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Innovation from Nature
Pichia pastoris:
Protein Production and More
Product
Substrate
Cofactor
Byproduct
Cosubstrate
Franz Hartner1, Liu Zhibin1, Beate Pscheidt1, Bettina Janesch1, Roland Weis1,2, Sandra Abad1, Karl
Gruber1 and Anton Glieder1
1Institute of Molecular Biotechnology, Research Centre Applied Biocatalysis , Petersgasse 14, A-8010,
Graz, AUSTRIA, [email protected]
2VTU Engineering GmbH, Grambach, Austria
Im Rahmen des K plus Programms gefördert durch:
The Making of
Industrial Biocatalysts
Organic, Food, Polymer Chemistry
Bioprocess Engineering
„Bio“Contributions
Identification,
Isolation
Expression
Adaptation
Production
(Expression)
Organic, Food, Polymer Chemistry
Bioprocess Engineering
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Natural Sources for Biocatalysts
* Most industrial enzymes
isolated from microorganisms
* Produced by microorganisms
* Enzymes from higher
eukaryotes
* Additional unique diversity
* Produced by microorganisms
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Is
Simple & fast
Should be
Simple & fast
Pichia pastoris – methylotrophic yeast
Excellent folding capabilities
Eukaryotic post-translational modifications
High Cell density, cheap media
High purity of secreted proteins
Plasmid cloning in E. coli before Pichia
transformation
High variations in screening (copy effects)
Slower than E. coli and labour intensive
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Pichia pastoris:
Expression, Engineering, Biotransformation
Reliable 96 well plate screening
Rational design of enzyme variants
Plasmid/E. coli independent library generation
Directed Evolution
Enhanced 2nd generation expression system
(vectors/platform strains)
Whole Cell Biocatalysis
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Pichia - Reliable Micro Cultivation
methanol inducible system
OD595 after 60 h = time of induction
14
12
10
8
6
4
2
0
1
0,2
1
2
2
3
3
4
Glucose concentration / %
enzyme activity after 75 h of induction
0,2
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
1
2
3
Glucose concentration / %
Weis, R., R. Luiten, W. Skranc, H. Schwab, M. Wubbolts, and A. Glieder. 2004. FEMS Yeast Research 5:179-189.
Apoptosis/Necrosis
Reporter for media/screening optimization
0.2% D
1% D
2% D
OD595 after 60 h = time of induction
14
12
10
8
6
4
2
0
1
0,2
3% D
negative
control
H2O2induced
32
43
1
Glucose concentration / %
2
enzyme activity after 75 h of induction
Tunel assay
Necrosis assay
© Angewandte
Biokatalyse-Kompetenzzentrum
GmbH H.
Weis,
R., R. Luiten, W. Skranc,
FEMS Yeast Research 5:179-189.
Glucose concentration / %
Schwab, M. Wubbolts, and A. Glieder. 2004.
300 µL
4000 L
4000 L
HRP
HbHNL
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Weis, R., R. Luiten, W. Skranc, H. Schwab, M.
Wubbolts, and A. Glieder. 2004. FEMS Yeast
Research 5:179-189.
Structural Design
HydroxyNitrileLyase HNL
(R)-2-chloro mandelonitrile
key intermediate for
(R)-2-chloro-mandelic acid
for production of cardiovascular drug
Glieder et al., Angew. Chem.
Int. Ed. (2003) 42; 4815-4818
(R)-2-hydroxy-4-phenylbutyronitrile
Intermediate for „-prils“
Weis et al., Angew. Chem. Int.
Ed. (2005) 44 (30), 4700-4704
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Designed for High Activity
600
500
µmol/min/mL
400
300
200
100
0
WT
A111G
V317G
Synthesis of
(R)-2-Chloro mandelonitrile
2-chloro mandelonitrile
in model of active centre of PaHNL5
6-7 fold increase in activity
ee > 95 %
Glieder, A., et al (2003) Angew Chem, Int Ed, 42, 4815-4818
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Designed Stereoselectivity
Low Stereoselectivity
with substrates with
CH2 spacers between
C=O and aromatic ring
OH
OH
R1
CN
O
CN
R2
2a
2b
2x
3-phenylpropenal
OH
OH
CN
O
1a
COOEt
Synthesis of precursors
of ACE (Angiotensin
Converting Enzyme)
