lipase, nitrilase technologyNov-9, 2006 2 The Energy Issue in Whole Cell Oxyfunctionalization Andreas Schmid Chair of Chemical Biotechnology, University of Dortmund & Institute for

1

S-MOIPAS-MOIPA

Outlook®

Geismar/USAcapacity: 2.500 t/a

OO

NHNH22

O

O

ClN

S

Prof. Dr. B. Hauer, BASF AG

(Herbicide)

lipase, nitrilase technology

Nov-9, 2006 2

The Energy Issue

in Whole Cell OxyfunctionalizationAndreas Schmid

Chair of Chemical Biotechnology, University of Dortmund

& Institute for Analytical Sciences (ISAS)

GreenChem Symposium,

Malmö, November 9, 2006

Nov-9, 2006 3

University of Dortmund

Oxidoreductases

Dehydrogenases(Addition of H2O,

LONZA AG)

Oxidases Peroxidases(vanadium-

chloroperoxidase)

Oxygenases

Heme oxygenases

(P450 enzymes)

Non-hemeiron-oxygenases

mononuclear

(e.g. a-ketoglutarate

dep. enzymes)

binuclear

(e.g. MMO,

AMO, XMO)

w/o cofactor

(quinone oxygenases)

Copper

oxygenases

Flavin

Oxygenases

(no hydroxyl. of sp3-C,

StyAB, Baeyer Villiger)

Iron

oxygenases

mono-

nuclear

(dopamine -

monooxygenase)

binuclear

(Cu-MMO)

Biocatalysts for Oxy-Functionalizations

Nov-9, 2006 4


sec. - hours( >> days)

μM -mM(M)

< 1 g L-1 h-1

(10 g L-1 h-1)μM-mM1-50 s-1

typicalparameters

stability[S, P]STYKmkcatCofactor dep.

enzymes

Bühler B. and Schmid A. (2004) J. Biotechnol. 2004, 113: 183-210

enzyme specificity multiple oxidation

uncoupling

effects of oxygenase

overexpression

product degradationcofactor recycling

enzyme activity

S, P toxicity

Oxygenase based

bioprocessesX

O

H C* OH

O

O

O

X*

O

OH

OH

OH

* *

*

**

Catalyst efficiency

Nov-9, 2006 5(source: Spiegel, 2006)

4 m python

and alligator

(Florida)

Redox - Biocatalyst design ... ... the need for break point analyses

Nov-9, 2006 6

System breakpoints

level effects

DNA mutations, regulation, � clonal

Enzyme inhibition, specificity, � proteome

Metabolism overflow, � fluxes, � energy

Cell membranes, permeabilisation

Population subpopulations (undefined in time & kind)

Process (Ecosystem) � productivity

Nov-9, 2006 7

System breakpoints

[Product]

Reaction time

Scale

DSP

[Biomass]�

TF

Toxicity (P)

TTN

Catalyst

stability

S-T-Yield


UUNIVERSITY OF NIVERSITY OF DDORTMUNDORTMUND

Styrene Monooxygenase StyABStyrene Monooxygenase StyAB

46 kDa;kcat = 1.6 s–1

Km = 0.38 �M

46 kDa;kcat = 1.6 s–1

Km = 0.38 �M

A two-component flavin- and NADH-dependent monooxygenaseA two-component flavin- and NADH-dependent monooxygenase

K. Otto, K. Hofstetter, M. Röthlisberger, B. Witholt, A. Schmid,

2004, J. Bact., 186 (16): 5292-5302)

H2O

O2

StyAStyA

FADH2

FAD

(CH2)n

R1

R2R3

R4

(CH2)n

OR1

R2R3

R4

NADH + H+

NAD+

FAD

FADH2

StyBStyBStyBStyB

R1 = H, Me

R2 = H, Me,

R3 = H, Me, Cl, NO2, F,R4 = H, Me, Cl, NO2, F,

n = 0-2

18 kDa;kcat = 47 s–1

Km = 11.6 �M

18 kDa;kcat = 47 s–1

Km = 11.6 �M

Homology model (StyA)

(phenol monooxygenas,

p-hydroxybenzoate hydroxylase.Feenstra, Venhorst, Vermeulen, Amsterdam


Large Scale ChromatographyLarge Scale Chromatography

Labomatic Chromatography Unit

1 - 400 bar

gradients, flow up to 1 L min-1Jochen Lutz


Styrene monooxygenase Sty AStyrene monooxygenase Sty AGramGram––ScaleScale ProductionProduction

CE Pool

90 kDa

64 kDa

43 kDa

30 kDa

20 kDa

14 kDa

Loading

Washing

Elution

K. Hofstetter, I. Lang, J. Lutz, B. Witholt, A. Schmid, 2004, Angew. Chem. Int. Ed., 43 (16): 2163-2166.

Crude cell

extract

59.7

24.3

100

StyA

pool

25.3

22.5

93

Protein [g]

Activitytot [kU]

Yield [%]

100 mL fractions

No fatty acids, DNA, RNA,

major protein impurities.

