Evaluation of S. cerevisiae promoters during
growth on xylose
L. Mande1; W.H. Van Zyl2; I. Ncube1;
D.C. La Grange1
Motivation
•Identify a promoter that is strongly induced during
growth on xylose
• Increase production of recombinant enzymes
needed during the degradation of lignocelluloses
•Recombinant enzymes such as xylanases, beta-
glucosidase, cellulases etc
Introduction
 S.cerevisiae has been used for years in the
production of recombinant proteins
 S.cerevisiae is a widely used organism
- fast cell growth
- tolerate high ethanol concentration
- tolerate wide spectrum of inhibitors
- well-characterised physiology & genetics
- GRAS status
Introduction
 Glucose is the most abundant sugar in
nature (cellulose)
 S. cerevisiae is a Crabtree positive
yeast
 Its produces ethanol on glucose under
aerobic conditions (little biomass)
 High expression level of recombinant
protein is linked to the amount of
biomass obtained during fermentation
(Ferndahl et al., 2010)
Introduction
 Xylose is the second most abundant sugar in nature
and considered a waste in most industries
 S. cerevisiae cannot utilise xylose as a carbon source
 S.cerevisiae engineered to grow on xylose has been
reported to produce more biomass on xylose
 Xylose is the more suitable carbon source in
recombinant protein production
Introduction
 Promoter
- ENO1, ENO2, YG100, PGK1, ADH2,GPD3
 Reporter gene
- Xylanase gene
 Terminator
- ENO1, ENO2, YG100, PGK1, ADH2,GPD3
 Episomal selectable marker
- URA3
promoter
Gene of
interest
Terminator
Promoter
Reporter
gene
Terminator
Marker
Engineering S.cerevisiae(Y294)
sXI gene
(B.thetaiotaomicron)
XYL3 (P. stipitis)
Fungi
Bacteria
D-xylose
D-xylose
NAD(P)H
Xylose reductase
NAD(P)+
Xylose isomerase
D-xylitol
GRE3
NAD+
Xylitol dehydrogenase
NADH
D-xylulose
ATP
Xylulokinase
D-xylulose-5-P
D-xylulose
Xylulokinase
ATP
D-xylulose-5-P
Restriction map of the plasmids
SalI (7668)
HindIII (7650)
BamHI (7680)
XhoI (7379)
BglII (7372)
XhoI (7229)
PGK1t 1
EcoRI (6592)
XYN2
PGK1p
pP GK1
7724bp
HindIII (5111)
URA3
ApaI (4393)
Figure 1. pPGK1 used as base for the construction of all
episomal plasmids.
Results and Discussion
A
B
Figure 2. Engineered S.cerevisiae on xylose and SC –URA3
with glucose.
Results and Discussion
Growth of engineered and wild type
S.cerevisiae on xylose
Growth of engineered and wild
type S.cerevisiae on glucose
16
20
18
14
16
12
12
OD600nm
OD600nm
14
10
8
10
8
6
6
4
4
2
2
0
0
10
20
30
40
50
Incubation Time
60
70
80
0
0
50
100
150
200
Incubation Time (Hour)
Figure 3.Growth curve of the wild type and engineered yeast
on glucose and on xylose
Results and Discussion
 Transformants spotted on RBB-xylan plates with different
carbon source
RBB-xylan xylose
RBB-xylan glucose
Figure 4. The RBB-xylan plates used to confirm xylanase
activity
Results and Discussion
 DNS ASSAY was done to determine the xylanase activity
following the method by (Bailey et al, 1992)
Figure 5. Determination of the amount of xylanase enzyme
produced using DNS assay
Results and Discussion
Enzyme activity
250
Control
200
PGK1
nkat/ml
150
ENO1
100
ENO2
50
ADH2
0
0
50
100
150
200
Time (h)
250
300
Figure 6.. .Determination of the amount of xylanase enzyme
produced using DNS assay
Construction of fur1::LEU2
strains
Glutamine
De novo
synthesis
Orotidine 5'-P
FUR1
Uridine 5'-P
Uracil
URA3
Uridine 5'-P
Cytidine 5'-PPP
DNA
Kern et al., 1990
Construction of fur1::LEU2 strains
pUC
plasmid
FUR1
1
FUR1 gene
disruption
3.27
pDF1
fur1'
LEU2
FUR1 gene
replacement
Yeast
chr 8R
FUR1
'fur1
1.33
2
3
FUTURE WORK
Evaluation of episomal promoters during growth
on xylose
Determine metabolic burden during growth on
xylose in the bioreactor
ACKNOWLEDGEMENT
Many thanks Dr. La Grange
Prof Ncube and Prof van Zyl
Dr V. Mbazima
Van Zyl lab (Stellenbosch University)
Department of BMBT (University of Limpopo)
RSES for financial assistance
Thank you …………………
Fur1 Disruption
 fur1::LEU2 allele was Isolated as 3.27kb from pDF1 plasmid
NcoI-NsiI restriction enzymes
Disrupted using the gene replacement method
Introduction
(Aristidou and Pentila,2000)
Transformation of integrated plasmids
 Transformation
- Transformation in S.
cerevisiae
S228c strain
- Transformants selected on
geneticin (G418)
NotI
1 40-ura
PGK1p
Amp
uPGK1x
XhoI (1583)
PGK1t
NotI
HindIII
(3515)
40-ura
G418
HindIII (2939)
Promoter
XYN2
PGK1
Marker
PacI (1044)
XYN2
5754bp
SalI (3497)
NotI (3490)
U
EcoRI (396)
NotI (424)
ApaI (473)
U
AscI (1723)
EcoRI (2007)
Integrating expression cassettes
 PGK1p and PGK1t isolated from plasmid by restriction
digest
 G418 isolated from plasmid by PCR
 40bp URA3 overhangs added to expression cassette
(target cassette to URA3-locus)
 Expression cassette cloned into pUC19
U
Promoter
XYN2
PGK1
Marker
U
Transformation
Rapid genomic DNA isolation,
“Bust and Grab” method
Construction of the strain that grows on xylose
 S. cerevisiae Y294 (Matα leu 2-3, 112 ura 3-52,his 3,trp1289)
 Transformation with pMJM121 with Synthetic codon
optimised xylose isomerase
 B. thetaiotaomicron XI
 Selective marker (zeocin)
 Disruption cassette (gre3::Xyl3Hygromycin) was used to
knock out GRE3 gene
RESULTS
U Promoter
XYN2
Marker
U
Promoter
XYN2
A
PGK1t
XYN2
B
10000
8000
6000
5000
ADH2
1735bp
4000
3500
3000
2500
2000
PFK2
1462bp
1500
XYN2
610 bp
1000
750
PFK1
909bp
GAL10
1228bp
ENO1
897bp
500
250
Figure 2: (A) Agarose gel electrophoresis of the PCR with XYN2 primers (B) Gel
electrophoresis of PCR with promoter specific primer and XYN2 right primer