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Im Rahmen des K plusProgramms gefrdert durch:

Pichia pastoris:Protein Production and More

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] Engineering GmbH, Grambach, Austria

Innovation from Nature

Cofactor

ProductSubstrate

Byproduct Cosubstrate

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Angewandte Biokatalyse-Kompetenzzentrum GmbH

Identification,

IsolationExpression

Adaptation

Production(Expression)

The Making ofIndustrial Biocatalysts

BioContributions

Organic, Food, Polymer Chemistry

Bioprocess Engineering

Organic, Food, Polymer Chemistry

Bioprocess Engineering

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* Most industrial enzymesisolated from microorganisms

* Produced by microorganisms

IsSimple & fast

* Enzymes from highereukaryotes

* Additional unique diversity

* Produced by microorganisms

Should be

Simple & fast

Natural Sources for Biocatalysts

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Excellent folding capabilitiesEukaryotic post-translational modifications

High Cell density, cheap media

High purity of secreted proteins

High variations in screening (copy effects)

Pichia pastorismethylotrophic yeast

Plasmid cloning inE. coli

beforePichia

transformation

Slower than E. coli and labour intensive

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Reliable 96 well plate screeningRational design of enzyme variants

Plasmid/E. coli independent library generation

Directed Evolution

Pichia pastoris:Expression, Engineering, Biotransformation

Enhanced 2nd generation expression system(vectors/platform strains)

Whole Cell Biocatalysis

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Pichia - Reliable Micro Cultivationmethanol inducible system

0

2

4

6

8

10

12

14

1 2 3 4

OD595 after 60 h = time of induction

0,2 1 2 3Glucose concentration / %

enzyme activity after 75 h of induction

Glucose concentration / %

0,2 1 2 3

0,2 1 2 3

Weis, R., R. Luiten, W. Skranc, H. Schwab, M. Wubbolts, and A. Glieder. 2004. FEMS Yeast Research 5:179-189.

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Apoptosis/NecrosisReporter for media/screening optimization

Weis, R., R. Luiten, W. Skranc, H. Schwab, M. Wubbolts, and A. Glieder. 2004.FEMS Yeast Research 5:179-189.

0

2

4

6

8

10

1214

1 2 3 4

OD595 after 60 h = time of induction

0,2 1 2 3Glucose concentration / %

enzyme activity after 75 h of induction

Glucose concentration / %

0.2% D 1% D 2% D

3% D negative

control

H2O2-

induced

Tunel assay

Necrosis assay

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300 L 4000 L

HRP HbHNL

4000 L

Weis, R., R. Luiten, W. Skranc, H. Schwab, M.

Wubbolts, and A. Glieder. 2004. FEMS Yeast

Research 5:179-189.

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(R)-2-chloro mandelonitrilekey intermediate for

(R)-2-chloro-mandelic acidfor production of cardiovascular drug

(R)-2-hydroxy-4-phenylbutyronitrileIntermediate for -prils

Glieder et al., Angew. Chem.Int. Ed. (2003) 42; 4815-4818

Weis et al., Angew. Chem. Int.Ed. (2005) 44 (30), 4700-4704

Structural DesignHydroxyNitrileLyase HNL

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2-chloro mandelonitrilein model of active centre of PaHNL5

ee > 95 %

Glieder, A., et al (2003) Angew Chem, Int Ed, 42, 4815-4818

Synthesis of

(R)-2-Chloro mandelonitrile6-7 fold increase in activity

0

100

200

300

400

500

600

WT A111G V317G

mol/min/mL

Designed for High Activity

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Synthesis of precursorsof ACE (Angiotensin

Converting Enzyme)inhibitors

DesignedStereoselectivity

Low Stereoselectivitywith substrates with

CH2 spacers between

C=O and aromatic ring

O

O

OH

CN

OH

CN

OH

CN

R

R

OH

COOEt

1a

2a

1b

2b

1

2

2x

3-phenylpropenal

3-phenylpropanal

R. Weis, R. Gaisberger, W. Skranc, K. Gruber, A.Glieder (2005), Angewandte Chemie Int. Ed., 44, 4700-4704

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OH

CN

Cl

OH

CN

Anticoagulant

ACE inhibitors

(R)-Hydroxynitrile

lyases

New R-HNLs for API Production

structureguided design

Directed evolutionFurther increasein ee and activity

Glieder, A., et al (2003) Angew

Chem, Int Ed, 42, 4815-4818

R. Weis, R. Gaisberger, W. Skranc, K.

Gruber, A.Glieder (2005)

