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 1 L 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.