Hansenula polymorpha
as a new promising organism for
high temperature alcoholic
fermentation of lignocellulose sugars
Andriy A. Sibirny1,2
1Institute
of Cell Biology, NAS of Ukraine, Lviv, Ukraine
2Department of Metabolic Engineering, Rzeszow University, Poland
Glucose and xylose are the main components
of agricultural residues
Corn fiber
glucose
Corn cobs
xylose
Soybean stalks
galactose
Peanut hulls
arabinose
mannose
Sunflower
stalk
Wheat straw
0
10
20
30
40
50
60
Percentage of total dry weight
70
80
Agricultural and wood wastes can be major
income sources
Harvest residues are significant resources.
Processing residues are particularly
economical.
Fermentation to ethanol is technically feasible.
The conversion process is becoming
economical.
Bioconversion adds value to farm products.
Reasons for research
Xylose is a prevalent sugar in fast growing
hardwoods and agricultural residues
Its utilization is essential for the conversion of
lignocellulosic residues to fuels and chemicals
Yeasts are among the best organisms for
producing ethanol and other products by
fermentation
Lignocelulose
Preliminary treatment:
grinding, steaming, weak acid
treatment
Simultaneous
saccharification and
fermentation
(enzymatic hydrolysis and
ethanol fermentation)
500С
Acid hydrolysis
Ethanol
fermentation
300С
Ethanol
Reasons for study of the yeast Hansenula
polymorpha as a fermenter of lignocellulose
sugars
H. polymorpha is a thermotolerant yeast. Its maximal
growth temperature is 49 – 50O C. It is able to
ferment xylose to ethanol at elevated temperatures.
Efficient fermentation at high temperatures is
important for development of the Simultaneous
Saccharification and Fermentation (SSF) technology.
H. polymorpha is among the best yeasts for
producing ethanol from xylose
Xylose
Xylose is the second most abundant
sugar in nature
This yeasts have a complete xylose
metabolic pathway
They can convert xylose to ethanol
XOR
NAD(P)H
[Xylitol]
XDH
NAD
Xylulose
XUK
ATP
Xylulose-5-P
Glucose
Xylose
Hxk1, Hxk2
Glk1
G6pd
Glucose-6-P
6-phosphoglucono-δ-lactone
Xor
Pgi1
Fructose-6-P
Pfk1, Pfk2
Fbp1
Ribulose-5-P
Rki1
Fba1
Ribose-5-P
Glyceraldehyde-3-P
G3pdh
Glyceraldehyde-3-P
Pgk
Fructose-6-P
+
Gpm1
2-P-glycerate
Eno1, Eno2
NAD
NADH
Phosphoenolpyruvate
Glyceraldehyde-3-P
Acetate
Pdc1, Pdc5
Acetaldehyde
Dha1
Xylulose-5-P
Sedoheptulose-7-P
Erythrose-4-P
Tkl1,Tkl2
Pathways of glucose and xylose
fermentation in yeasts
Pyk1
Acs1, Acs2
Xks
Tal1,Tal2
3-P-glycerate
Acetyl-CoA
Xylulose
Rpe1
Tkl1,Tkl2
1,3-bisphospho glycerate
Pyruvate
Xid
6Pg1, 6Pg2
Fructose-1,6-dP
Pdh
Xylitol
6-phosphogluconate
Adh1, Adh2
Ethanol
Scheme of mixed culture alcoholic fermetation
using pet- S. cerevisiae and gcr1 H. polymorpha
Saccaromyces yeast
culture
Ethanol
Fermenter
Catalytic convertor
Methylotrophic yeast
culture
Saccaromyces yeast
Methylotrophic yeast
Chemical catalysis
Glucose
Ethanol
Acetaldehyde
Acetaldehyde
Ethanol
Acetaldehyde
Ethanol
Medium
Glucose
Ethanol
Selection of the H. polymorpha gcr1 mutants
UVmutagenesis
Selection
33 (gcr1)
UVmutagenesis
Selection
7-4A
(gcr1 EAO)
33 (gcr1)
22 (leu10)
2-dGlc + Mth
Staining of the colonies for a highest
AO-activity in the presence of NaN3
Mutants H. polymorpha gcr1 defective in glucose
repression of alcohol oxidase
EAO2
Gcr1-GFP
AO activity, U/mg
1 мкм
6
5
4
3
2
1
0
Glc
Mth
Glc
Mth
Glc
22 wt
7-4А C-105
H.pol. strains - growth substrate
Concentration, g/l
Ethanol and acetaldehyde formation during
mixed cultivation of pet- S.cerevisiae and gcr1 H.
polymorpha in glucose (2%) medium
20
Glucose
15
Ethanol in medium
10
Acetaldehyde in
medium
Evaporated
ethanol
Evaporated
acetaldehyde
5
0
0
5
10
15
20
25
AOX activity i cells,
u/mg
Time, h.
