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One-Flow, kick off meeting 17-1-2017

Enzymatic synthesis of

Ursodeoxycholic acid

Prof. Isabel W.C.E. Arends

In collaboration with Dr. Frank Hollmann, and Prof. Ulf Hanefeld

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Department of Biotechnology

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From Industrial to Environmental Biotechnology

Biocatalysis: Green chemistry, enzymes as catalysts, novel enzymes, enzyme engineering

Cell Systems Engineering: Systems Biology, cell populations, analysis microbial cells

Industrial Microbiology: microbial performance.Integrating quantitative physiology and molecular biology; Metabolic-engineering.

Environmental Biotechnology: Waste water treatment; Resource recovery from waste streamsNon-aspectic and inherently stable systems; BiofilmsMicrobial ecology.

Bioprocess Engineering: HT measurements & modelling of biomolecular characteristics,hardware for bioconversion and bioseparation

Biotechnology and Society: Socially responsible innovation, communication andethical aspects of biorenewable production.

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http://cost-sysbiocat.fkit.hr/

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New

Enzymes

Tailored

Enzymes

Tailored

Processes

Integration in

Organic Synthesis

Biocatalysis Group at Delft

Turning an enzyme into a catalyst !Enzyme reactions not necessarily green

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Research Questions

A. Enzymes with novel reactivities• Hydrogenases

• Hydrolyases: C-O bond formation• Transketolases: C-C bond formation• Oxidoreductases and cofactor regeneration

B: Compatibility of enzymatic and/or chemocatalytic steps• Immobilization

• Compartmentalization

• Cascades

• Hybrid enzymes

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Ursodeoxycholic Acid

1. Secondary Bile Acid produced by intestinal bacteria

2. Used for treatment of cholestatic liver diseases, solubilizes cholesterol gallstones.

3. Current method multi-step chemical synthesis

4. Chemo-enzymatic synthesis promising alternative

T. Eggert, D. Bakonyi, W. Hummel, J. Biotechnol. 191 92014) 11-21.

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Synthesis UDCA from cholic acid and analogues

T. Eggert, D. Bakonyi, W. Hummel, J. Biotechnol. 191 92014) 11-21.

CDCA and LCA possible alternatives as starting material (back-ups)

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Chemical synthesis UDCA

(According to Hofmann 1963). Current chemical synthesis yield around 30%

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Possible route for Chemo-enzymatic synthesis.

R. Bovara,C. Carrea, S. Riva, F. Secundo, Biotechnol. Lett 18 (1996) 305.

• Two-pot reaction in membrane reactors, followed by WK-reduction, in resp. 90 and 88% yld.

• HSDHs from Clostridium

• Enzymatic NADP(H) regeneration by ketoglutarate and glucose

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Cascades and flow..

a marriage of convenience

• Cascade reactions

– Linear, orthogonal, coupled (cofactor regeneration) or cyclic

• Flow reactions can assist in compatibility issues

– Compartmentalization

– Immobilization

• S. Schoffelen, J.C.M. van Hest, Curr. Opin. Struct. Biol., 23, 613 (2013)

• V. Hessel et al., ChemSusChem, 6, 746-789 (2013)

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Enzyme catalysis in flow, benefits

• Expanded process windows

• Higher substrate loadings

• Lower product inhibition

• Shorter reaction times

• Increased enzyme lifetime

• Integrated product recovery

• Adjust reaction times for individual steps in time in order to respond to variation in enzyme activity.

R. Wohlgemuth, I. Plazl, P. Znidarsic-Plazl, K.Y. Gernaey and J.M. Woodley, Trends in Biotechn 33 (2015) 302.

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Work package description- 4yrs PD in Delft. Start apr 2017

WP1: Synthesis of UDCA

Production, and testing of enzymes in separate steps.

Choice for best enzyme preparations (stability in solvents etc.)

NAD(P) oxidative regeneration step

Stabilisation/immobilisation of enzyme

WP2: Shifting equilibrium in one-step epimerization step: One-pot two enzyme reaction. Choice of green spatial solvents

WP4: Integrated cascade in flow reactor.

Choice for concept in flow cascade

Coupling with Wolff-Kirschner-reduction (green alternatives ?)

In addition, explore fully enzymatic synthesis. This involves dehydroxylation at the 12-position; bioinformatics needed to find the corresponding enzymes

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Fully enzymatic synthesis ?

Dehydroxylation at C-12 has been observed in the whole cells preps with Bacteroides (Ebenharder 1982), but never reported afterwards.

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Selected examples of research at the

Biocatalysis group in Delft

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Synthetic Cascades which are enabled by combining

biocatalysts with artificial metalloenzymes

Cp*Ir(biot-p-L)Cl] within streptavidin affords an artificial transfer hydrogenase that is compatible with enzymes

V. Kohler, Y.M. Wilson, M. Duyrrenberger, D. Ghislieri, E. Chrakova, T. Quinto, L. Knorr, D. Haussinger, F. Hollmann, N.J. Turner, T.R. Ward, Nature Chemistry, 2012, 5, 93.