inhibitors
1b
3-phenylpropanal
R. Weis, R. Gaisberger, W. Skranc, K. Gruber, A.Glieder (2005), Angewandte Chemie Int. Ed., 44, 4700-4704
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
New R-HNLs for API Production
structure guided design
(R)-Hydroxynitrile
lyases
OH
OH
CN
CN
Cl
ACE inhibitors
R. Weis, R. Gaisberger, W. Skranc, K.
Gruber, A.Glieder (2005)
Angew. Chemie Int. Ed., 44, 4700-4704
Directed evolution
Further increase
in ee and activity
Liu, Z., Pscheidt, B., et al (2007)
ChemBioChem, in press
Anticoagulant
Glieder, A., et al (2003) Angew
Chem, Int Ed, 42, 4815-4818
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
New Strategy for Library Generation
Random & Site Directed mutagenesis
Linear Expression Cassette by OE-PCR:
• Random Mutagenesis – epPCR
Overlap 1
╳
Partial GAP promotor
╳
╳
╳
Mutated gene
Alpha-factor sequence
Zeocin cassette
Overlap 2
• Site-Saturation Mutagenesis
Overlap 1
╳
Partial GAP promotor
Alpha-factor sequence
Mutated gene
Zeocin cassette
Overlap 2
Partial gene
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
New Strategy for Library Generation
Recombination
• Single gene recombination: combining favorable mutations
• HNL variants of 1st round as templates
• Pool of fragments generated by PCR
• In vitro recombination
• Linear Expression Cassette by OE-PCR:
Overlap 1
linker
╳
promotor
╳
╳
╳
Mutated gene
Selection marker
Overlap 2
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Pichia pastoris
a host for screening and production
A260
DA280Dt-1/10-3min-1
Direct transformation of
linear integration cassette
400
350
300
250
200
150
100
50
0
0
10
20
30
40
50
Transformants
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
60
70
80
90
100
Synthesis of Cl-, Br- and Fsubstituted (R) -mandelonitriles
100
90
Conversion%
80
70
60
muteins
50
40
30
20
10
0
0,5 h
A111G
1h
2h
4h
4x A111G
Higher activity and higher ee by
Liu, Z., Pscheidt, B., et al (2008)
ChemBioChem,
Directed Evolution in Pichia pastoris
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
New Strategy for Library Generation
Random & Site Directed mutagenesis
Linear Expression Cassette by OE-PCR:
• Random Mutagenesis – epPCR
Overlap 1
╳
Partial GAP promotor
╳
╳
╳
Mutated gene
Alpha-factor sequence
Zeocin cassette
Overlap 2
• Site-Saturation Mutagenesis
Overlap 1
╳
Partial GAP promotor
Alpha-factor sequence
Mutated gene
Zeocin cassette
Overlap 2
Partial gene
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Site Saturation Approach
(sterically hindered aliphatic aldehydes)
H3C
CH3
CHO
OH
R-HNL/NaCN
R-HNL/NaCN
3M CitrateCitrate3M
phosphatephosphatebuffer (pH 2.4)
buffer
(pH 2.4)
Hydroxypivalaldehyde
Hydroxypivalaldehyde
H3C
OH
H3C
CN
OH
R-Hydroxypivalaldehyde(R)-Hydroxypivalaldehyde
cyanohydrine
cyanohydrin
precursor for panthotenic acid
•
Saturation of hydrophobic sites in substrate binding pocket
– ~200 transformants per site screened for improved conversion
•
•
•
~10 fold improved TOF value
enantiomeric excess ≥ 95% possible - reasonable amount of enzyme
For pivalaldehyde and hydroxypivalaldehyde conversion
All these enzymes produced by Pichia pastoris
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Engineered Promoter Libraries
delta5
delta6
delta7
delta8
delta1
delta2
Transcription Start
delta4
delta3
HAP1
HSF
STRE
ADR1
ABAA RAP1 (TUF1)
HAP234
HAP234
HSF
delta9
QA-1F
GCR1
MAT1MC
HAP234
Transcription factor binding sites:
e.g.
