A variety of microorganisms, including yeasts, are capable of utilizing n-
alkanes as carbon source (Schmitz et al., 2000; Watkinson & Morgan, 1990).
The over expression of P450 genes such as the CYP52 family coding for the
alkane hydroxylases may lead to an increase in activity and increased
formation of possible useful products from hydrocarbon metabolism (Iida et
al., 2000). Disruption of the -oxidation pathway by deleting the genes coding
for acyl CoA-oxidases, also leads to the accumulation of products that would
normally be broken down (Picataggio et al., 1991). The genetic engineering of
these two points of control opens up many possibilities for the accumulation
of different products from hydrocarbons. Although some work was done
concerning these systems in Candida tropicalis very little work has been done
in Yarrowia lipolytica.
It was the aim of the project to investigate the biotransformation of alkanes,
alkylbenzenes and their derivatives by different groups of genetically
engineered Y. lipolytica strains in order to investigate a number of questions.
The possible accumulation of monocarboxylic acids in Yarrowia lipolytica was
inestigated by using substrates such as undecene and hexylbenzene. Y.
lipolytica MTLY37 a -oxidation disrupted strain with POX2, POX3, POX4 and
POX5 genes deleted could not accumulate any monocarboxylic acid from
undecene. The undecene was however fully utilized indicating that this strain
still had some -oxidation activity. Little phenylacetic acid was formed (0.4
mM) from hexylbenzene. Another product that could not be positively
identified at the time, but which might have been phenylhexanoic acid
accumulated (4mM). No monocarboxylic acids other than phenylacetic acid
could also be accumulated from alkylbenzenes in strains with blocked -
oxidation expressing CPR and CYP genes, leading to the conclusion that Y.
lipolytica can not accumulate monocarboxylic acids. Y. lipolytica strains with disrupted -oxidation as well as a strain with
functional -oxidation expressing additional YlCPR and CYP52F1 genes
accumulated the full-length dioic acid from 5-methylundecane. All these
strains also sequentially broke down the 5-methylundecanedioic acid to 5-
methylnonanedioic acid, 3-methylheptanedioic acid and 3-methylpentanedioic
acid. Y. lipolytica MTLY76 was the only strain that did not degrade the 5-
methylundecanedioic acid completely.
Using hexylbenzene as substrate it was possible to establish that ethanol
delayed the induction of both the native ALK genes as well as the inserted
CYP genes. However, the cloned genes were later induced quite strongly
(probably by the phenylalkanoic acids formed from hexylbenzene) for an
extended period, while the native genes were only weekly induced. The
maximum activity of Y. lipolytica was slightly lower when ethanol was used as
inducer (13µmol.min -1 l -1 ) than when oleic acid was used as inducer
(19µmol.min -1 l -1 ). The alkane hydroxylase activity was however maintained for
a longer time when ethanol was used as inducer. When dodecane was used
as inducer native genes were strongly induced for a relatively long period, but
not as long as the cloned genes after ethanol.
Alkylbenzenes as substrate was also useful to distinguish between alkane
hydroxylase activity of native and cloned monooxygenases. A significant
difference in the activity of Y. lipolytica TVN356 expressing CPR together with
CYP557A1 (putative fatty acid hydroxylase from Rhodotorula retinophila) and
Y. lipolytica TVN91 expressing CPR together with CYP53 (benzoate para-
hydroxylase from R. minuta) could be observed (14µmol.min -1 l -1 and
8µmol.min -1 l -1 respectively) when decylbenzene was used as substrate. To
better study the hydroxylase activity of inserted P450s, it may be better to use
the ICL1 promoter to drive the expression of the inserted CYP genes and use
ethanol as inducer.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-03282006-085521 |
| Date | 28 March 2006 |
| Creators | van Rooyen, Newlandé |
| Contributors | Prof MS Smit, Dr ME Setati |
| 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-03282006-085521/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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