title: Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids.
abstract: An engineered reversal of the β-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6-10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned β-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (bktB) and thioesterase (ydiI) in combination with previously established core β-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C6-C10 carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes. Using this approach, the expression of the Pseudomonas putida alkane monooxygenase system, encoded by alkBGT, in combination with all β-oxidation reversal enzymes resulted in the production of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid. Following identification and characterization of potential alcohol and aldehyde dehydrogenases, chnD and chnE from Acinetobacter sp. strain SE19 were expressed in conjunction with alkBGT to demonstrate the synthesis of the C6-C10 dicarboxylic acids, adipic acid, suberic acid, and sebacic acid. The potential of a β-oxidation cycle with ω-oxidation termination pathways was further demonstrated through the production of greater than 0.8 g/L C6-C10 ω-hydroxyacids or about 0.5 g/L dicarboxylic acids of the same chain lengths from glycerol (an unrelated carbon source) using minimal media.
authors: Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, Houston, TX 77005, USA.
authors: James M
authors: JM
authors: Clomburg
authors: Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, Houston, TX 77005, USA.
authors: Matthew D
authors: MD
authors: Blankschien
authors: Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, Houston, TX 77005, USA.
authors: Jacob E
authors: JE
authors: Vick
authors: Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, Houston, TX 77005, USA.
authors: Alexander
authors: A
authors: Chou
authors: Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, Houston, TX 77005, USA.
authors: Seohyoung
authors: S
authors: Kim
authors: Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, Houston, TX 77005, USA; Department of Bioengineering, Rice University, 6100 Main Street, MS-142, Houston, TX 77005, USA. Electronic address: ramon.gonzalez@rice.edu.
authors: Ramon
authors: R
authors: Gonzalez
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002264:Carboxylic Acids; D003577:Cytochrome P-450 Enzyme System; D013869:Thiolester Hydrolases
keywords: Fuels and chemicals; Metabolic engineering; Synthetic biology; β-oxidation reversal; ω-oxidation