1,5
1
0,5
0
0
5
10
15
20
25
Time, h.
Acetaldehyde formation by Hansenula
polymorpha gcr1 mutant 7-4A in the glucose (2%)
containing medium
3
1
0,9
Concentration, g/l
0,7
Ethanol in
medium
0,6
Acetaldehyde
in medium
0,5
Evaporated
ethanol
Evaporated
acetaldehyde
0,4
0,3
0,2
Concentration, g/l
2,5
0,8
Ethanol in
medium
2
Acetaldehyd
e in medium
Evaporated
ethanol
1,5
Evaporated
acetaldehyde
1
0,5
0,1
0
0
0
10
20
300C
30
40 50
Time, h.
0
10
20
30
370C
40
50
Time, h.
2
0,5
0,45
0,4
0,35
0,3
0,25
0,2
0,15
0,1
0,05
0
1,8
Ethanol in
medium
Acetaldehyde
in medium
Evaporated
ethanol
Evaporated
acetaldehyde
1,6
Concentration, g/l
Concenration, g/l
Acetaldehyde formation by Hansenula
polymorpha gcr1 mutant 7-4A in the xylose (2%)
medium
1,4
Ethanol in
medium
1,2
Acetaldehyde
in medium
1
0,8
Evaporated
ethanol
0,6
Evaporated
acetaldehyde
0,4
0,2
0
0
10 20
300C
30 40 50
Time, h.
0
20
40
370C
60
Time, h.
Ethanol formation
by different Hansenula polymorpha strains (100h)
10
9
Glucose
Xylose
Maltose
Mannose
Cellobiose
Galactose
L-arabinose
Ethanol, mg/ml
8
7
6
5
4
3
2
1
0
KT2
356
ML6
ML3 ML8
ML9
CBS
4732
N95
1,2
14
12
1
Biomass, mg/ml
Productivity of fermentation, mg of
ethanol/mg of biomass
Growth and fermentation of glucose and xylose
of riboflavin-deficient H. polymorpha strain
10
0,8
Glucose
Xylose
0,6
0,4
0,2
0
0
20
40
60
80 100
Riboflavin concentration, mg/ml
8
Glucose
Xylose
6
4
2
0
0
20 40 60 80 100
Riboflavin concentration, mg/ml
Method of screening of mutants
with enhanced fermentation activity
S. cerevisiae
UV-treated H.
polymorpha or
P. stipitis yeast
colony
Mineral
medium
with 0,53% of
xylose S.
cerevisiae
suspension
Maximal ethanol production in the medium with
xylose (2%) of the H. polymorpha wild-type strain and
the mutants producing elevated amounts of ethanol
within 60 h. of incubation
2,5
Ethanol, mg/ml
2
1,5
1
0,5
0
WT
S1
S2
S3
S4
S5
S6
S7
S8
S9
Yeast strain
* WT – wild type CBS4732
Xylose fermentation rates by wild type strains of
H. polymorpha and P. stipitis
Ethanol, g/l
300C
370C
20
5
0
CBS6054 CBS6054
Pichia stipitis
KT2
#356
CBS4732
Hansenula polymorpha
ML3
Xylose fermentation by the Hansenula polymorpha
and Pichia stipitis strains at high temperatures
0.04
0.035
480C
370C
300C
500C
Hp – H. polymorpha
NCYC495 leu1-1
0.03
Fermentation productivity,
g ethanol/g biomass/hour.
Ps – P. stipitis
CBS6054
0.025
HpAAR - NCYC495
leu1-1 AllAlcRes
0.02
HpHSP - NCYC495
leu1-1(HSP104)
0.015
0.01
HpATH - - NCYC495
leu1-1(ΔATH1)
0.005
0
Ps
Hp
Ps
Hp
Ps
Hp
HpAAR
Ps
Hp
HpHSP
Hp
ATH-
Efficient fermentation at high temperatures is important for development of the
Simultaneous Saccharification and Fermentation (SSF) technology.
Linear schemes of constructs for deletions of the
XYL1, XYL2-A and XYL2-B genes of H. polymorpha
B Sm KSc RI
Sp P
H
5’-HpXYL1
K
RI
Sl
3’-HpXYL1
ScLEU2
ORI
P
Sc 5’-HpXYL2-A K
ORI
bla
p19L2LRA (~7.5 kb)
lacZ
Sp
H
Sl 3’-HpXYL2-B
lacZ
CaADE2
bla
pLV2 (~8.8 kb)
Sm
K
ORI
5’-Hp XYL2-B
Nt
Nt
ZeoR
Sl
H
3’- HpXYL2-B
lacZ
bla
pLR-260-Z (~7 т.п.н.)
