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Title: Evaluation of the bio-oxidation of alkanes
Author: Grant, C. R.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2012
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This thesis documents the progress made in utilising the alkane hydroxylase complex of P.putida GPo1 expressed in E.coli as a whole-cell biocatalyst for the oxidation of n-dodecane to 1-dodecanol. The process is of considerable interest due to the difficulty in performing the reaction using conventional chemistry and the large global market for fatty alcohols. The first results chapter compares the fermentative bio-oxidations using E.coli pGEc47ΔJ on n-octane and n-dodecane in a stirred tank reactor. The first reported conversion of n-dodecane in-vivo using this enzyme system in a recombinant host is reported. A number of bottlenecks were identified in this chapter; in particular, (i) poor induction of the alkS expression system with ndodecane, which controls the expression of the alk enzymes (ii) a suspected mass transport limitation (iii) substantial over-oxidation of the desired 1- dodecanol product to dodecanoic acid. The second results chapter firstly describes the development of a microwell platform in order to characterise the system more efficiently. Phase mixing limitations and organic phase spillage/evaporation were overcome in order to develop the microwell platform for the fermentative bio-oxidation which is the first reported microwell scale-down which matches the volumetric and specific rates achieved in a bioreactor for a substrate of such low solubility. Secondly, the microwell platform was used with design of experiments (DoE) methodology to rapidly and systematically characterise the overoxidation issue and identify appropriate solutions. Using this approach, substrate solubility was identified as the most critical factor affecting the tendency for overoxidation; the use of cosolvents to improve n-dodecane solubility in the aqueous phase was found to improve the 1-dodecanol yields and reduce dodecanoic acid yields. Oxygen availability and carbon source availability also proved important factors in the extent of overoxidation. Despite the improvements made the problem was only partially overcome and it was decided, based on the results, that biological engineering of the strain was necessary to remove the downstream aldehyde dehydrogenase alkH which was likely to be exacerbating overoxidation. The process of designing and constructing 3 new plasmids is described in results chapters four and five. These plasmids were designed with the aim of identifying the role of various alk proteins and ultimately identifying ways of improving substrate access to the enzyme and reducing overoxidation. It was found as a result of this work that overoxidation was reduced by removal of alkH but that the alkane-1-monooxygenase alkB was still capable of direct overoxidation to the dodecanoic acid even in the absence of alkH. More significantly, the function of an outer membrane protein of unknown function was also confirmed by this work. It was found to be essential for conversion of n-dodecane in-vivo but was also found to be toxic to the host organism when overexpressed. Finally, it was found that the alkane-1-monooxygenase enzyme system was also capable of C14 and C16 alkane oxidation; this has not previously been reported in literature in-vivo.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available