Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and
involve the interplay of a multitude of proteins. A unique collection of thermosensitive
secretory mutants allowed scientists to demonstrate that the secretory pathway of the
yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All
proteins commence their journey in the endoplasmic reticulum, where they undergo
amino-linked core glycosyl modification. After passage through the Golgi apparatus,
where the remodelling of the glycosyl chains is completed, proteins are transported to
their final destinations, which are either the cell surface, periplasmic space or the
vacuole.
Proteins destined for secretion are usually synthesised with a transient
amino-terminal secretion leader of varying length and hydrophobicity, which plays a
crucial role in the targeting and translocation of their protein cargo. Considerable
effort has been made to elucidate the molecular mechanisms involved in these
processes, especially due to their relevance in a rapidly expanding biotech industry.
The advantages of S. cerevisiae as a host for the expression of recombinant
proteins are well documented. Unfortunately, S. cerevisiae is also subject to a
number of drawbacks, with a relative low product yield being one of the major
disadvantages.
Bearing this in mind, different secretion leaders were compared with the aim of
improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the
S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for
its ability to degrade raw starch and an improved secretion of its amylase enzymes
from S. cerevisiae paves the way for a potential one-step starch utilisation process.
Three sets of constructs were prepared containing the LKA 1 and LKA2 genes
separately under secretory direction of either their native secretion leader, the
S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1
secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The
inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p
proteolytic processing of the leader/protein fusion. Strains expressing the amylase
genes under their native secretion leaders resulted in the highest saccharolytic
activity in the culture medium. In contrast to this, strains utilising the synthetic
secretion leader produced the highest fermentation yield, but had a lower than
expected extracellular activity. We hypothesise that the native amylase leaders may
function as intramolecular chaperones in the folding and processing of their
passenger proteins, thereby increasing processing efficiency and concomitant
enzyme activity. / AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en
betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke
versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat
gestelom die ooreenkoms tussen die sekresiepad van die gis
Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle
proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook
amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur
die Golgi-apparaat beweeg het, waar die laaste veranderings aan die
glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die
seloppervlak, die periplasmiese ruimte of die vakuool, vervoer.
Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike,
amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul
proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend
om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as
gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf.
Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante
proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die
relatiewe lae produkopbrengs die belangrikste is.
Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met
die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die
S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is
bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy
amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap
styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene
onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die
S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n
sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van
'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p
proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene
onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese
aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die
sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar
met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse
amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul
proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53364 |
| Date | 12 1900 |
| Creators | Kriel, Johan Hendrik |
| Contributors | Van Rensburg, P., Cordero Otero, R. R., Pretorius, I. S., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 69 pages : illustrations |
| Rights | Stellenbosch University |
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