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Enzyme catalysed oxidations

Cascades in order activate oxygen Enzyme instability issues Mass transfer limitations

Alcohol oxidations: oxidasesOxygen insertion: peroxidases/peroxygenases

D. Holtmann, M.W. Fraaije, I.W.C.E. Arends, D.J. Opperman and F. Hollmann, The taming of oxygen:

biocatalytic oxyfunctionalisations, Chem. Commun. 50 (2014) 13180-13200

F. Hollmann, I.W.C.E. Arends, K. Buehler, A. Schallmey and B. Bühler, Enzyme-mediated oxidations for

the chemist, Green Chem. 13 (2011) 226-265

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Non-conventional cofactor regeneration

F. Hollmann, I.W.C.E. Arends and K. Buehler, Biocatalytic redox reactions for organic synthesis: nonconventional regeneration methods, ChemCatChem 2 (2010) 762-782

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Laccase Enzyme

Activation by oxygen

messenger molecule: TEMPO

I. Matijosyte et al. J. Mol. Cat. B. 62 (2010) 142. S.A. Tromp, I. Matijosyte, et al. ChemCatChem 2 (2010) 827.

One-electron oxidant

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Alcohol Dehydrogenases for chiral oxidations

Oxidation of racemic profen aldehydes Nonsteroidal anti inflammatory drugs Shift of equilibrium towards acid by using NADH regeneration Cofactor regeneration with oxidase.

Konst et al. Angew. Chem. Int. Ed. 51 (2012) 914; ChemCatChem, 5 (2013) 3027.

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30405060

67

8 90

2

4

6

8

d[C

HO

n]/dt [m

Mh-1

]

T [oC]pH [-]

Laccase and NADH recycling

Cascading two enzymes:Laccase from Myceliophoria thermophilaADH from Thermus sp. ATN1

S. Aksu, I.W.C.E. Arends, F. Hollmann, Adv. Synth. Catal. 351 (2009) 1211.

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0

2

4

6

8

0 5

c(1-p

henyle

thanol)

(mM

)

time (h)

AOx alcohol oxidase from Pichia pastoris (PpAOx,

black squares, )

AOx alcohol oxidase from Candida boidinii (CbAOx,

red diamonds, )

ee 99%

Y Ni, et al. Angew. Chem. Int. Ed. 55 (2016) 798-801

Enzymatic cascade for in situ H2O2-generation with

methanol. Scale-up of peroxygenases from fungi.

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Methanol oxidation to formic acid: 2 H2O2

FDM: formaldehyde dismutase from Pseudomonas putida

AOx + FDM()

AOx ()

TN 485800

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From methanol to formic acid: 2 H2O2

From formic acid to CO2: 1 H2O2

Methanol oxidation to CO2: 3 H2O2

Y Ni, et al. Angew. Chem. Int. Ed. 55 (2016) 798-801

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Two-liquid-phase system

Higher substrate concentration

and product stability for enzymatic oxidation

Selectivity = [1-phenylethanol] (mol) / [total products] (mol).

Selectivity 97%; ee>98%

(R)-1-phenylethanol (,)

acetophenone (,)

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Photocatalytic NAD+ regeneration

0

0,04

0,08

0,12

0,16

0 30 60 90 120 150

time [min]

[NA

DH

] [m

M]

TF(FMN) = 237h-1

S. Gargiulo, I.W.C.E. Arends, F. Hollmann, Chemcatchem, 2011, 3, 338.

• Fast kinetics• Flavin true catalyst

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Photoenzymatic lactonization of diols

• Non-optimized conditions:– TTN(NAD)=170, TTN(FMN)=340

– Full conversion

S. Gargiulo, I.W.C.E. Arends, F. Hollmann, ChemCatchem, 2011, 3, 338.

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Photocatalytic oxidations – recent results

Carbonitrides active photocatalysts, even for hydroxylation.

W. Zhang, F. Hollmann

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Cascade with water splitting catalysts

Taking electrons from sunlight

M. Mifsud, S. Gargiulo, S. Iborra, I.W.C.E. Arends, F. Hollmann and A. Corma, Nature Commun. 5 (2014) 3145

Au-TiO2

V-TiO2

• OYE from Thermus scotoductus• 323 K• 10 g/l photocatalyst

30

0

25

50

75

100

0 2 4 6time [h]

conv

ersi

on [%

][FMN]=10 mM

[FMN]=50 mM

[FMN]=250 mM

[H2O2]= 1eq

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Sunlight promotes yield in chloroperoxidase catalyzed

sulfoxidation

Reaction on 25mL scale: (buffer pH5/tBuOH)=3:1; T=25oC; [Thioanisol] = 50 mM; [CPO]=3.9 μM; [EDTA]=50 mM.

D. Perez et al., Chem. Commun., (2009) 6848;E. Churakova et al., unpublished results.

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Chloroperoxidase loaded in polymersomes

polystyrene-

poly(isocyanoalanylthiophenylethylamide)

block copolymers

H.M. De Hoog et al. , Org. Biomol. Chem. 7 (2009) 4604

Collaboration Nijmegen-Delft (Nolte, Rowan, Cornelissen, van Hest)

Hans-Peter de Hoog

S

NH

O N

H

Nt-Bu

O

PS-PIAT

40n

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O

O

O

CALB

OH

O

O

H

HO

H

HOH

HO

OHH

H

OH

HOOH + glucoronic acid

GO

OO

O O

tetraguaiacol

CPO

Three-enzyme cascade

in polymersome-loaded

hydrogel reactor

H.M. De Hoog et al. Nanoscale 2 (2010) 709