Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Heterologous gene expression is of considerable interest for the production of proteins
of therapeutic and industrial importance. As the nature of recombinant proteins has
become more complex and as transformation systems have been established in more
species, so the variety of hosts available for expression has increased. Every system
available has both advantages and disadvantages. The research presented here
highlights the advantages of selecting the most appropriate expression system for
different recombinant proteins. Expression of different biocatalytically-relevant
enzymes, epoxide hydrolases, halohydrin dehalogenase, laccase and mannanase, in
different host systems is undertaken, and expression levels and activity are compared.
The development of Yarrowia lipolytica as a whole-cell biocatalyst is described.
Y. lipolytica is used for the functional expression of epoxide hydrolases (EHs) and
halohydrin dehalogenases. EHs are hydrolytic enzymes that convert epoxides to
vicinal diols by ring-opening. Two new fungal EHs from Rhodosporidium toruloides
NCYC 3181 and NCYC 3158 (a putative Cryptococcus curvatus strain) were
identified and cloned. Additional EHs from different sources, including bacteria,
yeasts, fungi and plants, were chosen for expression in Y. lipolytica, in order to
determine its suitability as the expression system of choice for the production of EHs.
Multi-copy integrants were developed, with the genes under control of the growthphase
dependent hp4d promoter. A Saccharomyces cerevisiae strain was developed,
expressing the EH from Rhodotorula araucariae1, to compare as a whole-cell
biocatalyst with Y. lipolytica. This strain proved to be an exceptionally poor wholecell
biocatalyst. All the Y. lipolytica strains developed showed varying levels of
activity towards different classes of epoxides. Some strains displayed opposite
enantioselectivities, allowing for potential complete conversions of racemic epoxides
to the desired enantiomeric product.
Halohydrins can be considered direct precursors of epoxides. Halohydrin
dehalogenases catalyse the nucleophilic displacement of a halogen ion in halohydrins by a vicinal hydroxyl group, yielding an epoxide, a proton and a halide ion. The
HheC gene from Agrobacterium radiobacter AD1, codon-optimised to match the
codon usage of Y. lipolytica, was over-expressed in Y. lipolytica by generation of
multi-copy integrants, further expanding the use of this organism as a host strain for
heterologous production of enzymes. Expression levels were maximised by creating
tandem repeats of the introduced HheC gene. The ring-closure activity with 2-chloro-
1-phenylethanol as substrate was demonstrated to be broadly dose-dependent.
The b-mannanase gene (man1) from Aspergillus aculeatus MRC11624 was expressed
in Y. lipolytica with effective secretion in the presence of its native secretion signal,
using the hp4d promoter. The same gene was expressed in Aspergillus niger2 under
control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpdP)
and the Aspergillus awamori glucoamylase terminator (glaT). Following optimisation
with copy numbers and culture conditions, maximal activity levels of 26,140 nkat.ml-1
for Y. lipolytica, and 16,596 nkat.ml-1 for A. niger were obtained.
Laccases are important enzymes for bioremediation, and the best characterised
enzymes are from the fungus Trametes versicolor. The objective of this research was
to optimise expression of T. versicolor laccases (lcc1 and lcc2) in A. niger D15 and
Pichia pastoris3. The Lcc1 enzyme was less active than Lcc2 in both hosts. P.
pastoris secreted 0.4 U.L-1 Lcc1 and 2.8 U.L-1 Lcc2, compared to 2,700 U.L-1
produced by A. niger. The Lcc2 enzyme from recombinant A. niger was subsequently
purified and characterised in terms of molecular weight and glycosylation, and
compared to the wild-type enzyme purified from T. versicolor.
The work presented underscores the requirement for experimentation before finalising
the choice of an expression system for the optimal production of the desired protein.
Every system available has both advantages and disadvantages, and when considering
which system to use for producing a recombinant protein, various factors must be
taken into consideration. However, the choice is broad and each decision needs to be
made empirically. 1 The construction of the S. cerevisiae epoxide hydrolase production strain was carried out by Dr
Neeresh Rohitlall of CSIR Biosciences. The Y. lipolytica epoxide hydrolase strains were constructed
by the author. 2 The construction of Man1-producing A. niger strain was done by Dr Shaunita Rose of the University
of Stellenbosch. The construction of Y. lipolytica Man1 production strains was done by the author.
3 The expression of T. versicolor laccases in P. pastoris was done by Christina Bohlin of Karlstad
University. A niger laccase production strains were created by the author. / AFRIKAANSE OPSOMMING: Heteroloë geen uitdrukking is van groot belang vir die produksie van proteïene wat
van terapeutiese en industriele belang is. Soos die aard van rekombinante proteïene
meer ingewikkeld raak en getransformasie-sisteme vir verskeie spesies gevestig raak,
is daar ’n groter verskeidenheid van gashere beskikbaar vir geenuitdrukking. Elke
sisteem het beide sy voor- en nadele. Hierdie navorsing beklemtoon die voordele
wanneer die mees gepaste uitdrukkingssiteem gekies word. Die uitdrukking van
verskeie ensieme van biokatalities belang, epoksiedhidrolases, halohidrien
dehalogenase, lakkase en mannanase in verskillende gasheersisteme is onderneem en
die uitdrukkingsvlakke en aktiwiteite vergelyk.
Die ontwikkeling van Yarrowia lipolytica as ’n heelsel biokatalis word beskryf.
