Y. lipolytica has the ability to utilise hydrophobic hydrocarbons as carbon
sources. It is also an attractive host for heterologous expression of
cytochrome P450 (CYP) genes. Y. lipolytica strains with CYP genes cloned
under control of two different promoters, pPOX2 and pICL were used in this
study. The purpose of this project was to detect the effect of cloned alkane
hydroxylases in Y. lipolytica. Alkylbenzenes and alkylcyclohexanes were used
to compare the hydroxylase activities of the genetically engineered strains
with control strains.
Butylbenzene and hexylbenzene were transformed to phenylacetic acid while
pentylbenzene, heptylbenene and nonylbenzene yielded benzoic acid as
product. Butylcyclohexane and limonene were transformed to
cyclohexylacetic acid and perillic acid, respectively, as major products. The
activity towards hexylbenzene was highest. Phenylhexanoic acid and
phenylbutanoic acid were also for the first time observed as intermediates in
the biotransformation of hexylbenzene.
Y. lipolytica strains expressing alkane hydroxylases under pPOX2 were
induced with oleic acid and harvested. A strain with multiple copies of a
proven alkane hydroxylase, cloned, had in one experiment higher activity than
the other strains, towards both hexylbenzene and nonylbenzene. However,
these results could not be confirmed, because in subsequent experiments the
resting cells had very low activities.
Ethanol and sodium acetate were used as inducers in the experiments
conducted with Y. lipolytica strains with CYP557A1, a putative alkane and/or
fatty acid hydroxylase, cloned under pICL. The substrates were added directly
to the cells. With single addition of ethanol, the strain with cloned CYP557A1
had in one whole cell experiment with butylbenzene as substrate higher
activity than the control strains. With multiple additions of sodium acetate the strain with cloned CYP557A1
showed higher activity in two shake flask experiments when hexylbenzene
was used as a substrate. However, when ethanol and sodium acetate were
used as inducers the alkylbenzenes were often consumed without the
equivalent formation of detectable products, complicating the interpretation of
results. This also happened in bioreactor experiments.
Biotransformation of alkylcyclohexanes also did not demonstrate the effect of
the cloned gene, since the activities of the test strain with CYP557A1 cloned
under pICL and the control strain were similar. Alkylcyclohexanes are not
promising substrates to distinguish between hydroxylase activity of the cloned
genes as they are very volatile and activity towards them is relatively low.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-09172008-150748 |
| Date | 17 September 2008 |
| Creators | Ramorobi, Limpho Martha |
| Contributors | 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-09172008-150748/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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