Cytochrome P450 monooxygenases (P450) are a diverse, ubiquitous family of
important enzymes which catalyze a variety of activities. Their outstanding abilities to
hydroxylate non-activated hydrocarbons make them attractive enzymes for applications
in the fields of chemical synthesis and bioremediation. Large scale applications of
P450s are however largely limited by: (i) their requirement for expensive cofactors; (ii)
dependence on co-proteins; and (iii) limited enzyme stability.
These problems are largely circumvented by using whole-cell biocatalysis. Therefore
the identification of appropriate hosts for heterologous expression is important. E. coli
has several limitations in its ability to express eukaryotic P450s, therefore yeasts are
attractive alternatives, but have scarcely been used for whole-cell applications (Zollner
et al, 2010).
The aim of this study was therefore to investigate several ascomycetous yeasts for their
potential as P450-expressing whole-cell biocatalysts. To perform parallel, unbiased
comparisons, the vector, cultivation conditions and assay conditions needed to be
consistent between the different yeasts. This was facilitated by a broad-range vector
system designed in our group, which allowed genomic chromosomal integration of
foreign DNA at the 18S rDNA regions, and expression using a constitutive TEF
promoter. Cultures of different yeasts were grown for the same duration at the same
temperature in a medium common among strains, prior to activity assays.
Using CYP53B1 as a reporter enzyme we demonstrated whole-cell activity for all of the
yeasts tested, except for H. polymorpha. The native reductase systems allowed
detectable activities of CYP53B1 in all of the other yeasts, with the highest activity (2.3
μmol.h-1 .gDCW
-1) found in A. adeninivorans. Coexpression of cytochrome P450
reductases (CPR) from Yarrowia lipolytica (YlCPR), Rhodotorula minuta (RmCPR) and
Ustilago maydis (UmCPR) all led to improvements of the CYP53B1 activities, with the highest activity (11.5 μmol.h-1.gDCW
-1) obtained with CYP53B1 and UmCPR
coexpressed in A. adeninivorans. The effects of the cloned UmCPR also differed
between the hosts, with the biggest improvements observed in A. adeninivorans and Y.
lipolytica. RmCPR and UmCPR improved the CYP53B1 activity more dramatically than
YlCPR, possibly because CYP53B1, RmCPR and UmCPR are all of basidiomycetous
origin.
The CYP53B1 activity achieved in this study was considerably better than results
obtained previously in our group. In the previous study, a Y. lipolytica strain with multiple
copies of CYP53B1 and an additional copy of YlCPR both under the regulation of strong
inducible promoters was used, under optimised and constant induction conditions.
(Shiningavamwe et al, 2006). The activity obtained in this study on the other hand was
achieved using an A. adeninivorans strain carrying presumably only one or two copies
of CYP53B1 and UmCPR, expressed under the control of a constitutive promoter, and
under non-optimised conditions.
Activity of the self-sufficient P450s CYP102A1 and CYP505A1 was assayed using
hexylbenzoic acid (HBA) as a non-natural substrate, since wild-type β-oxidation
pathways would not permit the use of fatty acids as substrates. HBA is hydroxylated at
the Ï-1 and Ï-2 positions of the alkyl chain by both CYP102A1 and CYP505A1 during
this study. Better expression of the bacterial CYP102A1 was obtained using K.
marxianus and S. cerevisiae, than in A. adeninivorans; whereas expression of the
eukaryotic CYP505A1 was better in Y. lipolytica and especially A. adeninivorans. The
best activity observed with a self-sufficient P450 was obtained once more with A.
adeninivorans expressing CYP505A1 (33 μmol.h-1 gDCW
-1).
Overall, the vector system allowed successful expression of P450s and CPRs from
bacterial, ascomycetous and basidiomycetous fungal origin, in multiple ascomycetous
yeast hosts. The differential effects of different CPRs on a class II P450 were
demonstrated, and the UmCPR was in this case found to be an excellent P450 reductase. We report heterologous P450 expression in A. adeninivorans for the first
time, and it proved to be, of the yeasts tested, the host with the highest potential for
efficient P450 expression and whole cell biocatalysis.
The findings of this study provide insight for the improvement of the field of eukaryotic
P450 research and particularly whole-cell biocatalysis, and could potentially assist in
enhancing the applications of these promising enzymes.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-05172013-153148 |
| Date | 17 May 2013 |
| Creators | Theron, Chrispian William |
| Contributors | Dr M Labuschagne, Prof J Albertyn, 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-05172013-153148/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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