Dissertation (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: To manipulate recombinant microorganisms for industrial processes, controllable genetic
systems are needed that can coordinate expression of recombinant metabolic pathways. All
components are sensitive to change and thus putative targets for modification and genetic
elements and regulatory systems need to be understood and determined. Central in gene
regulation is the transcription activators that mediate gene transcription mechanisms by
binding to promoters in response to environmental signals. Promoter engineering entails the
modification of transcription factors and their target promoters.
In this study, a metabolic control system in Saccharomyces cerevisiae was constructed that
would allow induction in response to physiological environment, specifically hypoxia and
low temperature conditions. Two approaches were undertaken to find such a system. Firstly,
a bi-directional reporter gene cloning vector was designed to search for novel hypoxiainducible
promoters. Secondly, a transcription regulatory circuit was built, consisting of an
inducible transcription regulator and promoter with a reporter gene through which it mediates
transcription. Advantage was taken of the modular nature of proteins and functional domains
originating from different transcriptional proteins were combined.
A search for promoter elements sensitive to hypoxia from a S. cerevisiae genomic DNA
(gDNA) library, using a bi-directional cloning vector, did not yield highly inducible
promoters. It was concluded that a multitude of signals overlap, rendering genetic induction
difficult to control. A synthetic regulatory system would minimize the impact of these
multiple interactions. Such a genetic circuit was constructed, consisting of a chimeric
transcription activator and a target fusion promoter. The chimeric transcription activator
consisted of the GAL4 DNA binding domain, ADR1 TADIII transactivation domain and three
domains of the MGA2 regulatory protein. The functional domains of Mga2p responsible for
unregulated expression (at high basal levels) under both aerobic and hypoxia conditions were
located, as well as a further upregulation under low temperature, and were mapped to the Nterminal
and mid-Mga2p regions. A target fusion promoter consisting of a partial GAL10/1 promoter sequence and a
Trichoderma reesei core xyn2 promoter were constructed as target for this chimeric
transactivator. This synthetic promoter was fused to the T. reesei xyn2 open reading frame
encoding for a readily assayable β-xylanase activity. Both the chimeric transactivator and
fusion promoter-reporter gene cassettes were expressed from the same episomal plasmid,
named pAR.
Transformed into S. cerevisiae Y294, this regulatory system induced transcription under
aerobic and hypoxia conditions. Furthermore, the reporter gene expression was upregulated
by the chimeric transactivator at low temperatures. The chimeric transactivator mediated a
seven-fold induction of the reporter gene under aerobic conditions in S. cerevisiae Y294
when transformed with plasmid AR. A two- to three-fold induction at 23ºC was reported
under anaerobic conditions, relative to a reference strain expressing a transcription activator
without the Mga2p domains. At 30ºC, a two- to three-fold induction under aerobic conditions
and similar induction under oxygen-limited conditions were observed.
Replacing the reporter gene with your favorite gene (for example a recombinant enzyme) and
incorporating such a pAR system into a recombinant yeast should induce expression of the
chosen gene under low temperatures, both aerobic and anaerobically (thus creating a
controllable system). The system also has wider application in identifying other transcription
factors’ signal-sensitive domains. The design of this system provides the ability to add a
linker to a transactivator and to either create specific signal sensitivity or relieve the regulator
of its signal dependence. It creates an easy system for assessing other transactivators and
their domains with unknown functions and thus provides a ”workhorse and prospector in
one”. / AFRIKAANSE OPSOMMING: Vir die manipulering van rekombinante mikroörganismes vir industriële prosesse word
beheerbare genetiese stelsels benodig om gekoördineerde uitdrukking van rekombinante
metaboliese weë teweeg te bring. Alle komponente van sulke stelsels is sensitief vir
verandering en genetiese elemente en reguleerbare sisteme moet dus deeglik verstaan of
bepaal word. Sentraal tot geenregulering is die transkripsie-aktiveerders wat geentranskripsie
beheer deur aan promoters te bind in reaksie op eksterne omgewingsfaktore. Promotoringenieurswese
behels wysigings van transkripsiefaktore en hul teikenpromotors.
In hierdie studie is 'n genetiese beheerstelsel vir Saccaromyces cerevisiae ontwikkel wat
induksie in reaksie tot spesifieke fisiologiese omgewingreaksies, naamlik hipoksie- en lae
temperatuur, toelaat. Twee benaderings is gevolg: eerstens is ‘n tweerigting verklikker-geen
vektor ontwikkel en gebruik om vir unieke induseerbare hipoksie-promoters te soek.