HSF......heat shock factor
HAP…..O2 and glucose regulation
STRE…..stress response element
GCR….glucose repressor
AOX1 promoter
953 bp
Deletions on many positions
Short deletions (5-60 bp), covering
putative transcription factor binding
sites
Hundreds of different variants
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
First Deletions Library
Next generation system commercialized by VTU Technology
relative GFP fluorescence after 72h of induction
160%
Strength
140%
Regulatory features
Promoter length
120%
100%
d1
WT
80%
d6*
60%
d2d6
40%
20%
0%
d2
(single copy clones)
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Basal (short) promoters
Transcription Initiation Site
TATA box
P(AOX1)
P(AOX1) basal
ScLeu2 basal or AOX1 basal promoter fragment
Basal promoters were cut 5’ of the TATA box
Different fractions of the promoter were added
Variants with small putative cis-acting elements added to the
basal promoters
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Schematic time course of GFP
expression
wild type promoter
d6* promoter
GFP
basal promoter
Growth phase
Derepression
phase
Methanol induction phase
time
Different promoters, different regulatory features, different levels of
GFP expression (growth, derepression, methanol phase)
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Platform Strains & Combinatorial Expression
expression enhancers
•
Models:
• Horseradish Peroxidase
• (Isoenzyme C)
• Candida antarctica lipase B
• (CALB)
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
HRP Overexpression
HRP activity [mOD 405/min]
1600
1400
HRP multi copy strain
HRP single copy strain
1200
51x
1000
800
600
400
200
0
pHRP-AOX1
1.6x
GAP-PDI
21x
AOX1∆6*-PDI
28x
30x
AOX1-PDI
11.6x
pHRP-AOX1
GAP: constitutive
∆6* : derepression, medium induction
AOX1: strong induction by methanol
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
18x
GAP-PDI
AOX1-PDI
AOX1∆6*-PDI
CALB Overexpression
low and high copy number
A combinatorial problem !!!
CALB activity [mOD 405/min]
CALB-AOX∆6*
low copy strain
CALB-AOX∆6*
high copy strain
1200
1000
3.3x
800
600
400
200
1.4x
1.6x
1.6x
0.1x
1.6x
2.4x
0
CALB-∆6* AOX1∆6*- AOX1-PDI GAP-PDI CALB-∆6* GAP-PDI AOX1∆6*- AOX1-PDI
PDI
PDI
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Alternative Oxidases
• Providing metabolic flexibility
– Developmental conditions (senescence, fruit ripening)
– Environmental fluctuations – stress answer, fungicide
resistance
• Minimize generation of ROS
• Maintain TCA cycle
– citric acid production (Aspergillus niger)
Antimycin A
Cyanide, azide
Hydroxamic acids, alkyl gallates
Introduction
AOD structure
• Nuclear encoded
• Plants: dimer, fungi and yeasts:
monomer
• 2 highly conserved E-X-X-H
motifs
→ di-iron di-carboxylate
proteins
• Model for C-terminal, membrane
embedded part → four helix
bundle
matrix
Structural model, Berthold, 2003
Inner mitochondrial membrane
Structure
Expression system
• Expression host: Pichia pastoris WT
• Enzymes
plant and fungal AODs
linker
• Expression constructs:
– AOD-linker-Streptag II
– AOD-EK-GFP-Streptag II
AOD
Strep-Tag II
AOD-StrepTag II
1125 bp
linker
AOD
linker
AOD-EK-GFP-StrepTag II
1 8 8 1 bp
• PCR based linear expression cassettes
• Structural genomics (membrane proteins)
Expression system
GFP
S trep-Tag II
Purification
Cells


Mitochondria
Cell debris


Matrix (3) Mitochondrial membranes (2)


Sol. Proteins (5) Residual membranes(4)


Unbound Proteins Strep-tagged proteins
(6-9)
(12-14)
MW 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
[kDa]
MW
[kDa]
191
97
191
97
64
64
51
51
39
39
28
19
14
28
19
14
 AOD verified by MALDI analysis (S. Deller)
 purified membrane enzyme = active
 CD spectra
Purification
Metabolic Engineering
Enoate
reductases
X
R' H
R'
X
Monooxygenases
R
R''
*
* R''
H R'
OH
R
R'
R
R'
Dehydrogenases
X
C-source(s)
R
XH
R'
R
R'
NAD(P)H
ATP
NAD(P)+
ADP
Kinases,
Transferases
R-OH
2-
R-OPO3
Central metabolism
Enzyme cascades
Optimized
cell growth
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
metabolites,
bulk chemicals
Engineering Central Metabolic
Pathways of P. pastoris
•
Bottlenecks in NAD+ regeneration and
ATP feedback inhibitions limit flux
through glycolysis and TCA cycle
Glucose
ADP, 2 NAD+
Pyruvate
•
Uncoupling of NAD+ regeneration from
ATP production by overexpressing
AOD
2 NAD+
2 CO2
 higher growth rate
 higher substrate uptake rate
 lower final biomass yield
AcetylCoA
TCA
cycle
→ higher flux through central
carbon metabolism
6 NAD+, 2 FAD
+PFK overexpression
→ improved NAD+ regeneration
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Methylotrophic Yeasts for
Whole Cell Biocatalysis
The lucky 4…..
methanol induces
expression of the
biocatalysts
Expression of
endogenous cofactor
regeneration system
induced by methanol
Back to the cell…..