EBA: expanded

bedchromatography


Asymmetric epoxidationsAsymmetric epoxidationsusing isolated monooxygenase subunitsusing isolated monooxygenase subunits

FADH2

FAD

A. Schmid, K. Hofstetter, H.-J. Feiten, F. Hollmann, B.Witholt, 2001, Adv. Synth. Catal., 343 (6-7): 732-737.

Recombinant bacterial cells expressing styAB.

Recombinant bacterial cells expressing styAB.

Multi-gram scale:

NADH+ H+

NAD+

StyBStyBcellscells

glucose

K. Hofstetter, I. Lang, J. Lutz, B. Witholt, A. Schmid,2004, Angew. Chem. Int. Ed., 43 (16): 2163-2166.

Gram scale:

Isolated StyAB coupled to NADH regeneration

system (formate/FDH)

Isolated StyAB coupled to NADH regeneration

system (formate/FDH)

NADH

+ H+

NAD+

StyBStyBFDHFDH

CO2

HCOO–

Med.Med.

CO2

HCOO–

F. Hollmann, P.-C. Lin, B. Witholt, A. Schmid, 2003, J.

Am. Chem. Soc., 125 (27): 8209-8217.

Milli-gram scale:

Isolated StyA combinedwith mediator-based

regeneration

Isolated StyA combinedwith mediator-based

regeneration

O2

H2O

StyAStyA

O


Electro-BiocatalyticElectro-Biocatalytic AsymmetricAsymmetric EpoxidationEpoxidation

F. Hollmann, K. Hofstetter, T. Habicher, B. Hauer, A. Schmid, 2005,

J. Am. Chem. Soc., 127 (18): 6540-6541.

A. Schmid, F. Hollmann, K. Hofstetter, T. Habicher, B. Hauer, 2004, Patent PF55587.

ee value > 99%

[epoxide] = f(T, [StyA], [O2])

Substrate ProductStyA

FAD

Airinsertion

Pt Anode

Cylindrical carbon felt

cathode

Magneticstirrer

Ag/AgClreferenceelectrode

catalase

H2O2

H2O+1/2O2

Starting reaction conditions:50 mM KPi pH 7.5; 50 �M FAD; 0.1 g L–1 StyA;

480 U mL–1 catalase; 14 cm2 carbon felt; 15

mL volume; 5 cm3 min–1 aeration; 30° C

temperature; Potential: –550 mV vs Ag/AgCl

electrode.

Reaction characteristics:


Electron Yield, Oxygen Supply, and Flavin Electron Yield, Oxygen Supply, and Flavin ReoxidationReoxidation

0

50

100

150

0 200 400 600 800

FAD [�M]

activity [

U/g

]

600 �M600 �M

2–20% FADred

CATH

ODE

H2O + 1/2 O2

H2O2

8% Electrons

25% Electrons

FADox

FADred

Styrene oxide

FADox + H2O2

O2


Stable Product Formation over TimeStable Product Formation over Time

0

50

100

150

200

250

0 100 200 300 400

time [min]

epoxid

e [

�M

]

+BSA

+Sucrose & BSA

StyA & buffer

BSA and sucrose prolonged product formation from 5 min to 6.5 h.

50 mL volume;

Carbon felt area/ volume quotient:

0.2 cm2 cm–3

50 mL volume;


0.2 cm2 cm–3

15 mL volume;


0.9 cm2 cm–3

15 mL volume;


0.9 cm2 cm–3

0

20

40

60

80

0.2 0.7 2.2

Carbon felt area-volume quotient [cm2 cm–3]

Activity [

U g

–1]


Enzyme and Regeneration EfficienciesEnzyme and Regeneration Efficiencies

� IInn vviivvoo bbiiooccaattaallyyttiicc pprroodduuccttiioonn ooff ssttyyrreennee ooxxiiddee

ccooffaaccttoorr ppeerr gglluuccoossee yyiieelldd:: �� 1100%%eeee:: >> 9999..99%%

RReeffeerreenncceess:: [1] S. Panke, M. Held, M. Wubbolts. B. Witholt, A. Schmid Biotechnol. Bioeng., 8800:33-41.

