Dissertation (PhD)--Stellenbosch University, 2007. / PCT patent registered: https://www.google.com/patents/WO2009034414A1?cl=en&dq=pct/ib2007/004098&hl=en&sa=X&ei=b7AxUsSZK4jB0gWi14HgCQ&ved=0CEkQ6AEwAg
USA: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en / USA patent registered: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en / ENGLISH ABSTRACT: The conversion of cellulosic biomass into fuels and chemicals has the potential to positively
impact the South African economy, but is reliant on the development of low-cost conversion
technology. Perhaps the most important progress to be made is the development of “consolidated
bioprocessing” (CBP). CBP refers to the conversion of pretreated biomass into desired
product(s) in a single process step with either a single organism or consortium of organisms and
without the addition of cellulase enzymes. Among the microbial hosts considered for CBP
development, Saccharomyces cerevisiae has received significant interest from the biotechnology
community as the yeast preferred for ethanol production. The major advantages of S. cerevisiae
include high ethanol productivity and tolerance, as well as a well-developed gene expression
system. Since S. cerevisiae is non-cellulolytic, the functional expression of at least three groups
of enzymes, namely endoglucanases (EC 3.2.1.4); exoglucanases (EC 3.2.1.91) and
β-glucosidases (EC 3.2.1.21) is a prerequisite for cellulose conversion via CBP. The endo- and
exoglucanases act synergistically to efficiently degrade cellulose to soluble cellodextrins and
cellobiose, whereas the β-glucosidases catalyze the conversion of the soluble cellulose hydrolysis
products to glucose. This study focuses on the efficient utilization of cellobiose by recombinant
S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize
cellobiose intracellularly.
Since it is generally accepted that S. cerevisiae do not produce a dedicated cellobiose
permease/transporter, the obvious strategy was to produce a secretable β-glucosidase that will
catalyze the hydrolysis of cellobiose to glucose extracellularly. β-Glucosidase genes of various
fungal origins were isolated and heterologously expressed in S. cerevisiae. The mature peptide
sequence of the respective β-glucosidases were fused to the secretion signal of the
Trichoderma reesei xyn2 gene and expressed constitutively from a multi-copy yeast expression
vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The
resulting recombinant enzymes were characterized with respect to pH and temperature optimum,
as well as kinetic properties. The maximum specific growth rates (μmax) of the recombinant
strains were compared during batch cultivation in high-performance bioreactors. S. cerevisiae
secreting the recombinant Saccharomycopsis fibuligera BGL1 enzyme was identified as the best
strain and grew at 0.23 h-1 on cellobiose (compared to 0.29 h-1 on glucose). More significantly, was the ability of this strain to anaerobically ferment cellobiose at 0.18 h-1 (compared to 0.25 h-1
on glucose).
However, extracellular cellobiose hydrolysis has two major disadvantages, namely glucose’s
inhibitory effect on the activity of cellulase enzymes as well as the increased risk of
contamination associated with external glucose release. In an alternative approach, the secretion
signal from the S. fibuligera β-glucosidase (BGL1) was removed and expressed constitutively
from the above-mentioned multi-copy yeast expression vector. Consequently, the BGL1 enzyme
was functionally produced within the intracellular space of the recombinant S. cerevisiae strain.
A strategy employing continuous selection pressure was used to adapt the native S. cerevisiae
disaccharide transport system(s) for cellobiose uptake and subsequent intracellular utilization.
