Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Cellulose, a glucose polymer, is considered the most abundant fermentable polymer
on earth. Agricultural waste is rich in cellulose and exploiting these renewable
sources as a substrate for ethanol production can assist in producing enough
bioethanol as a cost-effective replacement for currently used decreasing fossil fuels.
Saccharomyces cerevisiae is an excellent fermentative organism of hexoses;
however the inability of the yeast to utilize cellulose as a carbon source is a major
obstruction to overcome for its use in the production of bio-ethanol. Cellobiose, the
major-end product of cellulose hydrolysis, is hydrolyzed by -glucosidase or
cellobiose phosphorylase, the latter having a possible metabolic advantage over
-glucosidase. Recently, it has been showed that S. cerevisiae is able to transport
cellobiose. The construction of a cellulolytic yeast that can transport cellobiose has
the advantage that end-product inhibition of the extracellular cellulases by glucose
and cellobiose is relieved. Furthermore, the extracellular glucose concentration
remains low and the possibility of contamination is decreased.
In this study the cellobiose phosphorylase gene, cepA, of Clostridium stercorarium
was cloned and expressed under transcriptional control of the constitutive PGK1
promoter and terminator of S. cerevisiae on a multicopy episomal plasmid. The
enzyme was expressed intracellulary and thus required the transport of cellobiose
into the cell. The fur1 gene was disrupted for growth of the recombinant strain on
complex media without the loss of the plasmid. The recombinant strain,
S. cerevisiae[yCEPA], was able to sustain aerobic growth on cellobiose as sole
carbon source at 30°C with Vmax = 0.07 h-1 and yielded 0.05 g biomass per gram
cellobiose consumed. The recombinant enzyme had activity optima of 60°C and
pH 6-7. Using Michaelis-Menten kinetics, the Km values for the colorimetric substrate
p-nitrophenyl-b-D-glucopyranoside (pNPG) and cellobiose was estimated to be 1.69
and 92.85 mM respectively. Enzyme activity assays revealed that the recombinant
protein was localized in the membrane fraction and no activity was present in the
intracellular fraction. Due to an unfavourable codon bias in S. cerevisiae, CepA
activity was very low. Permeabilized S. cerevisiae[yCEPA] cells had much higher
CepA activity than whole cells indicating that the transport of cellobiose was
inadequate even after one year of selection. Low activity and insufficient cellobiose transport led to an inadequate glucose supply for the yeast resulting in low biomass
formation. Cellobiose utilization increased when combined with other sugars
(glucose, galactose, raffinose, maltose), as compared to using cellobiose alone. This
is possibly due to more ATP being available for the cell for cellobiose transport.
However, no cellobiose was utilized when grown with fructose indicating catabolite
repression by this sugar.
To our knowledge this is the first report of a heterologously expressed cellobiose
phosphorylase in yeast that conferred growth on cellobiose. Furthermore, this report
also reaffirms previous data that cellobiose can be utilized intracellularly in
S. cerevisiae. / AFRIKAANSE OPSOMMING: Sellulose, ‘n homopolimeer van glukose eenhede, word beskou as die volopste
suiker polimeer op aarde. Landbou afval produkte het ‘n hoë sellulose inhoud en
benutting van diè substraat vir bio-etanol produksie kan dien as ‘n koste-effektiewe
aanvulling en/of vervanging van dalende fossielbrandstof wat tans gebruik word. Die
gis, Saccharomyces cerevisiae, is ‘n uitmuntende organisme vir die fermentasie van
heksose suikers, maar die onvermoë van die gis om sellulose as koolstofbron te
benut is ‘n groot struikelblok in sy gebruik vir die produksie van bio-etanol.
Sellobiose, die hoof eindproduk van ensiematiese hidrolise van sellulose, word
afgebreek deur -glukosidase of sellobiose fosforilase. Laasgenoemde het ‘n
moontlike metaboliese voordeel bo die gebruik van -glukosidase vir sellobiose
hidrolise. Daar was onlangs gevind dat S. cerevisiae in staat is om sellobiose op te
neem. Die konstruksie van ‘n sellulolitiese gis wat sellobiose intrasellulêr kan benut,
het die voordeel dat eindproduk inhibisie van die ekstrasellulêre sellulases deur
sellobiose en glukose verlig word. Verder, wanneer die omsetting van glukose vanaf
sellobiose intrasellulêr plaasvind, word die ekstrasellulêre glukose konsentrasie laag
gehou en die moontlikheid van kontaminasie beperk.
In hierdie studie was die sellobiose fosforilase geen, cepA, van Clostridium
stercorarium gekloneer en uitgedruk onder transkripsionele beheer van die
konstitutiewe PGK1 promoter en termineerder van S. cerevisiae op ‘n multikopie
episomale plasmied. Die ensiem is as ‘n intrasellulêre proteïen uitgedruk en het dus
die opneem van die sellobiose molekuul benodig. Die disrupsie van die fur1 geen
het toegelaat dat die rekombinante ras op komplekse media kon groei sonder die
verlies van die plasmied. Die rekombinante ras, S. cerevisiae[yCEPA], het aërobiese
groei by 30°C op sellobiose as enigste koolstofbron onderhou met mmax = 0.07 h-1 en
‘n opbrengs van 0.05 gram selle droë gewig per gram sellobiose. Die rekombinante
ensiem het optima van 60°C en pH 6-7 gehad. Die K m waardes vir die kolorimetriese
substraat pNPG en sellobiose was 1.69 en 92.85 mM onderskeidelik. Ondersoek
van die ensiem aktiwiteit het getoon dat die rekombinante proteïen gelokaliseer was
in die membraan fraksie en geen aktiwiteit was teenwoordig in die intrasellulêre
fraksie nie. CepA aktiwiteit was laag as gevolg van ‘n lae kodon voorkeur in S.
cerevisiae. Verder het geperforeerde S. cerevisiae[yCEPA] selle aansienlik beter CepA aktiwiteit getoon as intakte selle. Hierdie aanduiding van onvoldoende
transport van sellobiose na binne in die sel tesame met die lae aktiwiteit van die
CepA ensiem het gelei tot onvoldoende glukose voorraad vir die sel en min biomassa
vorming. Sellobiose verbruik het toegeneem wanneer dit tesame met ander suikers
(glukose, galaktose, raffinose, maltose) gemeng was, heelwaarskynlik deur die
vorming van ekstra ATP’s vir die sel wat ‘n toename in sellobiose transport teweeg
gebring het. Fruktose het egter kataboliet onderdrukking veroorsaak en sellobiose
was nie benut nie.
Sover ons kennis strek, is hierdie die eerste verslag van ‘n heteroloë sellobiose
fosforilase wat in S. cerevisiae uitgedruk is en groei op sellobiose toegelaat het.
Verder, bewys die studie weereens dat S. cerevisiae wel sellobiose kan opneem.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/19862 |
| Date | 03 1900 |
| Creators | Sadie, Christa J. (Christiena Johanna) |
| 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 | iv, 89 leaves : ill. |
| Rights | Stellenbosch University |
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