Angew. Chemie Int. Ed., 44, 4700-4704

Liu, Z., Pscheidt, B., et al (2007)

ChemBioChem, in press

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Linear Expression Cassette by OE-PCR:

Random Mutagenesis epPCR

Site-Saturation Mutagenesis

New Strategy for Library GenerationRandom & Site Directed mutagenesis

Partial GAP promotor

Alpha-factor sequence

Partial gene

Mutated gene Zeocin cassette

Overlap 2

Overlap 1


Alpha-factor sequenceMutated gene Zeocin cassette

Overlap 2

Overlap 1

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Single gene recombination: combining favorable mutations

HNL variants of 1st round as templates

Pool of fragments generated by PCR

In vitrorecombination


promotorMutated gene Selection marker

Overlap 2

Overlap 1

linker

New Strategy for Library GenerationRecombination

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0

50

100

150

200

250

300

350

400

0 10 20 30 40 50 60 70 80 90 100

Transformants

A260

Pichia pastorisa host for screening and production

Direct transformation oflinear integration cassette

DA280Dt-1/10-3min-1

S h i f

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Synthesis of Cl-, Br- andF-substituted (R) -mandelonitriles

0

10

20

30

40

50

60

70

80

90

100

0,5 h 1 h 2 h 4 h

Conversion%

A111G 4x A111G

muteins

Higher activity and higher ee by

Directed Evolution inPichia pastorisLiu, Z., Pscheidt, B., et al (2008)ChemBioChem,

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Random Mutagenesis epPCR

Site-Saturation Mutagenesis

New Strategy for Library GenerationRandom & Site Directed mutagenesis


Alpha-factor sequence

Partial gene

Mutated gene Zeocin cassette

Overlap 2

Overlap 1


Alpha-factor sequenceMutated gene Zeocin cassette

Overlap 2

Overlap 1

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Site Saturation Approach(sterically hindered aliphatic aldehydes)

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

CHO

OH

CH3CH3

Hydroxypivalaldehyde

R-HNL/NaCN

3M Citrate-phosphate-

buffer (pH 2.4)CN

OH

OHCH3

CH3

R-Hydroxypivalaldehyde-cyanohydrine

R-HNL/NaCN

(R)-Hydroxypivalaldehydecyanohydrin

Hydroxypivalaldehyde

3M Citrate-phosphate-

buffer (pH 2.4)

precursor for panthotenic acid

All these enzymes produced by Pichia pastoris

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Engineered Promoter Libraries

Transcription factor binding sites:e.g.HSF......heat shock factorHAP..O2 and glucose regulationSTRE..stress response element

GCR.glucose repressorDeletions on many positions

Short deletions (5-60 bp), coveringputative transcription factor bindingsites

Hundreds of different variants

AOX1 promoter953 bp

delta1

delta2

delta3 delta4

delta5delta6

delta7delta8

delta9

HSF GCR1HAP234

ADR1RAP1 (TUF1)

HAP1

HAP234HAP234

STREHSF

QA-1F

MAT1MCABAA

Transcription Start

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First Deletions Library

d2

d2d6

d6*

d1Strength

Regulatory features

Promoter length

0%

20%

40%

60%

80%

100%

120%

140%

160%

relativeGFPfluorescenceafter7

2hofinduction

WT

(single copy clones)

Next generation system commercialized by VTU Technology

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Basal (short) promoters

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 thebasal promoters

ScLeu2basal or AOX1 basal promoter fragment

P(AOX1) P(AOX1) basal

TATA boxTranscription Initiation Site

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Schematic time course of GFP

expression

time

G

FP

wild type promoter

d6* promoter

basal promoter

Different promoters, different regulatory features, different levels ofGFP expression (growth, derepression, methanol phase)