RNA Bio-Dot results revealed the induction of the native α-glucoside (AGT1) and maltose
(MAL) transporters in the adapted strain, capable of transporting and utilizing cellobiose
intracellularly. Aerobic batch cultivation of the strain resulted in a μmax of 0.17 h-1 and 0.30 h-1
when grown in cellobiose- and cellobiose/maltose-medium, respectively. The addition of
maltose significantly improved the uptake of cellobiose, suggesting that cellobiose transport (via
the combined action of the maltose permease and α-glucosidase transporter) is the rate-limiting
step when the adapted strain is grown on cellobiose as sole carbon source. In agreement with the
increased μmax value, the substrate consumption rate also improved significantly from
0.25 g.g DW-1.h-1 when grown on cellobiose to 0.37 g.g DW-1.h-1 upon addition of maltose to the
medium. The adapted strain also displayed several interesting phenotypical characteristics, for
example, flocculation, pseudohyphal growth and biofilm-formation. These features resemble
some of the properties associated with the highly efficient cellulase enzyme systems of
cellulosome-producing anaerobes.
Recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport
and utilize cellobiose intracellularly. Both recombinant strains are of particular interest when the
final goal of industrial-scale ethanol production from cellulosic waste is considered. However,
the latter strain’s ability to efficiently remove cellobiose from the extracellular space together
with its flocculating, pseudohyphae- and biofilm-forming properties can be an additional
advantage when the recombinant S. cerevisiae strain is considered as a potential host for future
CBP technology. / AFRIKAANSE OPSOMMING: Die omskakeling van sellulose-bevattende biomassa na brandstof en chemikalieë beskik oor die
potensiaal om die Suid-Afrikaanse ekonomie positief te beïnvloed, indien bekostigbare
tegnologie ontwikkel word. Die merkwaardigste vordering tot dusvêr kon in die ontwikkeling
van “gekonsolideerde bioprosessering” (CBP) wees. CBP verwys na die eenstap-omskakeling
van voorafbehandelde biomassa na gewenste produkte met behulp van ‘n enkele organisme of ‘n
konsortium van organismes sonder die byvoeging van sellulase ensieme. Onder die mikrobiese
gashere wat oorweeg word vir CBP-ontwikkeling, het Saccharomyces cerevisiae as die voorkeur
gis vir etanolproduksie troot belangstelling by die biotegnologie-gemeenskap ontlok. Die
voordele van S. cerevisiae sluit in hoë etanol-produktiwiteit en toleransie, tesame met ‘n goed
ontwikkelde geen-uitdrukkingsisteem. Aangesien S. cerevisiae nie sellulose kan benut nie, is die
funksionele uitdrukking van ten minste drie groepe ensieme, naamlik endoglukanases (EC
3.2.1.4); eksoglukanases (EC 3.2.1.91) en β-glukosidases (EC 3.2.1.21), ‘n voorvereiste vir die
omskakeling van sellulose via CBP. Die sinergistiese werking van endo- en eksoglukanases
word benodig vir die effektiewe afbraak van sellulose tot oplosbare sello-oligosakkariede en
sellobiose, waarna β-glukosidases die finale omskakeling van die oplosbare sellulose-afbraak
produkte na glukose kataliseer. Hierdie studie fokus op die effektiewe benutting van sellobiose
m.b.v. rekombinante S. cerevisiae-rasse met die vermoeë om sellobiose ekstrasellulêr af te breek
of dit op te neem en intrasellulêr te benut.
Aangesien dit algemeen aanvaar word dat S. cerevisiae nie ‘n toegewyde sellobiosepermease/
transporter produseer nie, was die mees voor-die-hand-liggende strategie die produksie
van ‘n β-glukosidase wat uitgeskei word om sodoende die ekstrasellulêre hidroliese van
sellobiose na glukose te kataliseer. β-Glukosidase gene is vanaf verskeie fungi geïsoleer en
daaropvolgend in S. cerevisiae uitgedruk. Die geprosesseerde peptiedvolgorde van die
onderskeie β-glukosidases is met die sekresiesein van die Trichoderma reesei xyn2-geen verenig
en konstitutief vanaf ‘n multikopie-gisuitdrukkingsvektor onder transkripsionele beheer van die
S. cerevisiae PGK1 promotor en termineerder uitgedruk. Die gevolglike rekombinante ensieme
is op grond van hul pH en temperatuur optima, asook kinetiese eienskappe, gekarakteriseer. Die
maksimum spesifieke groeitempos (μmax) van die rekombinante rasse is gedurende aankweking in
hoë-verrigting bioreaktors vergelyk. Die S. cerevisiae ras wat die rekombinante Saccharomycopsis fibuligera BGL1 ensiem uitskei, was as the beste ras geïdentifiseer en kon teen
0.23 h-1 op sellobiose (vergeleke met 0.29 h-1 op glukose) groei. Meer noemenswaardig is the ras
se vermoë om sellobiose anaërobies teen 0.18 h-1 (vergeleke met 0.25 h-1 op glukose) te
fermenteer.