Y. lipolytica word gebruik vir die funksionele uitdrukking van epoksiedhidrolases
(EHs) en halohidrien dehalogenases. EHs is hidroliseringsensieme wat die epoksiede
omskakel na aangrensende diole deur middel van ring-opening. Twee nuwe fungi
EHs vanaf Rhodosporidiom toruloides NCYC 3181 en NCYC 3158 (’n moontlike
Cryptococcus curvatus) is geïdentifiseer en gekloneer. Verdere EHs van verskillende
bronne, insluitend bakterieë, giste, fungi en plante, is gekies vir uitdrukking in
Y. lipolytica ten einde sy geskiktheid vir die produksie van EHs te bepaal. Multikopie
integrante is ook ontwikkel met gene onder beheer van die groei-fase afhanklike hp4d
promotor. ’n Saccharomyces cerevisiae ras is ook ontwikkel vir die uitdrukking van
die EH van Rhodotorula araucariae4 sodat dit met Y. lipolytica as ’n heelsel
biokatalis vergelyk kan word. Hierdie ras was ‘n buitengewone swak heelsel biokatalis. Al die Y. lipolytica rasse wat ontwikkel is het wisselende aktiwiteitsvlakke
teenoor verskillende klasse van epoksiede getoon. Sommige rasse het teenoorgestelde
enantio-selektiwiteit getoon en het die potensiaal om rasemiese epoksiede volledig na
die gewensde enantiomeriese produk om te skakeling.
Halohidriene kan as direkte voorgangers van epoksiede beskou word. Halohidrien
dehalogenases kataliseer die nukleofiliese vervanging van ’n halogeen-ioon in
halohidriene deur ’n aangrensende hidroksiel groep, wat ’n epoksied, ’n proton en ’n
halied-ioon lewer. Die HheC geen van Agrobacterium radiobacter AD1 is kodon–
geöptimiseer om te pas by die kodon gebruik van Y. lipolytica en was uitgedruk in
Y. lipolytica deur die skep van mulitkopie integrante, ’n verdere verbreeding van die
toepaslikheid van die organisme as gasheerras vir die heteroloë produksie van
ensieme. Maksimum uitdrukkingsvlakke is bereik deur die skep van opeenvolgende
herhalings van die ingevoegde HheC-geen. Daar is ook gewys dat die ring-sluitingsaktiwiteit
met 2-chloro-1-pheniel-etanol as substraat meestal dosis-afhanklik is.
Die -mannanase geen (man1) van Aspergillus aculeatus MRC11624 is uitgedruk en
effektief in Y. lipolytica mbv sy eie uitskeidings sein uitgeskei, met die gebruik van
die groei-fase afhanklike hp4d promotor. Dieselfde geen is uitgedruk in Aspergillus
niger5 onder beheer van die A. niger gliseraldehied-3-fosfaat dehidrogenase promotor
(gpdp) en die Aspergillus awamori glikoamilase termineerder (glaT). Verdere
optimisering van kopiegetal en voedingskondisies het gelei tot maksimum
aktiwiteitsvlakke van 26,140 nkat.ml-1 vir Y. lipolytica en 16,596 nkat.ml-1 vir
A. niger. Lakkases is belangrike ensieme vir bio-remediëring, en die ensieme van die fungus
Trametes versicolor is die beste gekarateriseer. Die doelwit van hierdie navorsing
was die optimisering van die uitdrukking van T. versicolor lakkases (lcc1 en lcc2) in
A. niger en Pichia pastoris6. Die Lcc1 ensiem was minder aktief as Lcc2 in altwee
die gashere. P. pastoris het 0.4 U.L-1 Lcc1 en 2.8 U.L-1 Lcc2 onderskeidelik
uitgeskei, in vergelyking met 2,700 U.L-1 Lcc2 wat deur A. niger geproduseer is. Die
Lcc2 ensiem afkomstig van die rekombinante A. niger is vervolgens gesuiwer en
gekarakteriseer met betrekking tot molekulêre massa en glikosilering, en daarna
vergelyk met die wilde-tipe ensiem wat deur T. versicolor geproduseer word.
Die werk wat hier aangebied word, beklemtoon die vereistes vir eksperimentering
voor die finale keuse met betrekking tot ’n gepaste uitdrukkingsisteem gemaak kan
word vir die optimale produksie van die gewensde proteïen. Elke sisteem het beide
voordele en nadele, en wanneer ’n sisteem oorweeg word is daar verskeie faktore wat
in ag geneem moet word. ’n wye verskeidenheid van keuses is beskikbaar en elke
besluit moet empiries gemaak word. 4 Die konstruksie van die S. cerevisiae epoksiedhidrolase-produserende ras is deur Dr Neeresh Rohitlall
van CSIR Biosciences gedoen. Die Y. lipolytica epoxied hydrolase rasse is deur die outeur gemaak. 5 Die konstruksie van die Man1-produserende A. niger ras is deur Dr Shaunita Rose van die
Universiteit van Stellenbosch gemaak. Die Y. lipolytica Man1 ras is gemaak deur die outeur. 6 Die uitdrukking van T. versicolor lakkases in P. pastoris is gedoen deur Christina Bohlin van Karlstad
University. Die A niger lakkase produksie ras is geskep deur die outeur.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/21733 |
| Date | 03 1900 |
| Creators | Roth, Robyn Lindsay |
| Contributors | Van Zyl, W. H., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xiii, 280 leaves : ill. |
| Rights | Stellenbosch University |
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