Cytochrome P450 monooxygenases are enzymes capable of efficiently
hydroxylating hydrophobic substrates with high regio-, stereo-, and
enantioselectivies; under mild reaction conditions. They are therefore attractive
alternatives to traditional chemistry for the synthesis of hydroxylated products.
Their use in large scale applications has however been hindered by their
requirement for continuous supply of reducing cofactors. Self-sufficient P450s
occur, containing fused hydroxylase and reductase domains in a single
polypeptide. They exhibit higher activities than any other reported P450s.
Yarrowia lipolytica is a yeast capable of efficient degradation of hydrophobic
substrates and growth on alkanes as sole carbon source. The intricate pathways
involved in hydrophobic substrate metabolism within this yeast involve initial
hydroxylation by P450s. Y. lipolytica contains 12 alkane and fatty acid
hydroxlyase encoding genes CYP52F1 to CYP52F12. This yeast has been
widely tested for biotransformations of inexpensive substrates to more valuable
products. Y. lipolytica has also been investigated as a host for the heterologous
expression of diverse foreign proteins, including cytochrome P450s. Various
genetic tools are available for this purpose, including strong, inducible promoters,
(e.g. POX2 and ICL); the defective selection marker ura3d4 for integration of
cloned genes in higher copy numbers; specific targets for integration; and
customized host strains.
Heterologous expression of two CYP genes, by Y.lipolytica has previously been
investigated in our research group. These were CYP53B1, encoding a benzoatepara-
hydroxylase, and CYP557A1, a putative alkane and fatty acid hydroxylase.
They were cloned in single and multiple copies under the control of the POX2
promoter. Problems were encountered with the POX2 promoter when whole cells
were used for biotransformations, since native P450s were also induced by the
fatty acids used as inducers for the POX2 promoter, and thus interfered with activity determination of cloned P450s. A further limitation to this study was the
lack of appropriate negative control strains for accurate comparisons.
In the current study, the same P450s were cloned into Y. lipolytica under the
control of the ICL promoter. More appropriate negative controls strains were also
constructed. Both ethanol and oleic acid can induce the ICL promoter. Ethanol
induction of the CYP53 expressing strains resulted in lower whole cell activities
than oleic acid induction. It has been reported in the literature that activities of
enzymes expressed under the ICL promoter in cell-free extracts from cells
induced with either oleic acid or ethanol were comparable. It therefore seemed
that ethanol was repressing other components of the metabolic pathways for the
degradation of hydrophobic substrates in the cells.
Additionally no convincing activity was discernable for the ICL regulated CYP557
containing strains compared to the controls under POX2 regulation, and negative
control strains. Continuous ethanol addition again led to reduced activities
compared to situations where ethanol was added only once, confirming the
metabolic inhibition by ethanol.
It can be concluded that ethanol induction of the ICL promoter is not viable for
whole cell biotransformations of hydrophobic substrates. Different inducers
which will repress induction of the endogenous CYP genes but will allow
induction of hydrophobic substrate uptake systems must be identified if the ICL
promoter is to be used for the expression of cloned CYP genes.
The CYP102A1 gene encoding the self-sufficient subterminal fatty acid
hydroxylase from Bacillus megaterium was also cloned into Y. lipolytica under
the POX2 and ICL promoters. Strains containing this gene did not display
detectable subterminal hydroxylation of fatty acids or 4-nonyloxy-benzoic acid
which was used as a fatty acid analogue. Significantly increased cytochrome c
reductase activities were however detected in the soluble fraction of cell free extracts from test strains compared to the negative control strains. Microsomal
activities were comparable in cells with and without cloned CYP102A1. This
indicated that the CYP102A1 was functionally expressed, but located in the
soluble fraction, where it was unlikely to interact with the hydrophobic substrates.
It was therefore concluded that Y. lipolytica is not a suitable host for whole cell
biotransformations using the CYP102A1 enzyme.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-10092009-161253 |
| Date | 09 October 2009 |
| Creators | Theron, Chrispian Willilam |
| 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-10092009-161253/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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