Cytochrome P450 monooxygenases are a superfamily of heme-containing enzymes that are
found in all domains of life. P450s catalyse diverse reactions, many of which are difficult
reactions to accomplish, even with the use of chemical catalysts. One such reaction is the
terminal hydroxylation of alkanes, the first step in alkane degradation. The CYP153 family, found
in alkane-utilising bacteria, is one of only two P450 families that can catalyse this reaction. One
of the long-term goals of our groupâs research is the directed evolution of terminal alkane
hydroxylases, using preferably a self-sufficient terminal alkane hydroxylase as the starting point.
There are, however, no naturally self-sufficient CYP153s. Therefore, the first aim of this study
was to create a self-sufficient CYP153 by fusing a CYP153 heme domain to the reductase
(PFOR) domain of a self-sufficient P450.
The gene encoding the heme domain of CYP153A6 from Mycobacterium sp. HXN-1500 was
ligated to the DNA encoding the PFOR reductase domain of CYP116B3 from Rhodococcus
ruber DSM 44319. The fusion gene was expressed in E. coli using a pET28a plasmid. The
resulting protein was misfolded and expressed mainly in the insoluble fraction in the form of
inclusion bodies. Factors possibly responsible for this were investigated including the expression
conditions, the effect of an N-terminal His-tag on protein folding, the effect of the linker region
sequence on protein folding, and the possibility of rapid expression resulting in protein
misfolding, but with all of these experiments only low levels of P420s were observed in the
soluble fraction and no P450 forms were detected. A whole-cell octane bioconversion
experiment conducted using the expressed fusion revealed the presence of P450 forms of the
protein, but no 1-octanol was produced, indicating that octane possibly facilitated the correct
folding of the CYP153A6 heme domain but that the heme domain and the PFOR reductase
domain were unable to form a functional complex. This theory does, however, require further
research.
In this study, CYP153A6 and its redox partners, ferredoxin reductase and ferredoxin were
expressed in E. coli using the pET28b plasmid. Expression of CYP153A6 in E. coli using this
plasmid has not previously been reported in literature. Whole-cell octane bioconversions
conducted using the expressed CYP153A6 resulted in the production of 42 mM of 1-octanol after 24 hours, with the P450 concentration increasing during this time, a trend which was also
observed with the fusion.
The second aim of this study was to apply cassette PCR to the fusion to generate diverse selfsufficient
terminal alkane hydroxylases, which would provide the genetic diversity required for
directed evolution. Degenerate primers designed according to conserved N- and C-terminal
regions of CYP153A amino acid sequences were used to amplify internal CYP153A gene
fragments from environmental DNA extracted from enrichments of soil sampled at a dieselcontaminated
site in the Eastern Cape. Three different sequences were identified, one of them
being CYP153A6, which was excluded from the rest of the study. The two remaining sequences
and two sequences originating from another project using environmental DNA from samples
from the Beatrix Goldmine in the Free State were linked to the 5â- and 3â-ends of the CYP153A6
gene, generating full-length chimeric CYP153A genes. Because of the fact that the expression of
the fusion was unsuccessful, the functionality of these chimeric genes was tested using the
above-mentioned functional CYP153A6 operon. Expression was observed in the insoluble
fraction in the form of inclusion bodies, with the proteins being misfolded. A whole-cell octane
bioconversion did not result in P450 forms of the proteins and no 1-octanol was produced,
indicating that these chimeras were non-functional.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-11192010-152722 |
| Date | 19 November 2010 |
| Creators | Randall, Charlene |
| Contributors | Prof J Albertyn, Prof MS Smit |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-11192010-152722/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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