Alkane and fatty acid hydroxylating cytochrome P450 monooxygenases in yeast
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Date
2008-11
Authors
Shuping, Daniel Sechaba Skake
Journal Title
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Publisher
University of the Free State
Abstract
Yarrowia lypolytica, Candida tropicalis, Candida maltosa and Candida cloacae are extensively studied n-alkane degrading yeasts and are widely used in various industrial processes (Madzek et al., 2004, Mobley, 1999). Cytochrome P450 monooxygenases belonging to the CYP52 family are responsible for the terminal hydroxylation of n-alkanes and fatty acids. Candida species have been successfully used in the synthesis of long-chain α,ω-dicarboxylic acids (DCAs) which are difficult to produce using chemical processes (Wache et al., 2006 Mobley, 1999). These processes, which yield more than 100 g l-1 DCA at volumetric rates up to 1.9 g l-1h-1, are regarded as the most successful P450 dependent biotransformation processes developed thus far (Julsing et al., 2008). Because C. tropicalis and C. maltosa are related to Candida albicans (Eschenfeldt et al., 2003), they are regarded as potential pathogens. However, Y. lipolytica has GRAS status for a number of processes and the entire genome sequence is known (Fickers et al., 2005). Although Y. lipolytica has been shown to produce DCA, none of its mutant strains have produced DCA concentrations close to the DCA produced by Candida sp. (Kogure et al., 2007, Smit et al., 2005).
In order to understand the differences in terminal hydroxylase activity that contribute to differences in DCA production, we compared the wild-type strains Y. lipolytica W29 and C. tropicalis ATTC20336 by using alkylbenzenes, 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates. We also cloned and expressed CYP52A13 and CYP52A17 from Candida tropicalis ATTC20336 into Y. lipolytica CTY021 and CTY022, two β-oxidation disrupted strains. Dodecane, tetradecane, hexadecane and 4-hexylbenzoic acid were used as substrates. One experiment was done in bioreactors using a control strain and a strain expressing CYP52A13.
The experiments with the wild-type strains showed that Yarrowia lipolytica W29 and Candida tropicalis ATTC20336 responded differently to hydrocarbons. These differences are probably due to differences in the alkane and fatty acid hydroxylases of the two yeasts. In the biotransformation of alkylbenzenes by Y. lipolytica W29, nonylbenzene was the only substrate significantly converted to benzoic acid. The highest product formation (20.6 mM benzoic acid) was observed when the cultures were not induced with oleic acid or n-alkanes (C12, C14). In the case of C. tropicalis ATTC20336, both nonylbenzene and hexylbenzene were accepted as substrates and n-dodecane and oleic acid, reported inducers of CYP52 genes (Craft et al., 2003), significantly enhanced biotransformation of these substrates. Biotransformation of 4-hexylbenzoic acid by Y. lipolytica also occurred in the absence of an inducer, while in C. tropicalis it was only converted to the corresponding DCA after induction with oleic acid. This meant 4-hexylbenzoic acid could possibly be used as a marker substrate for monitoring the expression of C. tropicalis CYP52 genes in Y. lipolytica W29. Biotransformation of 4-nonyloxybenzoic acid by both these strains occurred when cultures were induced with oleic acid.
The CYP52A17 and CYP52A13 were cloned under the pPOX2 promoter into Yarrowia lipolytica strains with β-oxidation disrupted. A transformant with at least three copies of the CYP52A17 gene cloned had relatively low activity towards alkanes when compared to the control strain. However, the CTY021:CYP52A13 transformant with at least two copies of the CYP52A13 gene showed improved activity towards n-alkanes. The biggest improvement (92 %) was observed with n-tetradecane. TLC analyses showed that after induction with oleic acid, palmitic acid and n-hexadecane slightly more product was formed from 4-hexylbenzoic acid by the strain with CYP52A13 cloned than by the control strain. However, this improvement was much less than anticipated and 4-hexylbenzoic acid was in the end not used to screen for strains expressing the cloned CYP52 genes.
A bioreactor study was conducted in triplicate in a Sixfors multireactor to compare C16DCA production form hexadecane by the control strain and the strain with CYP52A13 cloned. The control strain (CTY026) and test strain CTY021:CYP52A13 grew differently. Although there was an indication that the strain with CYP52A13 cloned produced DCA faster, results were too varaible to reach a firm conclusion that cloning of the CYP52A13 gene significantly improved DCA production by Y. lipolytica.
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Keywords
Dissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2008, Yarrowia lipolytica, Candida tropicalis, Alkylbenzene, n-alkanes, Fatty acids, DCA, 4-hexylbenzoic acid, 4-nonyloxybenoic acid, CYP52A13, CYP52A17, Cytochrome P-450, Hydroxylation, Monooxygenases