Pichia pastoris is
designed to tolerate
methanol and methanol
can act as a solvent for
the substrate
Methanol is a nutrient
and Cosubstrate for the
Cofactorregeneration
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Co-factor Regeneration
!using the whole pathway!
CH3OH
CH3OH
Toxic intermediates
O2
AOX1/2
GSH
2 NADH
per MeOH
H2O2
CTA
NAD+
NADH
O2 + H2O
DAS
Peroxisome
CO2
rearrangement
reactions
DAK1
FBA
TPI
FBP
F1,6BP
GAP
CO2 as
byproduct

irreversible
reaction
F6P
Pi
P. pastoris on methanol
S. cerevisiae on glucose
GAP
cell
constituents
DHAP
ATP
ADP
FMD1
NAD+
NADH
Xu5P
GAP
DHA
DHA
FLD1
HCOOH
GS-CH2OH
HCHO
HCHO
Cytosol
qS,max 10-17 mmol g-1 h-1 Jahic et al., 2002
qS,max 15 mmol g-1 h-1 Blank et al., 2004
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Growth of DAS Knockout Strains
0.35
glucose
methanol
0.30
µ / h-1
0.25
0.20
0.15
0.10
0.05
0.00
wild
wild type
type
Daox1
aox1
Ddas1
das1
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Ddas2
das2 Ddas1Ddas2
das1 das2
Butanediol DH catalysed
Biotransformtion
•
•
•
•
Increased cell density (60 g/L)
25 g/L substrate concentration
6% methanol
Shake flasks
O
OH
3S-acetoine
O
OH
3R-acetoine
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
OH
OH
meso-2,3-butanediol
OH
OH
2R,3R-butanediol
Synthetic ADH3 and ADH4 genes
ADH3_WT
ADH3_synthetic_high expression
ADH3, ADH4
are
NADPH dependent
(BASF)
ADH3 ADH4
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Yields in Shake flasks
O
conversion/[%]
Acetophenone
100
80
60
40
20
0
0
1
2
3
4
time/[h]
CAP
conversion/[%]
CBS 7435
ADH3 B4
ADH4 G5
O
100
80
60
40
20
0
0
2
4
6
8
10
12
14
16
18
20
22
24
time/[h]
ADH3 B4
ADH4 G5
• shake flask cultures, 1 g/L substrate
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Cl
Pichia pastoris based
Synthetic biotechnology
Engineered enzymes
laboratory evolved & designed
400
350
300
A260
250
200
150
100
50
0
0
10
20
30
40
50
Transformants
Optimized synthetic genes
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
60
70
80
90
100
Synthetic Biotechnology
160%
140%
120%
100% WT
80%
60%
40%
d2
20%
0%
Synthetic (modular)
promoter libraries
d1
d6*
d2d6
License free and advanced expression system
Overlap
1
╳
promotor
╳
╳
Mutated gene
╳
Overlap 2
Selection marker
PCR based
linear expression cassettes
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Engineered
platform hosts
Synthetic Biotechnology in
Whole Cell Biocatalysis
Classic: Growing Cells
By-product: biomass
Carbon metabolism and
biomass production
5
O2
1
CH3OH
Minimal Cell for
NAD(P)H
dependent catalysis
2
HCHO
+
NADH
+
H
recomb.
4
2O2
reduced
chiral
product
+ H2O
peroxisomes
OH
OH
+
R2
R1
R1 H
X
*
oxidised
Substrate
Reductase/
Dehydrogenase
O
R1
R2
R1
R2
Oxygenase
Enoate-Reductase X
R2
R1
R3
+
O2
* R3
H R2
Classic: Resting Cells
No catalyst regeneration
enzyme
R2
R1
H2O
CO2
NAD+
H2O2
1/
3
HCOOH
P.methylotrophic
pastoris cells Yeast
Engineered
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
Glieder group
Pichia Team (Graz)
Roland Weis
Hannelore Mandl
Beate Pscheidt
(Karl Gruber)
Liu Zhibin
Franz Hartner
Sandra Abad
Claudia Ruth
Ulrike Schreiner
Kerstin Kitz
Bettina Janesch
Manuel Peter
Astrid Hoermann
Maria Freigassner
Andrea Mellitzer
GLIEDER GROUP
© Angewandte Biokatalyse-Kompetenzzentrum GmbH
July 2007
&
DSM, BASF, VTU
Project Ideas
Strain engineering by promoter
replacements
Design of fully synthetic
promoters for different yeast
starins
© Angewandte Biokatalyse-Kompetenzzentrum GmbH