Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: QUANTITATIVE YEAST PHYSIOLOGY AND NITROGEN METABOLISM
DURING HETEROLOGOUS PROTEIN PRODUCTION
By Johann F. Görgens
The physiology and nitrogen metabolism of the yeast, Saccharomyces cerevisiae,
during heterologous xylanase production in a defined medium was quantified by the
comparison of isogenic yeast strains, whereby several potential limitations in the
production of the heterologous xylanase could be identified. The presence of global
sensing and regulatory mechanisms, by which the yeast is able to actively regulate
both heterologous gene expression and the physiological response to the process, was
also investigated.
The deleterious effects of heterologous xylanase production on the physiology of the
recombinant host were disproportionately large with respect to the amount of foreign
protein produced. The cellular processes involved in this response were identified by
the transcriptional profiling of isogenic recombinant strains, in a novel analytical
approach to investigating foreign protein production by S. cerevisiae. Heterologous
gene expression affected a combination of cellular processes and induced the yeast
stringent stress response. The corresponding loss of metabolic functionality resulted
in the disproportionate physiological effects of foreign protein production, similar to
previous observations in recombinant Escherichia coli, and a possible reduction in
attainable production levels. Reducing the propensity of recombinant gene expression
to introduce metabolic stress may therefore increase production levels of foreign
proteins by yeast. The metabolic vitality of transformed strains was also reduced by
the presence of multiple copies of active, plasmid-based PGK1-promoters in the cell
without expression of the heterologous gene. The negative effect was caused by an
increase in the biosynthetic and glycolytic capacity of the strain at the expense of
other processes.
Production levels of heterologous xylanase were influenced by expression vector
selection and the presence of auxotrophic mutations in transformed strains of S.
cerevisiae. The increased transcription levels obtained with the multicopy plasmidbased YEp-type expression system, compared to the integrative YIp-type expression
system, resulted in higher levels of xylanase production. Heterologous xylanase
production thus did not saturate the secretory capacity of the host strain. The genetic
stability of the autoselective YEp-type expression system in long-term chemostat
culture was also demonstrated. High levels of heterologous xylanase production by
transformed S. cerevisiae strains containing auxotrophic markers required the
stabilisation of nitrogen metabolism via saturation of yeast cells with an excess of
imported amino acids. By the removal of excessive auxotrophic markers, high levels
of xylanase production by a prototrophic transformant in defined medium without
amino acid addition could be obtained. Heterologous xylanase production by the
prototrophic transformant was further enhanced by increasing the availability of
preferred amino acids or succinate in the defined medium, indicating an additional
requirement for metabolic precursors and building blocks for foreign protein
synthesis. Comparable levels of heterologous xylanase production were obtained in
high cell density cultures of the alternative yeast, Pichia stipitis, by the proper
induction of the native ADH2-promoter, the control of oxygenation, and addition of
an amino acid mixture to the defined medium, indicating the presence of generic
limitations in transcription, nutrient availability and the yeast biosynthetic capacity for
foreign protein production by various yeasts.
The presence of global sensing and regulatory mechanisms was confirmed by the
physiological response of S. cerevisiae to heterologous protein production, which
included the downregulation of biosynthesis and growth, and the induction of various
processes involved in the stringent stress response. Additionally, heterologous
xylanase production was actively regulated on a posttranscriptional level by the
auxotrophic transformants in response to the level of amino acid availability. The
biosynthetic capacity for foreign protein production by both recombinant S. cerevisiae
and P. stiptis was also regulated in response to the physiological state of the yeast and
the availability of nutrients. The presence of these regulatory mechanisms
complicated the manipulation of cellular biosynthesis at will. / AFRIKAANSE OPSOMMING: KWANTITATIEWE GIS-FISIOLOGIE EN -STIKSTOF METABOLISME
GEDURENDE HETEROLOË PROTEÏEN PRODUKSIE
Deur Johann Ferdinand Görgens
Die fisiologie en stikstof-metabolisme van die gis, Saccharomyces cerevisiae,
gedurende heteroloë xilanase produksie in ‘n gedefiniëerde medium is gekarakteriseer
deur isogeniese gis-rasse te vergelyk, waardeur verskeie moontlike beperkings in die
produksie van die heteroloë xilanase uitgewys kon word. Die teenwoordigheid van
globale sensoriese- en beheer-meganismes, wat die gis in staat stel om beide heteroloë
geen uitdrukking en die fisiologiese respons op die proses aktief te reguleer, is ook
ondersoek.
