Combination of type II fatty acid biosynthesis enzymes and thiolases supports a functional β-oxidation reversal

James M. Clomburg, Stephanie C. Contreras, Alexander Chou, Justin Siegel, Ramon Gonzalez

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

An engineered reversal of the β-oxidation cycle (r-BOX) and the fatty acid biosynthesis (FAB) pathway are promising biological platforms for advanced fuel and chemical production in part due to their iterative nature supporting the synthesis of various chain length products. While diverging in their carbon-carbon elongation reaction mechanism, iterative operation of each pathway relies on common chemical conversions (reduction, dehydration, and reduction) differing only in the attached moiety (acyl carrier protein (ACP) in FAB vs Coenzyme A in r-BOX). Given this similarity, we sought to determine whether FAB enzymes can be used in the context of r-BOX as a means of expanding available r-BOX components with a ubiquitous set of well characterized enzymes. Using enzymes from the type II FAB pathway (FabG, FabZ, and FabI) in conjunction with a thiolase catalyzing a non-decarboxylative condensation, we demonstrate that FAB enzymes support a functional r-BOX. Pathway operation with FAB enzymes was improved through computationally directed protein design to develop FabZ variants with amino acid substitutions designed to disrupt hydrogen bonding at the FabZ-ACP interface and introduce steric and electrostatic repulsion between the FabZ and ACP. FabZ with R126W and R121E substitutions resulted in improved carboxylic acid and alcohol production from one- and multiple-turn r-BOX compared to the wild-type enzyme. Furthermore, the ability for FAB enzymes to operate on functionalized intermediates was exploited to produce branched chain carboxylic acids through an r-BOX with functionalized priming. These results not only provide an expanded set of enzymes within the modular r-BOX pathway, but can also potentially expand the scope of products targeted through this pathway by operating with CoA intermediates containing various functional groups.

Original languageEnglish (US)
Pages (from-to)11-19
Number of pages9
JournalMetabolic Engineering
Volume45
DOIs
StatePublished - Jan 1 2018

Fingerprint

Biosynthesis
Fatty acids
Catalyst supports
Fatty Acids
Enzymes
Oxidation
Acyl Carrier Protein
Coenzyme A
Carboxylic Acids
Carboxylic acids
Substitution reactions
Carbon
Coenzymes
Amino Acid Substitution
Hydrogen Bonding
Static Electricity
Dehydration
Chain length
Functional groups
Amino acids

Keywords

  • Fatty acid biosynthesis
  • Fuels and chemicals
  • Metabolic engineering
  • Synthetic biology
  • β-oxidation reversal

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Combination of type II fatty acid biosynthesis enzymes and thiolases supports a functional β-oxidation reversal. / Clomburg, James M.; Contreras, Stephanie C.; Chou, Alexander; Siegel, Justin; Gonzalez, Ramon.

In: Metabolic Engineering, Vol. 45, 01.01.2018, p. 11-19.

Research output: Contribution to journalArticle

Clomburg, James M. ; Contreras, Stephanie C. ; Chou, Alexander ; Siegel, Justin ; Gonzalez, Ramon. / Combination of type II fatty acid biosynthesis enzymes and thiolases supports a functional β-oxidation reversal. In: Metabolic Engineering. 2018 ; Vol. 45. pp. 11-19.
@article{aecf1a77274b4c66ba218ab1b69e16f7,
title = "Combination of type II fatty acid biosynthesis enzymes and thiolases supports a functional β-oxidation reversal",
abstract = "An engineered reversal of the β-oxidation cycle (r-BOX) and the fatty acid biosynthesis (FAB) pathway are promising biological platforms for advanced fuel and chemical production in part due to their iterative nature supporting the synthesis of various chain length products. While diverging in their carbon-carbon elongation reaction mechanism, iterative operation of each pathway relies on common chemical conversions (reduction, dehydration, and reduction) differing only in the attached moiety (acyl carrier protein (ACP) in FAB vs Coenzyme A in r-BOX). Given this similarity, we sought to determine whether FAB enzymes can be used in the context of r-BOX as a means of expanding available r-BOX components with a ubiquitous set of well characterized enzymes. Using enzymes from the type II FAB pathway (FabG, FabZ, and FabI) in conjunction with a thiolase catalyzing a non-decarboxylative condensation, we demonstrate that FAB enzymes support a functional r-BOX. Pathway operation with FAB enzymes was improved through computationally directed protein design to develop FabZ variants with amino acid substitutions designed to disrupt hydrogen bonding at the FabZ-ACP interface and introduce steric and electrostatic repulsion between the FabZ and ACP. FabZ with R126W and R121E substitutions resulted in improved carboxylic acid and alcohol production from one- and multiple-turn r-BOX compared to the wild-type enzyme. Furthermore, the ability for FAB enzymes to operate on functionalized intermediates was exploited to produce branched chain carboxylic acids through an r-BOX with functionalized priming. These results not only provide an expanded set of enzymes within the modular r-BOX pathway, but can also potentially expand the scope of products targeted through this pathway by operating with CoA intermediates containing various functional groups.",
keywords = "Fatty acid biosynthesis, Fuels and chemicals, Metabolic engineering, Synthetic biology, β-oxidation reversal",
author = "Clomburg, {James M.} and Contreras, {Stephanie C.} and Alexander Chou and Justin Siegel and Ramon Gonzalez",
year = "2018",
month = "1",
day = "1",
doi = "10.1016/j.ymben.2017.11.003",
language = "English (US)",
volume = "45",
pages = "11--19",
journal = "Metabolic Engineering",
issn = "1096-7176",
publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Combination of type II fatty acid biosynthesis enzymes and thiolases supports a functional β-oxidation reversal