Linear schemes of plasmids carrying bacterial xylA
genes under the H. polymorpha GAP promoter
B
HpGAPpr
ORI
H
H
Sc Bg
HpAOXtr
Ec XylA ORF
K
RI
LEU2
Err
pScoel (9 kb)
1.3 kb
3.2 kb
B
ORI
H
H
HpGAPpr
Scoel XylA ORF
1.2 kb
3.1 kb
Sc Bg
HpAOXtr
K
RI
LEU2
Err
pEc ( 8.9 kb )
Linear scheme of the plasmid pGLG61_xylAEc for
multicopy integration into H. polymorpha genome
H
H
Xb RI
B
bla HpAOX tr Ec xylA ORF Hp GAP pr
Sc LEU2
GAP
pr
P
APH
TEL188
ORI
pGLG61_XylAEc ~ 9 т.п.н.
S. cerevisiae genome fragment containing the LEU2 gene is shown as grey box;
fragment containing the HpGAP promoter: yellow box; fragment containing the HpAOX
terminator: blue box; fragment containing the ORF of E. coli xylA gene: red box; fragment
containing the TEL188 telomeric fragment: black box; the APH gene of E. coli under control
of the truncated GAP promoter of H. polymorpha are shown as violet and green boxes,
respectively; the bacterial part: thin line.
Restriction sites: H, Hind III;; RI, EcoR I; B, BamH I; Xb, XbaI; P, PstI.
Growth and xylose isomerase activity of the H.
polymorpha Δxyl1 transformants containing
bacterial E. coli and S. coelicolor xylA genes
70
XI activity, U/mg prot.
Biomass, mg/ml
3
2,5
2
1,5
1
0,5
0
1
2
3
4
5
6
Growth in the liquid minimal medium with
4% xylose as a sole carbon source:
1-Δxyl1
2, 3 - Δxyl1(pEc)
4,5 - Δxyl1(pScoel)
6 – CBS4732 leu2-2
60
50
40
30
20
10
0
1
2
3
4
5
Xylose isomerase activity:
1-Δxyl1
2, 3 - Δxyl1(pEc)
4,5 - Δxyl1(pScoel)
6 – E. coli
6
Growth and xylose isomerase activity of the H.
polymorpha Δxyl1 Δxyl2-A transformants containing
bacterial E. coli xylA gene
3,5
XI activity, U/mg prot.
35
Biomass, mg/ml
3
2,5
2
1,5
1
0,5
0
1
2
3
4
5
30
25
20
15
10
5
0
6
strain
Growth in the liquid minimal medium with 4%
xylose as a sole carbon source:
1 - Δxyl1,
2 - Δxyl1 Δxyl2А
3, 4, 5 - transformants
Δxyl1Δxyl2А(pGLG_xylAEc)
6 - CBS 4732 leu2-2
1
2
3
4
5
The xylose isomerase activity:
1 - Δxyl1,
2 - Δxyl1 Δxyl2А
3, 4, 5 - transformants
Δxyl1Δxyl2А(pGLG_xylAEc)
6 - E. coli
strain
Isolation of the spontaneous large colonies among the
H. polymorpha transformants Δxyl1Δxyl2A(pGLG_xylAEc)
Growth analysis of the
large colonies in the
liquid medium with 2 %
xylose
YNB +
1 % xylose
Spontaneous large colonies of the
transformants Δxyl1Δxyl2A(pGLG_xylAEc)
Xylose isomerase
activity assay
Determination of
ethanol production
during xylose
fermentation
Growth of the H. polymorpha strains in the minimal
medium with 2 % xylose at 37о С; 5 days
Biomass,
mg/ml
16
14
12
10
8
6
4
2
0
1
2
3
4
5
6
7
8
9
10
11
strain
1 - CBS 4732 leu2-2
2 - Δxyl1 Δxyl2-A
3 - transformant Δxyl1Δxyl2-A(pGLG61_xylAEc)
4 – 11 – large clones of the transformant Δxyl1Δxyl2-A(pGLG61_xylAEc)
Alcoholic xylose fermentation by the H.
polymorpha Δxyl1 Δxyl2-A(pGLG_xylAEc)
strains at 370C
CBS4732 leu2-2
0,6
Ethanol, g/l
0,5
Δxyl1Δxyl2-A
0,4
Δxyl1 Δxyl2-A/pGLG-xylAEc
#4S
0,3
Δxyl1 Δxyl2A(pGLG_xylAEc) #4L3
0,2
0,1
Δxyl1 Δxyl2-A/pGLG-xylAEc
#4L4
0
0
0,5
1
Days
1,5
2
Δxyl1 Δxyl2-A/pGLG-xylAEc
#4L5
Xylitol dehydrogenase activities of the H.