Die nadelige effekte van heteroloë xilanase produksie op die fisiologie van die
rekombinante gasheer-organisme was uitermatig groot in vergelyking met die
hoeveelheid vreemde proteïen wat geproduseer is. Die sellulêre prosesse
verantwoordelik vir hierdie respons is identifiseer deur die transkripsionele profiele van
isogeniese rekombinante rasse te vergelyk, in ‘n nuwe analitiese benadering tot die
bestudering van vreemde proteïen produksie deur S. cerevisiae. Heteroloë geen
uitdrukking het ‘n kombinasie van sellulêre prosesse geaffekteer en die gis se algemene
voedingstres-respons geaktiveer. Die gepaardgaande verlies aan metaboliese funksie
het die uitermatige fisiologiese effek van vreemde proteïen produksie veroorsaak,
soortgelyk aan vorige waarnemings met rekombinante Escherichia coli. Die haalbare
produksie-vlakke is moontlik ook verlaag deur hierdie respons. ‘n Verlaging van die
geneigdheid van rekombinante geen uitdrukking om metaboliese stres te veroorsaak,
mag dus die produksievlakke van vreemde proteïene in gis verbeter. Die metaboliese
groei-potensiaal van die getransformeerde rasse is ook verlaag deur die teenwoordigheid
van etlike aktiewe kopieë van plasmied-gebaseerde PGK1-promotors in die sel, sonder
uitdrukking van die heteroloë geen, deur ‘n toename in die biosintetiese en glikolitiese
kapasiteit ten koste van die ander sellulêre prosesse.
Die produksievlakke van heteroloë xilanase is deur die keuse van uitdrukkings-sisteem
en die teenwoordigheid van autotrofiese mutasies in die getransformeerde rasse van S.cerevisiae beïnvloed. Die verhoogde transkripsie vlakke wat met die multi-kopie,
plasmied-gebaseerde YEp-tipe uitdrukkingsisteem, eerder as die geïntegreerde YIp-tipe
sisteem, verkry is, het tot verhoogde xilanase produksie gelei. Heteroloë xilanase
produksie het dus nie die uitskeidingskapasiteit van die gasheer versadig nie. Die
genetiese stabiliteit van die autoselektiewe, YEp-tipe uitdrukkingsisteem in langtermyn
chemostaat-kulture is ook gedemonstreer. Hoë vlakke van xilanase produksie deur
getransformeerde S. cerevisiae rasse met autotrofiese merkers het die stabilisering van
die stikstof metabolisme, deur die versadiging van die sel met ingevoerde aminosure,
vereis. Die verwydering van oormatige autotrofiese merkers het tot hoë vlakke van
xilanase produksie deur die prototrofiese transformant in gedefinieerde medium sonder
aminosuur byvoeging gelei. Heteroloë xilanase produksie deur die prototrofiese
transformant kon verder verbeter word deur die byvoeging van voorkeur-aminosure of
suksinaat tot die gedefinieerde medium, en ‘n addisionele behoefte aan metaboliese
voorloper-molekules en bou-blokke vir vreemde proteïensintese het dus bestaan.
Vergelykbare vlakke van heteroloë xilanase produksie is in kulture met hoë sel-digthede
van die alternatiewe gis, Pichia stipitis, verkry deur die doeltreffende induksie van die
eiesoortige ADH2-promotor en die byvoeging van ‘n aminosuur-mengsel tot die
gedefinieerde medium, wat die teenwoordigheid van generiese beperkinge in
transkripsie, voedingstof-beskikbaarheid en biosintetiese kapasiteit van die gis vir
vreemde proteïen produksie deur verskeie giste uitgewys het.
Die teenwoordigheid van globale sensoriese- en beheer-meganismes is bevestig deur die
fisiologiese respons van S. cerevisiae tot heteroloë proteïen produksie, wat die afwaartse
regulering van biosintese en groei, en die induksie van verskeie prosesse betrokke by
die algemene voedingstres-respons, ingesluit het. Heteroloë xilanase produksie is ook
op ‘n na-transkripsionele vlak aktief gereguleer deur die autotrofiese transformante in
reaksie tot die vlak van aminosuur beskikbaarheid. Die biosintetiese kapasiteit vir
vreemde proteïen-produksie van beide rekombinante S. cerevisiae en P. stipitis is ook in
reaksie tot die fisiologiese toestand van die gis en die beskikbaarheid van
voedingstowwe gereguleer. Die teenwoordigheid van hierdie regulatoriese meganismes
het die willekeurige manipulasie van sellulêre proteïen-biosintese bemoeilik.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/16051 |
| Date | 04 1900 |
| Creators | Gorgens, Johann Ferdinand |
| Contributors | Knoetze, J. H., Van Zyl, W. H., Hahn-Hagerdal, B., University of Stellenbosch. Faculty of Engineering. Dept. of Process Engineering. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xviii, 246 leaves : ill. |
| Rights | University of Stellenbosch |
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