NAD+

NADH+ H+ FAD

FADH2

O

StyB

Glucose

CO2

CCeelllluullaarr MMeettaabboolliissmm[[11]]::5500 UU**ggCCDDWW--11 ffoorr 1100hh

O

H2O

O2

StyAMetabolism


� IIn vitro electroenzymatic production of styrene oxide

References: [1] S. Panke, M. Held, M. Wubbolts. B. Witholt, A. Schmid Biotechnol. Bioeng., 880:33-41.

[2] F. Hollmann, K. Hofstetter, unpublished data

electron transfer yield: 0.02%ee: > 99.9%


FAD

FADH2

O

H2O

O2

StyA

Cath

ode

IIndirectregeneration[2]:70 U*g(StyA)-1 for0.2h

CCellular Metabolism[1]:50 U*gCDW-1 for 10h[Cp*Rh(bpy)(H2O)2+]

[Cp*Rh(bpy)(H)+]


[3] K. Hofstetter, unpublished data

DDirect regeneration[3]:35 U*g(StyA)-1 for 6h

� In vitro electroenzymatic production of styrene oxide

References: [1] S. Panke, M. Held, M. Wubbolts. B. Witholt, A. Schmid Biotechnol. Bioeng., 880:33-41.

[2] F. Hollmann, K. Hofstetter, unpublished data

FAD

FADH2

O

H2O

O2

StyA

Cath

ode

electron transfer yield: 1%ee: > 99.9%

IIndirectregeneration[2]:70 U*g(StyA)-1 for0.2h

CCellular Metabolism[1]:50 U*gCDW-1 for 10h


Nov-9, 2006 18

> 99% e.e.

StyAB

O2 H2O

NADH NAD+

O

StyAB

O2 H2O

NADH NAD+

O

kcat

Regeneration of NADH

Oxygen transfer rate

Toxicity of substrate/product

• High enantio-, stereo- and chemoselectivity

• Example: Oxygenation of Styrene to (S)-Styrene oxide by StyAB in whole cells

Redox reactions in biocatalysis

Limitations Improvement by

Enzyme engineering

Overexpression in recombinant cells

Whole cells, metabolic engineering

Optimization of aeration system

Two-liquid phase system

Metabolism and Biocatalysis: QuestionsMetabolism and Biocatalysis: Questions


OutlookOutlook

Reactor design:Bubble-free aeration?Continuous reactor?Two-liquid phase system?

Application of the basic conceptto other monooxygenases(Cytochrome P450’s, etc.)?

Specific product

formation rate

20 U g–1 180 U g–1

1.9 U mg–1?

Reaction

volume

15 mL 50 mL

Liters?

Electron

yield

0.5% 2-3%

25%?

Reaction time(stable product

formation rate)

5 min 6.5 h

Days?

Start

Status

Vision

Nov-9, 2006 20

Organic phase

BEHP: Bis(2-ethylhexyl)phthalate

Biocatalyst: recombinant E. coli

Substrate: styrene

Product: styrene oxide

Aqueous phase (medium)

Reaction:

Glucose feed

O

O

O

O

O

O

The organic / aqueous bioreactorsate of the art, but still an extreme environment


Nov-9, 2006 21

2-L-P Bioprocess DesignBis(2-ethylhexyl)phthalate (BEHP) as Carrier Solvent

200+/-29900.7Styrene

17+/-1702.3Styrene oxide

Aqueous concentrations

for �max/2 [mM]Partition coefficients, KpSubstance

Inoculation Start of theFed-Batch

Addition of organic

phase (induction)

Batch ~8-9 h Biotransformation ~7-14 h1-2 h

FeedInoculum

Organic phase (BEHP)

containing substrate

and octane as inducer

Witholt B. et al., Favre-Bulle O., Wubbolts, M; Panke, S.; Schmid A, …

Nov-9, 2006 22

Process leveloverflow metabolism, productivity

Ø-Productivity: 5.3 g/Ltot/h (max.: 8.2 g/Ltot/h)

Product toxicity limits achievable product concentration

0

200

400

600

800

Co

ncen

trati

on

s [

mM

]

-2 0 2 4 6 8

Time [h]

0

10

20

30

40

50

60

Sp

ec. A

cti

vit

y [

U/g

CD

W]Styrene

Styrene oxide2-PhenylethanolTotal

0

10

20

30

40

50

60

0

2

4

6

8

10

12

14

Feed

rate

[g

glu

co

se/h

]

Cell c

on

c. [g

CD

W/L

]

-2 0 2 4 6 8

Time [h]

Glu

co

se &

a

ceta

te c

on

c. [g

/L]

O

> 99% e.e.