polymorpha Δxyl1 Δxy2-A and Δxyl1 Δxy2-A
Δxy2-B mutants
XDH activity, U/mg
0,3
0,25
0,2
0,15
0,1
0,05
0
1
2
3
1- СBS 4732 leu2-2 (control)
2- Δxyl1
3- Δxyl1 Δxy2-A
4,5- Δxyl1 Δxy2-A Δxy2-B
4
5
Xylose
Glucose
The aim:
To clone the H.polymorpha
PDC1 gene coding for pyruvate
decarboxylase and put the gene
under the strong constitutive
promoter (HpGAPpr)
Pyruvate
PDH
complex
PDC
Acetyl-CoA
Acetaldehyde
CO2
CO2
Ethanol
Linear scheme of the plasmid carrying the
HpPDC1 gene under the HpGAP promoter
B
Nt H
Nd
ORI HpGAP_pr
HpPDC1_ORF
HpAOX_tr
ScBg
K RI
Er r
ScLEU2
pKO8_prGAP_PDC1Hp ~8.6 kb
The linear scheme of the plasmid pKO8_prGAP_PDC1Hp (~ 8.6 kb). The LEU2 gene of S.
cerevisiae is shown as grey box; fragment containing the promoter of the HpGAPDH gene is
shown as yellow box; fragment containing the terminator of the HpAOX gene: blue box; the ORF
of the H. polymorpha PDC1 gene is shown as red box.
Restriction sites: B, BamH I; K, Kpn I; RI, EcoR I; Bg, BglII; Sc, Sac I; Nd, NdeI; Nt, NotI; H,
Hind III.
Effect of the PDC1 gene overexpression on
ethanol production during xylose fermentation
of protein
PDC activity, u/mg
Specific activity of pyruvate decarboxylase (Pdc1p) and ethanol production
16
14
12
10
8
6
4
2
0
1-495-3Leu+
1
2
2-495-PDC1Hp-4
1,2
Ethanol, g/l
1
0,8
YNB + 8 % xylose
0,6
37 ºC, 100 rpm
0,4
54 hours
0,2
0
1
2
The double replacement (Lys Arg Asn Asp)
results in preference of the modified xylose reductase
for NADH over NADPH
Ctn 233 ALNTPTLFAHDTIKAIAAKYNKTPAEVLLRWAAQRGIAVIP K S N LPERLVQNRSFNTFDL
Dhn 228 ALDTPTLFEHKTIKSIANKNKKTPAQVLLRWASQRNIAVIP K S N NPDRLLQNLEVNDFDL
Hpl 300 AKNTVSLLKHDLINSIASAHKVTPAQVLLRWATQRDILVIP K S N QKERLVQNLKVNDFNL
341 K (Lys)
R(Arg)
343 N(Asn)
D(Asp)
Ctn – Candida tenuis
Dhn – Debaryomyces hansenii
Hpl – Hansenula polymorpha
341
343
Linear scheme of the plasmid pX1M carrying the
modified HpXYL1 gene under the HpGAP
promoter
APH
B
HpGAPpr
lacZ
MCS
Sc
ORF HpXYL1M
HpAOXtr
MCS
lacZpr
Ethanol production by the CBS 4732 strain and
Δxyl1(pX1M) transformants;
2 day fermentation, min. medium, 370C
0,133
Ethanol production,
mg/mg biomass
0,14
0,108
0,12
0,1
0,08
0,05
0,06
0,04
0,02
0
CBS4732
1a
1b
Strain H. polymorpha
Alcoholic xylose fermentation by the H.
рolymorpha strains unable to utilize ethanol
3
Ethanol, g/l
2,5
2
NCYC495leu1-1 2Eth495 2-3Eth-(AllAlc)
495 2-4Eth-(AllAlc)
NCYC495leu1-1
1,5
1
0,5
0
0
20
40
60
80
100
Time, h.
Minimal medium 12 % xylose; 48ºС.
The strains were selected as resistant to allyl alcohol (0.2мМ).
Strategies for improvement
of fermentation characteristics in H. polymorpha
Overexpression
of genes conferring
ethanol and
temperature
tolerance
HSP
genes
OLE1
OLE2
ATH1
Overexpression
of genes for
pentose metabolism
Evolutionary
engineering
and direct selection
of ethanol
overproducers
XYL3
RPE1
TAL1
TKL1
RKI1
Acknowledgement
Dr. Andriy Voronovsky
Dr. Oleh Stasyk
Kostyantyn Dmutruk
Olena Ryabova
Olena Ishchuk
Olena Verba
Olga Rohulya
Yuriy Dem’yanchuk
Institute of Cell Biology, Nat. Acad. Sci., Lviv,
UKRAINE
Dr. Charles A. Abbas
Archer Daniels Midland Company Research
Center, Decatur, IL, USA.
Thank you
for your attention!