Growth phaseDerepression

phase

Methanol induction phase

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Platform Strains & Combinatorial Expressionexpression enhancers

Models:

Horseradish Peroxidase (Isoenzyme C)

Candida antarctica lipase B (CALB)

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0

200

400

600

800

1000

1200

1400

1600

pHRP-AOX1 GAP-PDI AOX16*-PDI AOX1-PDI pHRP-AOX1 GAP-PDI AOX1-PDI AOX16*-PDI

HRPactivity

[mOD405/min]

HRP Overexpression

HRP single copy strain HRP multi copy strain

1.6x30x

21x 11.6x

51x

18x

28x

GAP: constitutive6* : derepression, medium inductionAOX1: strong induction by methanol

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0

200

400

600

800

1000

1200

CALB-6* AOX16*-

PDI

AOX1-PDI GAP-PDI CALB-6* GAP-PDI AOX16*-

PDI

AOX1-PDI

CALBactivity[mOD405/min]

CALB Overexpressionlow and high copy number

CALB-AOX6*low copy strain

CALB-AOX6*high copy strain

1.4x

1.6x 1.6x

0.1x1.6x

2.4x

3.3x

A combinatorial problem !!!

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Introduction

Alternative Oxidases

Providing metabolic flexibility

Developmental conditions (senescence, fruit ripening)

Environmental fluctuations stress answer, fungicideresistance

Minimize generation of ROS

Maintain TCA cycle

citric acid production (Aspergillus niger)Antimycin A

Cyanide, azide

Hydroxamic acids, alkyl gallates

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Structure

AOD structure

Nuclear encoded

Plants: dimer, fungi and yeasts:monomer

2 highly conserved E-X-X-Hmotifs di-iron di-carboxylate

proteins Model for C-terminal, membrane

embedded part four helixbundle

matrix

Inner mitochondrial membrane

Structural model, Berthold, 2003

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Expression system

Expression system

Expression host: Pichia pastorisWT

Enzymes plant and fungal AODs

Expression constructs:

AOD-linker-Streptag II

AOD-EK-GFP-Streptag II

PCR based linear expression cassettes

Structural genomics (membrane proteins)

AOD-StrepTag II

AOD

linker

Strep-Tag II

AO D-EK-GFP-StrepTag II

GF PAOD linker

linker

Strep-Tag I

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Purification

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)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15MW[kDa]

19197

64

51

39

28

1914

AOD verified by MALDI analysis (S. Deller)

MW[kDa]

19197

64

51

39

28

1914

purified membrane enzyme = active

CD spectra

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Optimizedcell growth

NAD(P)HATP

NAD(P)+

ADP

Dehydrogenases

Monooxygenases

Enoatereductases

Kinases,

TransferasesR R'

X

R R'

XH

R R' R R'

OH

R'

H

R''

R'

H

X

R'

R''

X

R

* *

Central metabolism

Enzyme cascades

R-OH R-OPO32-

C-s

ource(s)

metabolites,

bulk chemicals

Metabolic Engineering

Engineering Central Metabolic

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Engineering Central MetabolicPathways ofP. pastoris

Bottlenecks in NAD+

regeneration andATP feedback inhibitions limit fluxthrough glycolysis and TCA cycle

Uncoupling of NAD+ regeneration fromATP production by overexpressing

AOD

higher growth rate

higher substrate uptake rate

lower final biomass yield

higher flux through centralcarbon metabolism

improved NAD+ regeneration

Glucose

Pyruvate

AcetylCoA

ADP, 2 NAD+

2 CO2

TCA

cycle

6 NAD+, 2 FAD

2 NAD+

+PFK overexpression

Methylotrophic Yeasts for

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The lucky 4.. methanol inducesexpression of the

biocatalysts

Pichia pastoris isdesigned to tolerate

methanol and methanol

can act as a solvent for

the substrate

Methanol is a nutrient

and Cosubstrate for the

Cofactorregeneration

Methylotrophic Yeasts for


Expression of

endogenous cofactor

regeneration system

induced by methanol

Back to the cell..