AU - Clomburg, James M.

AU - Contreras, Stephanie C.

AU - Chou, Alexander

AU - Siegel, Justin

AU - Gonzalez, Ramon

PY - 2018/1/1

Y1 - 2018/1/1

N2 - An engineered reversal of the β-oxidation cycle (r-BOX) and the fatty acid biosynthesis (FAB) pathway are promising biological platforms for advanced fuel and chemical production in part due to their iterative nature supporting the synthesis of various chain length products. While diverging in their carbon-carbon elongation reaction mechanism, iterative operation of each pathway relies on common chemical conversions (reduction, dehydration, and reduction) differing only in the attached moiety (acyl carrier protein (ACP) in FAB vs Coenzyme A in r-BOX). Given this similarity, we sought to determine whether FAB enzymes can be used in the context of r-BOX as a means of expanding available r-BOX components with a ubiquitous set of well characterized enzymes. Using enzymes from the type II FAB pathway (FabG, FabZ, and FabI) in conjunction with a thiolase catalyzing a non-decarboxylative condensation, we demonstrate that FAB enzymes support a functional r-BOX. Pathway operation with FAB enzymes was improved through computationally directed protein design to develop FabZ variants with amino acid substitutions designed to disrupt hydrogen bonding at the FabZ-ACP interface and introduce steric and electrostatic repulsion between the FabZ and ACP. FabZ with R126W and R121E substitutions resulted in improved carboxylic acid and alcohol production from one- and multiple-turn r-BOX compared to the wild-type enzyme. Furthermore, the ability for FAB enzymes to operate on functionalized intermediates was exploited to produce branched chain carboxylic acids through an r-BOX with functionalized priming. These results not only provide an expanded set of enzymes within the modular r-BOX pathway, but can also potentially expand the scope of products targeted through this pathway by operating with CoA intermediates containing various functional groups.

AB - An engineered reversal of the β-oxidation cycle (r-BOX) and the fatty acid biosynthesis (FAB) pathway are promising biological platforms for advanced fuel and chemical production in part due to their iterative nature supporting the synthesis of various chain length products. While diverging in their carbon-carbon elongation reaction mechanism, iterative operation of each pathway relies on common chemical conversions (reduction, dehydration, and reduction) differing only in the attached moiety (acyl carrier protein (ACP) in FAB vs Coenzyme A in r-BOX). Given this similarity, we sought to determine whether FAB enzymes can be used in the context of r-BOX as a means of expanding available r-BOX components with a ubiquitous set of well characterized enzymes. Using enzymes from the type II FAB pathway (FabG, FabZ, and FabI) in conjunction with a thiolase catalyzing a non-decarboxylative condensation, we demonstrate that FAB enzymes support a functional r-BOX. Pathway operation with FAB enzymes was improved through computationally directed protein design to develop FabZ variants with amino acid substitutions designed to disrupt hydrogen bonding at the FabZ-ACP interface and introduce steric and electrostatic repulsion between the FabZ and ACP. FabZ with R126W and R121E substitutions resulted in improved carboxylic acid and alcohol production from one- and multiple-turn r-BOX compared to the wild-type enzyme. Furthermore, the ability for FAB enzymes to operate on functionalized intermediates was exploited to produce branched chain carboxylic acids through an r-BOX with functionalized priming. These results not only provide an expanded set of enzymes within the modular r-BOX pathway, but can also potentially expand the scope of products targeted through this pathway by operating with CoA intermediates containing various functional groups.

KW - Fatty acid biosynthesis

KW - Fuels and chemicals

KW - Metabolic engineering

KW - Synthetic biology

KW - β-oxidation reversal

UR - http://www.scopus.com/inward/record.url?scp=85034771851&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85034771851&partnerID=8YFLogxK

U2 - 10.1016/j.ymben.2017.11.003

DO - 10.1016/j.ymben.2017.11.003

M3 - Article

C2 - 29146470

AN - SCOPUS:85034771851

VL - 45

SP - 11

EP - 19

JO - Metabolic Engineering

JF - Metabolic Engineering

SN - 1096-7176

ER -