Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Starch-containing agricultural crops are widely available as feedstocks for the production
of fuel ethanol, potable spirits or beer, single-cell protein (animal feed) and high-fructose
corn syrups (sweeteners). Starch-rich crops, such as maize, rye, barley and wheat, are
usually used for the production of whisky. One of the first steps in the production of whisky
is to boil the raw starch at temperatures exceeding 100°C. This gelatinisation step is
performed to disrupt and solubilise the starch granules to make them more accessible for
enzymatic hydrolysis. After this cooking process, the starch is liquefied by a-amylase and
then saccharified by glucoamylase and a debranching enzyme.
Lipomyces kononenkoae and Saccharomycopsis fibuligera secrete highly effective
a-amylases and glucoamylases, making them two of the most efficient raw-starchdegrading
yeasts known. However, L. kononenkoae and S. fibuligera cannot be used in
existing industrial fermentations because of their low ethanol tolerance, slow growth rate,
catabolite repression, poorly characterised genetics and lack of GRAS (Generally
Regarded As Safe) status.
This study is divided into two sections. The aim of the first section was to clone a gene
(LKA2) encoding a novel starch-degrading enzyme, a second a-amylase (Lka2p) from
L. kononenkoae. LKA2 was cloned into a multicopy plasmid, the yeast episomal plasmid,
YEp352, under the control of the phosphoglycerate kinase promoter (PGK1 p) and
terminator (PGKh) expression cassette. This recombinant plasmid was designated
pJUL3 and transformed into a laboratory strain of S. cerevisiae, I1278b. Plate and liquid
assays revealed that the recombinant yeast secreted active a-amylase into the medium.
The optimum pH for Lka2p was pH 3.5 and the optimum temperature 60°C.
The aim of the second part of the study was to construct recombinant strains of
S. cerevisiae secreting a-amylase and/or glucoamylase. The individual genes were cloned
into a yeast-integrating plasmid, Ylp5, under the control of the PGK1p-PGK1.,-expression
cassette. Two indigenous yeasts were selected on the basis of their ability to utilise raw
starch, L. kononenkoae and S. fibuligera, as gene donors. Eight constructs containing the
L. kononenkoae a-amylase genes, LKA 1 and LKA2, and the S. fibuligera a-amylase
(SFA 1) and glucoamylase (SFG1) genes were prepared: four single-cassette plasmids
expressing the individual coding sequences under the control of the PGK1 p-PGK1.,-
expression cassette, resulting in plPLKA 1, pIPLKA2, plPSFA 1 and pIPSFG1, respectively;
two double-cassette plasm ids (expressing both LKA 1 and LKA2 under the control of the
PGK1p-PGK1 .,-expression cassette, and SFA 1 and SFG1 under their respective native
promoters and terminators), resulting in pIPLKA1/2 and pIPSFAG, respectively, and two
single-cassette plasmids expressing SFA 1 and SFG1 with their native promoters and terminators, resulting in pSFA 1 and pSFG1, respectively. The respective constructs were
transformed into a laboratory strain of S. cere visiae , L1278b. By homologous
recombination, each plasmid was integrated into the yeast genome at the ura3 locus.
S. cerevisiae L:1278b that had been transformed with plPLKA 1/2, LKA 1 and LKA2 under
the control of the PGK1 rrPGK1,expression cassette resulted in the highest levels of
a-amylase activity when assayed for amylolytic activity in a liquid medium. This
recombinant strain resulted in the most efficient starch utilisation in batch fermentations,
consuming 80% of starch and producing 6 gIL of ethanol after 156 hours of fermentation.
The strain expressing SFG1 under the control of the PGK1rrPGK1,expression cassette
gave the highest levels of glucoamylase activity.' These results confirmed that
co-expression of a-amylase and/or glucoamylase synergistically enhance starch
degradation.
This study paves the way for the development of efficient starch-degrading strains of
S. cerevisiae for the production of whisky, beer and biofuel ethanol. / AFRIKAANSE OPSOMMING: Styselbevattende landbougewasse kom wydverspreid voor as die substraat vir die
produksie van brandstofetanol, drinkbare spiritualië of bier, enkelselproteïen en hoëfruktose
graanstroop. Styselbevattende gewasse, soos mielies, rog, gars en koring, word
gewoonlik vir die produksie van whisky gebruik. Die eerste stap in die produksie van
whisky is om die stysel by temperature bo 1DOOG te kook. Hierdie jelatinisasie stap word
uitgevoer om die styselkorrels te versteur en vloeibaar te maak sodat hulle meer toeganklik
vir ensimatiese hidrolise is. Na dié kookproses word die stysel deur o-arnilases vervloei en
dan deur glukoamilases en 'n vertakkingsensiem versuiker.