Ekstrasellulêre sellobiose-hidroliese het twee groot nadele, naamlik glukose se onderdrukkende
effek op die aktiwiteit van sellulase ensieme, asook die verhoogde risiko van kontaminasie wat
gepaard gaan met die glukose wat ekstern vrygestel word. ’n Alternatiewe benadering waarin die
sekresiesein van die S. fibuligera β-glucosidase (BGL1) verwyder en konstitutief uitgedruk is
vanaf die bogenoemde multi-kopie gisuitrukkingsvektor, is gevolg. Die funksionele BGL1
ensiem is gevolglik binne-in die intrasellulêre ruimte van die rekombinante S. cerevisiae ras
geproduseer. Kontinûe selektiewe druk is gebruik om die oorspronklike S. cerevisiae
disakkaried-transportsisteme vir sellobiose-opname and daaropvolgende intrasellulêre benutting
aan te pas. RNA Bio-Dot resultate het gewys dat die oorspronklike α-glukosied (AGT1) en
maltose (MAL) transporters in die aangepaste ras, wat in staat is om sellobiose op te neem en
intrasellulêr te benut, geïnduseer is. Aërobiese kweking van die geselekteerde ras het gedui dat
die ras teen 0.17 h-1 en 0.30 h-1 groei in onderskeidelik sellobiose en sellobiose/maltose-medium.
Die byvoeging van maltose het die opname van sellobiose betekenisvol verbeter, waarna
aangeneem is dat sellobiose transport (via die gekombineerde werking van die maltose permease
en α-glukosidase transporter) die beperkende stap gedurende groei van die geselekteerde ras op
sellobiose as enigste koolstofbron is. In ooreenstemming hiermee, het die substraatbenuttingstempo
ook betekenisvol toegeneem van 0.25 g.g DW-1.h-1, gedurende groei op
sellobiose, tot 0.37 g.g DW-1.h-1 wanneer maltose by die medium gevoeg word. Die
geselekteerde ras het ook verskeie interessante fenotipiese kenmerke getoon, byvoorbeeld
flokkulasie, pseudohife- en biofilm-vorming. Hierdie eienskappe kom ooreen met sommige van
die kenmerke wat met die hoogs effektiewe sellulase ensiem-sisteme van sellulosomeproduserende
anaerobe geassosieer word.
Hierdie studie beskryf die suksesvolle konstruksie van ‘n rekombinante S. cerevisiae ras met die
vermoë om sellobiose ekstrasellulêr af te breek of om dit op te neem en intrasellulêr te benut.
Beide rekombinante rasse is van wesenlike belang indien die einddoel van industriële-skaal
etanolproduksie vanaf selluloseafval oorweeg word. Die laasgenoemde ras se vermoë om
sellobiose effektief uit die ekstrasellulêre ruimte te verwyder tesame met die flokkulasie, pseudohife- en biofilm-vormings eienskappe kan ‘n addisionele voordeel inhou, indien die
rekombinante S. cerevisiae ras as ‘n potensiële gasheer vir toekomstige CBP-tegnologie oorweeg
word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/19455 |
| Date | 03 1900 |
| Creators | Van Rooyen, Ronel, 1976- |
| Contributors | Van Zyl, H. W., 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 | xv, 172 leaves : ill. |
| Rights | Stellenbosch University |
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