Heterologous expression of cytochrome P450 monooxygenases by the yeast Yarrowia lipolytica
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Date
2007-05
Authors
Theron, Chrispian William
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Publisher
University of the Free State
Abstract
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.
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Keywords
Dissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2007, Aromatase, Hydroxylation, Ascomycetes, Yarrowia lipolytica, Heterologous expression, Cytochrome P450 monooxygenase, ICL promotor, Substrate hydroxylation, Hydrophobic substrates, Whole cell biotransformations, Benzoate-para-hydroxylase, Self-sufficient P450s, CYP102A1