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Title: Rhodococcus aetherivorans I24 as a platform for the biocatalytic reduction of ketones
Author: Duffield, S. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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Rhodococcus aetherivorans I24 is a saprophytic actinomycete bacteria originally isolated from a toluene-contaminated aquifer. Here, two alcohol dehydrogenases capable of enantioselectively reducing halomethyl ketones were isolated by chromatographic techniques, and their corresponding DNA sequences identified by comparison with experimentally determined amino acid sequence data. Attempts were made to overexpress these genes in the standard cloning host Escherichia coli. One enzyme, Adh1, expressed well, and was characterised successfully in a recombinant form. The second, Adh2, was difficult to express recombinantly in an active form in this system. Concurrently, tools for rhodococcal gene expression were developed, including characterisation of the ohp promoter region of Rhodococcus ruber V49, and creation of a chromosomal expression system. Use of the ohp promoter for expression of recombinant proteins in Rhodococcus was investigated, using the luciferase genes from the marine bacterium Vibrio fischeri as a model. In concert, the mode of action of OhpR, the regulator of the operon was elucidated, and its binding site identified. Protein folding of highly expressed gene products is frequently problematic. To address this potential bottleneck, the GroE chaperone system of R. aetherivorans I24 was investigated through a bioinformatics approach coupled with mutational analyses. Of the three GroEL homologs present in R. aetherivorans I24, two were found to respond to heat shocks by increased levels of transcription. This response was observed to correlate with the presence of binding sites for the heat shock regulatory protein, HrcA, upstream of the homologs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available