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Co-factor Regeneration!using the whole pathway!

CH3OH CH3OHO

2

H2O

2

GSH

GS-CH2OH HCOOH

CO2

F6P

HCHO

O2

+ H2O

GAP

HCHO

Xu5P

DHA

Peroxisome

DHA

NAD+

NADH

NAD+

NADH

ATPADP

DHAP

F1,6BP

PiGAP

GAP

cellconstituents

Cytosol

rearrangementreactions

AOX1/2

CTA

FMD1

DAK1

FLD1

FBA FBP

TPI

DAS

Toxic intermediates 2 NADHper MeOH

CO2 as

byproduct

irreversiblereaction

qS,max 10-17 mmol g-1 h-1

qS,max 15 mmol g-1 h-1S. cerevisiaeon glucose

P. pastorison methanol

Blank et al., 2004

Jahic et al., 2002

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Growth of DAS Knockout Strains

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

wild type aox1 das1 das2 das1 das2

/h-1

glucosemethanol

wild type Daox1 Ddas1 Ddas2 Ddas1Ddas2

B t di l DH t l d

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Butanediol DH catalysedBiotransformtion

Increased cell density (60 g/L)

25 g/L substrate concentration

6% methanol

Shake flasks

O

OH

O

OH

OH

OH

OH

OH

3S-acetoine

3R-acetoine 2R,3R-butanediol

meso-2,3-butanediol

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Synthetic ADH3 and ADH4 genes

ADH3_WT

ADH3_synthetic_high expression

ADH3 ADH4

ADH3, ADH4

are

NADPH dependent(BASF)

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Acetophenone

0

2040

6080

100

0 1 2 3 4

time/[h]

conversion/[%]

CBS 7435 ADH3 B4 ADH4 G5

Yields in Shake flasks

shake flask cultures, 1 g/L substrate

CAP

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16 18 20 22 24

time/[h]

conversion/[%]

ADH3 B4 ADH4 G5

O

O

Cl

i hi i b d

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Pichia pastoris basedSynthetic biotechnology

Optimized synthetic genes

Engineered enzymeslaboratory evolved & designed

0

50

100

150

200

250

300

350

400

0 10 20 30 40 50 60 70 80 90 100

Transformants

A260

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Synthetic Biotechnology

PCR basedlinear expression cassettes

d2

d2d6

d6*

d1

0%20%

40%60%80%

100%120%140%

160%

WT

Synthetic (modular)

promoter libraries

promotor Mutated gene Selection markerOverlap 2

Overlap1

Engineeredplatform hostsLicense free and advanced expression system

S th ti Bi t h l i

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Synthetic Biotechnology in


Minimal Cell forNAD(P)Hdependent catalysis

CH3OH

O2

H2O2

HCOOH CO2HCHO

O2 + H2O

R1 R2

O

R1 R2

OH

R1 R2

OH

R1 R2 O2

R2

R3

X

R1R2

H

R3

R1

H

X

NAD+ NADH + H+

1

1/2

4

2 3

5

Carbon metabolism andbiomass production

reducedchiral

product

oxidisedSubstrate

Reductase/Dehydrogenase

Oxygenase

Enoate-Reductase

+ +H2O

* *

recomb.

enzyme

peroxisomes

P. pastoriscells

Classic: Growing CellsBy-product: biomass

Classic: Resting CellsNo catalyst regeneration

Engineered methylotrophic Yeast

Glieder group

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Glieder groupPichia Team (Graz)

GLIEDER GROUP July 2007

DSM, BASF, VTU

Roland Weis

Hannelore MandlBeate Pscheidt(Karl Gruber)Liu ZhibinFranz Hartner

Sandra AbadClaudia RuthUlrike SchreinerKerstin KitzBettina Janesch

Manuel PeterAstrid HoermannMaria FreigassnerAndrea Mellitzer

&

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Project Ideas

Strain engineering by promoterreplacements

Design of fully synthetic

promoters for different yeaststarins

799606_20080521_TGL_AIBY

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