StyAB

O2H2O

NADH NAD+

E. coli JM101 (pSPZ10) J.B. Park, PhD Thesis 2004

Nov-9, 2006 23

qC

O2[m

mo

l/g

CD

W/h

]

qO

2[m

mo

l/g

CD

W/h

]

CD

W [

g/L

]

Aceta

te c

on

c. [g

/L]

-50 -25 0 25 50 75 100

3

4

5

6

7

0

3

6

9

12I II III IV V VI

1

2

3

4

5

Sty

ren

e o

xid

e c

on

c. [m

M]

Sp

ecif

icaciv

ity

[U

/g C

DW

]

Sty

ren

e f

eed

co

nc. [m

M]

-50 -25 0 25 50 75 1000

20

40

60

0

10

20

30

40

Sty

ren

e c

on

c. [m

M]

0

50

100

150

Continuous org./aq. BiotransformationE. coli -metabolism - energy (NADH)

Culture time [h]

III-V: Styrene limitation

VI: no limitation by

Styrene O2

Stepwise decrease of Yx/glu

increasing energy demand

Increasing NADH-requirement

Saturation of TCA cycle (V-VI)Based on:a stoichiometric model of metabolismacetic acid formation

=> NADH-Limitation (VI)

D = 0.1 h-1

E. coli JM101 (pSPZ10) J.B. Park, PhD Thesis 2004

Nov-9, 2006 24

What is the maximal NADH synthesis rate?

NAD(P)HFormation in

Biomass synthesis

Biocatalysis

Solvent „detoxification“

Glucose catabolism

Research Questions

Quantify the metabolic impact of octanol and toluol.

What is the metabolic impact of an additional energy/redox sink?

Nov-9, 2006 25

1313CC-tracer

CellCell

MSMS

Stable isotope tracer experiments

Sample ID:

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 11000

100

%

10.725747

8.393488

6.089232

4.40545 5.639

182

4.56763

4.64872 5.126

125

5.720191

6.413268

6.719302

7.736415 8.114

457

9.177575

8.906545

9.501611

9.969663

9.897655

10.266696

11.805867

11.391821

10.959773

12.813979

11.859873 12.579

953

12.111901

13.5971066

13.3801042 13.921

1102

A

GV

L

I

P Y

H

K

M

S

T E1313CC-pattern(protein-boundamino acids)

Nov-9, 2006 26

1 2 3 4 5 6

1 2 3 1 2 31 2 3

Glucose

Entner-

Doudoroff

Pyruvate(inferred from

Alanine)Glycolysis Pentose-

Phosphate

Fischer and Sauer, J. Eur. Biochem. 2003

METAFoR: Metabolic flux ratio analysis

Glucose-6-P/

6-P-Gluconate

Fructose-6-P

Trioses

Pentose-5-P

Glucose

8%92%

Pyruvate

Pen

tose-P

ho

sp

hate

-Path

wayE

ntn

er-

Do

ud

oro

ff-P

ath

way

Flux analysis based on 13C-labeling experiments

Pseudomonas sp.

Nov-9, 2006 27

Input:

– Reaction network

– Physiological data

– Biomass composition

– Metabolic flux ratios

Output:

– In vivo reaction velocities

e.g. mmol/g/h

Assumptions:

– Steady state

– Amino acid = central

carbon metabolites

6.0 mmol/g/h

Fischer et al., Anal. Biochem. 2004

Glucose-6-P/

6-P-Gluconate

Fructose-6-P

Trioses

Pentose-5-P

Glucose

Pyruvate

Pen

tose-P

ho

sp

hate

-Path

wayE

ntn

er-

Do

ud

oro

ff-P

ath

way

5.5 mmol/g/h 0.5 mmol/g/h

8%92%

13C-constrained net flux analysis

Nov-9, 2006 28

Prediction is very difficult,

especially about the future.Niels Bohr


OutlookOutlook

- cell free biocatalytic oxyfunctionalizations- cell free biocatalytic oxyfunctionalizations

From: Dr. H. Pütter, BASF AG,

Ammonia Laboratory, personal communication.

Undivided and flow

through stack-plate

cells for continuous

paired electro-

synthesis of

phthalids and

benzaldehydes.

BASF AG, Germany.

