title: Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli.
abstract: The alkane-1-monoxygenase (alkB) complex of Pseudomonas putida GPo1 has been extensively studied in the past and shown to be capable of oxidising aliphatic C(5)-C(12) alkanes to primary alcohols both in the wild-type organism by growth on C(5)-C(12) alkanes as sole carbon source and in vitro. Despite this, successful n-dodecane oxidation for the production of 1-dodecanol or dodecanoic acid has proven elusive in the past when using alkB-expressing recombinants. This article demonstrates, for the first time in vivo, by using the Escherichia coli GEC137 pGEc47ΔJ strain, that n-dodecane oxidation using this enzyme for the production of primary alcohols and carboxylic acids is feasible and in fact potentially more promising than n-octane oxidation due to lower product and substrate toxicity. Yields are reported of 1-dodecanol of up to 2 g/L(organic) and dodecanoic acid up to 19.7 g/L(organic) in a 2 L stirred tank reactor with 1L aqueous phase and 200 mL of n-dodecane as a second phase. The maximum volumetric rate of combined alcohol and acid production achieved was 1.9 g/L(organic)/h (0.35 g/L(total)/h). The maximum specific activity of combined alcohol and acid production was 7-fold lower on n-dodecane (3.5 μmol/min/g(dcw)) than on n-octane (21 μmol/min/g(dcw)); similar to the 5-fold difference observed between wild-type growth rates using the two respective alkanes as sole carbon source. Despite this, both total volumetric rate and final yield exceeded n-octane oxidation by 3.5-fold under the same conditions, due to the lower toxicity of n-dodecane and its oxidation products to E. coli compared to the 8-carbon equivalents. Substrate access limitations and the overoxidation of 1-dodecanol to dodecanoic acid were identified as the most important limitations to be addressed.
authors: Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. christopher.grant@ucl.ac.uk
authors: Chris
authors: C
authors: Grant
authors: John M
authors: JM
authors: Woodley
authors: Frank
authors: F
authors: Baganz
mesh_terms: D020003:1-Octanol / Q000378:metabolism; D000438:Alcohols / Q000378:metabolism*; D000473:Alkanes / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002264:Carboxylic Acids / Q000378:metabolism*; D002384:Catalysis; D042926:Cytochrome P-450 CYP4A / Q000235:genetics / Q000378:metabolism*; D007851:Dodecanol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005285:Fermentation; D005798:Genes, Bacterial; D007850:Lauric Acids / Q000378:metabolism; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D000438:Alcohols; D000473:Alkanes; D001426:Bacterial Proteins; D002264:Carboxylic Acids; D007850:Lauric Acids; D011993:Recombinant Fusion Proteins; C030358:lauric acid; C007548:n-dodecane; D007851:Dodecanol; D042926:Cytochrome P-450 CYP4A; D020003:1-Octanol