The dimorphic fungus Yarrowia lipolytica is an n-alkane assimilating yeast. During nalkane
oxidation toxic fatty aldehydes are formed that are further oxidized by fatty
aldehyde dehydrogenases (FALDH) to carboxylic acids that then enter the β-oxidation
pathway.
Very little research emphasis has been placed on FALDHs in yeasts and their precise role
in n-alkane metabolism. This study aimed at contributing to the limited knowledge of
yeast FALDHs and in particular the four putative Y. lipolytica FALDHs (YlFALDH1 - 4)
that were recently identified in the fully sequenced genome of Y. lipolytica E150. The
contribution made from this study to YlFALDHs was with reference to their promoter
expression and subcellular localization.
The promoter and terminator of each YlFALDH was initially used to construct reporter
cassettes in conjunction with β-galactosidase (lacZ) by utilizing the Sticky-end PCR
(SEP) and Enzyme-free cloning methods. These two methods proved to be unsuitable for
the expression study. The promoter region of each YlFALDH was then cloned into the
pINA781 expression vector containing lacZ to study the expression further. With the aid
the pINA781 integrative vector and qualitative plate assays, with 5-bromo-4-chloro-3-
indolyl-β-D-galactosidase (X-gal), it was observed that the promoters of YlFALDH1 and
2 were inducible by dodecane and hexadecane but not by oleic acid, glucose or glycerol.
The promoter of YlFALDH2 also seemed to display the same level of transcriptional
strength as the inducible POX2 promoter. Induction of the YlFALDH3 and 4 promoters
was not observed.
Localization of the YlFALDH proteins was studied with the aid of green fluorescent
protein (GFP) from Aequorea victoria and putative localization sequences (LS) from each YlFALDH isozyme. The putative Y. lipolytica LSs comprised of the last 150 bp of
the COOH-terminal of the YlFALDH proteins. These LSs were modeled from Rattus
norvegicus FALDH that possesses a 35 amino acid hydrophobic protein anchor at its
COOH-terminal.
For localization studies, chimerical JMP5 molecules were created with an inducible ICL1
promoter, GFP and putative Y. lipolytica LS from each isozyme. Chimerical molecules
were also constructed with a pKOV136 vector that contained a constitutive TEF
promoter, a GFP-LS fragment (from a JMP5-chimera) and LIP2 terminator. No
fluorescence was observed with epifluorescence or confocal laser microscopy when
either of the JMP5- or pKOV136-chimeras were transformed into Y. lipolytica E150 and
Po1g respectively. Consequently the subcellular localization could not be identified.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-10122006-113238 |
| Date | 12 October 2006 |
| Creators | Müller, Walter Joseph |
| Contributors | Prof MS Smit, Prof J Albertyn |
| 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-10122006-113238/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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