10-100 mA cm–2; 0-60ºC; 1 atm;

< 50 V; electrolyte solution

containing conducting salts (e.g.

methyltributylammonium

metasulfate); cosolvents (e.g.

dimethyl carbonate);

Challenging the scale issue !

single cell microbiology ...

nL �L mL L

Scale Down Scale Up

Nov-9, 2006 31

Questions: Single Cell Analysis

=?

How can the metabolic repertoire

of a microbial cell be studied at the

single cell level?

Does the population average

reflect single cell behaviour?

How large is the population

heterogeneity of a pure microbial

population?

How does one engineer a none

heterogenic population - one cell ?

= ?

Challenging the average assumption!

= ?

Challenging the average assumption!

Nov-9, 2006 32

Single cell analysesSingle cell analyses(Evotec Cytocon(Evotec Cytocon400400))

MS LTQ FT

Thermo

detection limit: sub fmol

mass accuracy: 2 ppm

data acquisition rate: 1 s

Peptide & small molecule analyses

Technical Details

reactor volume: ca. 40 nL

pump rate: 0.1 to 10 �L/h

product amount: pmol/h

macro-level (kg of Product)

molecular-level

gene

enzyme

cell / tissue / organ

catalyst design

(enzyme, cell, … )

reaction medium

reaction conditions

metabolic network

reactor

product recovery & purification

process theory / modeling

catalyst

process

efficiency

productivity

Biocatalyst & Bioprocess design:

the need for an integrated approach

Nov-9, 2006 34

Acknowledgements

Bernhard Hauer (BASF) Lars Blank

Uwe Sauer (ETHZ) Hendrik Kortmann

Bruno Bühler

Andreas Manz (ISAS) Jin-Byung Park

Birgitta Ebert

Georgios Ionidis

BASF AG, ISAS/Leibniz, DBU, ETHZ, EU

Nov-9, 2006 35

Chair of Chemical BiotechnologyUniversity of Dortmund & ISAS, Dortmund, Germany

Nov-9, 2006 36

Thank You

Nov-9, 2006 37http://www.bci.uni-dortmund.de/bt

PostDoc Vacancies:Quantitative Metabolomics

Catalytic Biofilms

Biocatalyst design

Electroenzymology

Please contact:[email protected]

or

[email protected]


Chair of Chemical

Biotechnology&

ISAS Dortmund

Nov-9, 2006 38

StyA

2

O

+H O

FADH

NADH

NAD

FAD

FADH

O2

StyB

FAD

StyAStyA

2

OO

+H O

FADH

NADH

NAD

FAD

FADH

O2O2

StyB

FAD

OOOO OO

OO

StyAB

OO

OO

O2

O2 H2O

Glukose

Glukose

NADH NAD+

G6P

TCA

Cycle

Pyr

OAA

NADH

NADH

NADH

NADPH

NADPH

NADH

P5P

En

tne

r-

Do

ud

oro

ff

Pa

thw

ay

PP

Pa

thw

ay

From Gene

to Product

Economy, Ecology

Market &Substrate Product

Bühler et al. JBC (2000)

Panke et al. AEM (1998)

Otto et al. JBact (2004)

Bühler et al. AEM (2002)Suske et al. JBC (1997)

Suske et al. JBC (1996)

Hollmann et al. JACS (2005)Hofstetter et al. Angew. (2004)

Hollmann et al. JACS (2003)

Bühler et al. B&B (2003b)

Panke et al. B&B (2002)

Held et al. B&B (1999)

Meyer et al. AEM (2005)

Meyer et al. B&B (2003)Panke et al. B&B (2000)

Yildirim et al. ASC (2004)

Ionidis et al. (in prep.)

Schmid et al. ASC (2001)

Bühler et al. B&B (2003a)

Schmid et al. Nature (2001)

Blank et al. Genome Biol (2005)

Yildirim et al. ASC (2005)

Meyer et al. JBC (2002a)

Meyer et al. JBC (2002b)

Panke et al. AEM (1999)

O

R

R

OH

R

R

R

R

OOH

R

R

R RO

(CH2)0-2

N

N

OOH

OH

OH

R

OH

OH

R

alkS xylMA

Wubbolts et al. B&B (1996)

OH

O

nOHn

n = 1-8

cath

od

e FADH2

FAD

O

H2O

O2

StyAe–

process

biocatalystselection

biocatalystcharacterization

biocatalystdesign

bioprocessdesign

product

recovery

Chemical Biotechnology

Documents

lipase, nitrilase technologyNov-9, 2006 2 The Energy Issue in Whole Cell Oxyfunctionalization Andreas Schmid Chair of Chemical Biotechnology, University of Dortmund & Institute for