Biohydroxylation reactions are catalyzed by various types of hydroxylating
enzymes (Ayala and Torres, 2004) which include dioxygenases, lipooxygenases
as well as CYP450 monooxygenases. These particular hydroxylation reactions
have several advantages over chemical synthesis. Several microorganisms
including yeasts have the ability to hydroxylate various substrates. The
exploitation of microbial hydroxylations for the production of industrially useful
products such as pharmaceuticals is a more recent development (Holland et al.,
2000).
Yeasts from the genera Schizosaccharomyces, Pichia, Saccharomyces
and Yarrowia have all been used to express foreign CYP450 genes (Mukarami et
al., 1990; Nthangeni et al., 2004) since they offer an advantage especially when
a eukaryotic environment is required for the functional expression of the
heterologous gene (Blanquet et al., 2003). A recent evaluation of several yeasts
revealed that Y. lipolytica is, a highly attractive alternative host for secretion and
expression cloning (Muller et al., 1998; Juretzek et al., 2001). However, a
literature search on successful expression of CYP450s in Y. lipolytica yielded
only six cases. Three of these were done in our laboratory. In most of the
reported cases, the recombinant CYP450 activities were never evaluated in
terms of whole cell biotransformations.
It was therefore the aim of this study to evaluate Y. lipolytica as a
recombinant whole-cell biocatalyst for hydroxylation reactions by using available
Y. lipolytica strains overexpressing different CYP450s which were (i) CYP1A1
coding for polyaromatic hydrocarbon hydroxylase (ii) CYP53B1 coding for
benzoate para-hydroxylase (iii) CYP52F1 coding for alkane hydroxylase and (iv)
CYP557A1 coding for putative fatty acid hydroxylase. Hydroxylase activities of
the genetically engineered strains were compared with activities in wild type yeasts expressing the relevant CYP450s. A variety of substrates used for
biotransformation reactions included, acetanilide, benzoic acid , phenylnonane,
trans-cinnamic acid, 4-nitrophenyl octyl ether and 4-nonyloxybenzoic acid.
Experiments using Y. lipolytica overexpressing CYP1A1 illustrated the
limitation of using Y. lipolytica for the biotransformation of substrates such as AA
since the endogenous enzymes degraded this substrate within only 12 h after
substrate addition.
In an attempt to distinguish the activities of the putative fatty acid
hydroxylase and the alkane hydroxylase overexpressed in Y. lipolytica from the
endogenous CYP450s, 4-nitrophenyl octyl ether, 4-nonyloxybenzoic acid and
phenylnonane were used as substrates. 4-nitrophenyl octyl ether proved to be
expensive and less sensitive to TLC, GC and GC-MS analyses. It has been used
in other studies because it yielded p-nitrophenol which can be assayed
colourimetrically by measuring absorbance at 420 nm. However, in our
experiments, intermediates accumulated that were not completely transformed
into p-nitrophenol.
Further biotransformation experiments were carried out using 4-
nonyloxybenzoic acid as the substrate. Biotransformation experiments were done
using Y. lipolytica strains with intact and partially disrupted -oxidation pathway
overexpressing CYP52F1 and CYP557A1. Additional experiments were carried
out using wild type Y. lipolytica W29, R. minuta and R. retinophila strains. The
results demonstrated that, the wild type Y. lipolytica W29 demonstrated the
highest specific hydroxylase activity when 4-nonyloxybenzoic acid was used as
the substrate. The main limitation here was the inability to selectively induce the
overexpressed CYP450 genes alone without the background endogenous
CYP450 activity. Due to the limitations above, the next strain used was Y. lipolytica TVN91
(overexpressing benzoate-para hydroxylase from R. minuta). In this case, the
host strain lacked the specific CYP450 to perform the same hydroxylation
reaction. The substrate used here was BA. Different growth and induction
conditions were evaluated to optimize benzoate-para-hydroxylase activities. A
comparison of the hydroxylase activities indicated that the activity of the
recombinant Y. lipolytica strain overexpressing the CYP53B1 was about 30 times
slower than that of the wild type R. minuta from which the gene was cloned.
Continuous addition of stearic acid resulted in the best activity with Y. lipolytica
TVN91, because the hydroxylase activity was maintained for a longer duration.
When PN and CA were used to evaluate substrate transport limitation, the results
demonstrated that substrate transport was not limiting and the specific
hydroxylase activity was not increased. PN was initially converted to BA before
hydroxylation to form pHBA. These results further demonstrated that hydroxylase
activity of PN was much faster than that of BA.
The results from the bioreactor study demonstrated that an improved
aeration and mixing led to an increase in the benzoate para-hydroxylase activity.
The possibility of using a chemically defined media (YNB) supplemented with
yeast extract and casamino acid for biotransformation was also demonstrated.
The results of this study also demonstrated that it is possible to use
harvested recombinant cells for biotransformation without significant loss of
activity. This makes it possible to study in detail the kinetics of the overexpressed
CYP450s. It was, however apparent that the hydroxylase activities were
significantly increased by both aeration and cell concentration.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-09122007-100400 |
| Date | 12 September 2007 |
| Creators | Obiero, George Ogello |
| 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-09122007-100400/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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