Lipomyces kononenkoae en Saccharomycopsis filuligera skei hoogs effektiewe a-amilases
en glukoamilases uit, wat dit twee van die effektiefste rou-stysel-afbrekende giste bekend,
maak. L. kononenkoae en S. fibuligera kan egter nie in reeds bestaande industriële
fermentasies gebruik word nie, as gevolg van hulle lae etanoltoleransie, stadige
groeitempo, katabolietonderdrukking, swak gekarakteriseerde genetika en gebrek aan
ABAV (Algemeen Beskou As Veilig) status.
Hierdie tesis is in twee afdelings verdeel. Die doel van die eerste deel was om 'n geen
(LKA2) wat vir 'n nuwe, unieke styselafbrekende ensiem kodeer, te kloneer, 'n tweede
a-amilase (Lka2p) van L. kononenkoae. LKA2 is in 'n multikopie plasmied, die gis
episomale plasmied, YEp352, onder beheer van die fosfogliseraatkinasepromotor- en
termineerder-kasset (PGK1 p-PGK1 r), gekloneer. Hierdie rekornbinante plasmied is pJUL3
genoem en in 'n laboratoriumras van Saccharomyces cerevisiae, L:1278b, getransformeer.
Plaat- en vloeibare-ensiem toetse het getoon dat die rekombinante gis aktiewe a-amilase
in die medium uitskei. Die optimum pH vir Lka2p is 3.5, is en die optimum temperatuur
60oG.
Die doel van die tweede deel van die studie was om rekombinante rasse van S. cerevisiae
te konstrueer wat a-amilases en/of glukoamilases uitskei. Die individuele gene is toe in 'n
gis-integreringsplasmied, Ylp5, onder beheer van die PGK1p-PGK1,ekspressiekasset,
gekloneer. Twee inheemse giste is op grond van hulle vermoë om stysel te benut
geselekteer, L. kononenkoae en S. filuIigera, as geen donors. Agt konstrukte bevattende
die L. kononenkoae se a-amilasegene, LKA 1 en LKA2, en S. filuligera se a-amilasegeen
(SFA 1) en glukoamilasegeen (SFG1), moes gekonstrueer word: vier _enkel-kasset
plasmiede wat die individuele koderende sekwense onder beheer van die PGK1 p-PGK1,
ekspressiekasset uitdruk, wat onderskeidelik plPLKA 1, pIPLKA2, plPSFA 1 en plPSFG1
lewer; twee dubbel-kasset plasmiede (wat beide LKA 1 en LKA2 onder beheer van die
PGK1 p-PGK1,ekspressiekasset, en SFA 1 en SFG1 met hulle onderskeie inheemse promotors en termineerders) uitdruk, wat onderskeidelik pIPLKA1/2 en plPSFAG lewer, en
twee enkel-kasset plasmiede wat SFA 1 and SFG1 met hulonderskeie inheemse
promotors en termineerders, en wat onderskeidelik pSFA 1 en pSFG1 lewer. Die
onderskeie konstrukte is in 'n laboratoriumras van S. cerevisiae, L1278b, getransformeer.
Deur middel van homoloë rekombinasie, is die onderskeie plasmiede in die ura3-lokus van
die gisgenoom geïntegreer. S. cerevisiae L1278b, getransformeer met plPLKA 1/2, LKA 1
en LKA2 onder die beheer van die PGK1 ~PGK1 ïekspressiekasset, het die hoogste
vlakke van a-amilase aktiwiteit gelewer toe dit vir amilolitiese aktiwiteit in vloeibare medium
getoets is. Hierdie rekombinante ras het stysel die effektiefste benut, nl. 80% van die
stysel en 'n opbrengs van 6 gIL etanol na 156 ure in lotfermentasies. Die ras wat SFG1
onder beheer van die PGK1~PGK1ïekspressiekasset uitdruk, het die hoogste vlakke van
glukoamilase-aktiwiteit gelewer. Hierdie resultate bevestig dat die gesamentlike
uitdrukking van a-amilase- en/of glukoamilase-ensieme styselafbreking sinergisties
. bevorder.
Hierdie studie baan die weg vir die ontwikkeling van 'n effektiewe styselfermenterende ras
van S. cerevisiae wat moontlik gebruik kan word vir die produksie van whisky en
biobrandstofalkohol.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52745 |
| Date | 12 1900 |
| Creators | Eksteen, Jeremy Michael |
| Contributors | Pretorius, I. S., Van Rensburg, P., 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 | English |
| Type | Thesis |
| Format | 91 p. : ill. |
| Rights | Stellenbosch University |
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