Tweedens is ‘n transkripsie reguleringstelsel gebou wat uit ‘n induseerbare transkripsiereguleerder
and promotor met ‘n verklikkergeen bestaan, waardeur transkripsie bemiddel kan
word. Hierdie benadering benut die modulêre onderbou van proteïene en funksionele
domeine afkomstig vanaf verskillende transkripsiefaktore is gekombineer.
'n Soektog na hipoksie-sensitiewe promotors vanuit 'n Saccharomyces cerevisiae-genoom-
DNA (gDNA), deur van ‘n tweerigting verklikker-vektor gebruik te maak, het ongelukkig nie
hoogs-induseerbare promotors opgelewer nie. Die gevolgtrekking was dat ‘n veelvoud van
seine met mekaar oorvleuel en die beheer van genetiese induksie dus bemoeilik. Die
ontwikkeling van ‘n sintetiese regulering-sisteem kan die impak van die veelvuldige
interaksies verminder. Vir dié doel is ‘n sintetiese reguleringstelsel ontwerp, bestaande uit ‘n
chimeriese transkripsie-aktiveerder met ‘n teiken fusie-promotor. Die chimeriese
transaktiveerder bestaan uit die GAL4 DNA bindingsdomein, die ADR1 TAD III
transaktiveringsdomein en drie domeine van die Mga2 reguleringsproteïen. In die studie is
die funksionele domeins van Mga2p betrokke by lae temperatuur-respons en ongereguleerde uitdrukking (teen hoë basale vlakke) onder beide aërobiese en anaërobiese toestande
aangedui en is tot die N-terminaal en middel-Mga2p areas gekarteer.
‘n Teiken-fusie-promoter, bestaande uit 'n gedeeltelike GAL1/10 DNA promotoropeenvolging
en ‘n Trichoderma reesei kern xyn2-promoter, is as teiken vir hierdie
chimeriese transaktiveerder saamgestel. Hierdie sintetiese promotor is aan die T. reesei xyn2
oopleesraam, wat vir ‘n maklik meetbare β-xylanase aktiwiteit kodeer, gekoppel. Beide die
chimeriese transaktiveerder and fusie-promoter-verklikker-geenkaset word vanaf dieselfde
episomale plasmied, bekend as pAR, uitgedruk.
Hierdie reguleringsisteem induseer transkripsie onder aërobiese en hipoksie toestande in
S. cerevisiae Y294. Verder word die verklikkergeen se uitdrukking deur die chimeriese
transaktiveerder by lae temperature verhoog. Die chimeriese transaktiveerder induseer ‘n
sewe-voudige induksie van die verklikkergeen onder aërobiese toestande by 23ºC vanaf die
pAR-stelsel in S. cerevisiae Y294. ‘n Twee- tot drie-voudige induksie teen 23ºC is onder
hipoksie toestande gevind, relatief tot induksievlakke van ‘n verwysingstam met ‘n
transaktiveerder sonder die Mga2 domeine. By 30ºC is ‘n twee- tot drie-voudige induksie
onder aërobiese en lae suurstofvlakke waargeneem.
Deur die verklikker geen met ‘n jou-gunsteling-geen te vervang (bv. ‘n rekombinante
ensiem) en so 'n pAR-sisteem in ‘n rekombinante gis te inkorporeer, word uitdrukking onder
lae temperature onder beide aërobiese- en anaërobiese toestande geïnduseer (en sodoende
word ‘n reguleerbare sisteem geskep). Die sisteem het wyer toepassing om sein-sensitiewe
domeine van ander transkripsiefaktore te identifiseer. Die ontwerp van die stelsel maak dit
moontlik om 'n skakel tot die transaktiveerder by te voeg wat óf sensitiwiteit tot 'n spesifieke
sein skep, óf die reguleerder vanaf seinafhanklikheid verlos. So word ‘n bruikbare stelsel vir
die bestudering van ander transaktivators en hul domeine met onbekende funksie geskep – ‘n
“werksesel en prospekteerder in een”.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/16512 |
| Date | 10 1900 |
| Creators | Conradie, E. C. (Elizabeth Cornelia) |
| Contributors | Van Zyl, W.H., University of Stellenbosch. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xviii, 221 leaves : ill. |
| Rights | University of Stellenbosch |
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