Construction of self-sufficient CYP153 chimeras

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Date
2010
Authors
Randall, Charlene
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Publisher
University of the Free State
Abstract
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 group’s 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 5’- and 3’-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.
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Keywords
Dissertation (M.Sc. (Microbial, Biochomical and Food Biotechnology))--University of the Free State, 2010, Cytochrome P-450, Enzymes, Qxidoreductases, Chimeras, CYP153A6, CYP116B3, Self-sufficient, Fusion, Cassette PCR, Terminal alkane hydroxylase, Octane bioconversion, PFOR
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