hydrolyzing fatty acids of different chain lengths from the ACP. (b) Ribbon model of TE dimer (PDB accession no. 2OWN). Numbering of residues corresponds to that in Fig. 2, based on an alignment of 2OWN, CpFatB1, and CpFatB2 (see Fig. S1 in the supplemental material). Each monomer (light and dark gray) exhibits the characteristic hotdog fold of two α-helices partially wrapped in a β-sheet. The proposed fatty acid pocket is indicated by an electron density that was identified in the crystal structure (space filling, orange). Side chains are shown for the putative catalytic residues Asp219, Asn221, His223, and Glu257 (orange side chains) (13), as are the key positive patch residues Arg165, Arg166, Arg192, Arg195, and Arg253 (green), which may be involved in ACP binding (18). The CpFatB2-specific amino acid deletion (red) is also highlighted.« less

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