Enzymatic synthesis of alkyl oligoxylosides from isolated xylans and from lignocellulosic biomass

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Enzymatic synthesis of alkyl oligoxylosides from isolated xylans and from lignocellulosic biomass

Marjorie Ochs, Murielle Muzard, Richard Plantier-Royon, B. Estrine, Caroline Rémond

To cite this version:

Marjorie Ochs, Murielle Muzard, Richard Plantier-Royon, B. Estrine, Caroline Rémond. Enzymatic synthesis of alkyl oligoxylosides from isolated xylans and from lignocellulosic biomass. Biotrans 2011:

10. International Symposium on Biocatalysis, Oct 2011, Messina, Italy. 1 p. �hal-02805236�

Biotrans 2011 Italy

10th International Symposium on Biocatalysis

October 2 - 6, 2011

Giardini Naxos (ME), Sicily

2 Introduction 3

Venue address

Atahotel Naxos Beach Via Recanati, 26

98035 Giardini Naxos (Messina) Italy

Permanent Steering Committee

Herfried GRIENGL Robert AZERAD Stefano SERVI

Wolf-Dieter FESSNER Vladimir KREN Maurice FRANSSEN Vicente GOTOR Jean-Louis REYMOND

Scientific Committee

Sergio RIVA (Italy) Yasuhisa ASANO (Japan) Andrea BOMMARIUS (USA)

Marco FRAAIJE (the Netherlands) Lucia GARDOSSI (Italy)

Marina LOTTI (Italy) Mosè ROSSI (Italy) Georg SPRENGER (Germany)

Organizing Committee

Sergio RIVA Stefano SERVI Francesco MOLINARI Giovanni NICOLOSI Nicola D’ANTONA Paola D’ARRIGO Daniela MONTI Gianluca OTTOLINA

Dear Biotrans2011 Participants,

On behalf of the Biotrans Steering and Scientific Committees and of the local Organizing Committee I wish to welcome you all to the 10th International Symposium on Biocatalysis and Biotransformations taking place from October 2nd to 6th, 2011, at Atahotel Naxos Beach in Giardini Naxos, Sicily.

The Symposium will be attended by more than 640 industrial and academic participants, making Biotrans2011 probably the largest scientific event on Biocatalysis ever organized.

This Meeting will continue the series of Symposia successfully held every two years since 1993. The first edi- tion took place in Graz (Austria), following the brilliant intuition of Herfried Griengl to have also in Europe an international forum to discuss topics that nowadays are more and more related to “sustainable chemistry”, involving competences in the areas of Chemistry, Biochemistry, Biology and Engineering.

More than 50 lectures and 400 poster communications will be presented, focusing on the following topics:

• Discovery and design of new biocatalysts

• Enzymes structure & mechanism, bioinformatics & modelling

• Biotransformations in organic synthesis

• Oxidative biocatalysis

• Biocatalysis for polymer and material chemistry

• Cascade chemo-enzymatic processes

• Biocatalysis and biorefineries

• Industrial processes research & development

As in some of the previous editions, a COST Workshop – related to the activities of the COST Action CM0701

“CASCAT” – is fully embedded in the Conference Program.

We thank the COST organization for the support given to our Conference and, similarly, we gratefully ac- knowledge the support by numerous Sponsors and Institutions, whose names are listed in this book.

I wish all the participants a successful scientific meeting and an enjoyable stay in this wonderful part of Italy.

Sergio Riva

Chairman of the Biotrans2011 Symposium

Sponsor Exhibitors

Under the patronage of

13 Conference Program

KL - Keynote Lecture PL - Plenary Lecture

IL - Invited Lecture OC - Oral Communication PC - Poster Communication

Protein engineering on the move from directed evolution to in silico approaches

Uwe T. Bornscheuer

Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Haus- dorff-Str. 4, D-17487 Greifswald, Germany

E-mail: uwe.bornscheuer@uni-greifswald.de

Protein engineering has developed in the past decade to a highly important technology

as it is a useful tool to create enzymes with desired properties (with respect to e.g., substrate specificity, stereoselectivity or thermostability), but also helps to understand how proteins evolved and how they function.

Whereas initially rational protein design based on detailed analysis of three-dimensional structures was the method of choice, directed evolution – in essence a random mutagenesis followed by screening or selec- tion of desired mutants – became an important alternative. More recently, researchers used combinations of both methods. In this lecture, the principle strategies and current challenges in protein engineering will be highlighted. Examples will include the creation of an epoxide hydrolase from an esterase scaffold within the α/β-hydrolase fold enzyme family

and the inversion of enantioselectivity of an esterase active towards sterically demanding tertiary alcohols.

The vast number of protein sequences available from databases substantially facilitates protein engineering.

We used this plethora of information to develop a method for an “in silico neutral drift” based on the analysis of >2.800 sequences of enzymes from the α/β-hydrolase fold family using the 3DM database.

This resulted in ‘small, but smart’ focused protein libraries, from which enzyme variants with substantially enhanced ther- mostability, enantioselectivity and altered substrate range could be identified.

Finally, a detailed in silico analysis enabled the identification of a toolbox of novel (R)-selective transaminases.

[1] Kazlauskas, R.J., Bornscheuer, U.T. (2009) Nature Chem. Biol., 5, 526-529.

[2] Lutz, S., Bornscheuer, U.T. (Eds.) (2009) Protein Engineering Handbook, Wiley-VCH, Weinheim

[3] Jochens, H., Stiba, K., Savile, C., Fujii, R., Yu., J.-G., Gerassenkov, T., Kazlauskas, R.J., Bornscheuer, U.T. (2009) Angew.

Chem. Int. Ed., 48, 3532-3535.

[4] Bartsch, S., Kourist, R., Bornscheuer, U.T. (2008) Angew. Chem. Int. Ed., 47, 1508-1511.

[5] Kourist, R., Jochens, H., Bartsch, S., Kuipers, R., Padhi, S.K., Gall, M., Böttcher, D., Joosten, H.-J., Bornscheuer, U.T.

(2010), ChemBioChem, 11, 1635-1643.

KL

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Enzymologists in wonderland: new biotech applications from evolved D-amino acid oxidases

Loredano Pollegioni

“The Protein Factory“, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano (via Mancinelli 7, Milano) and Università degli Studi dell’Insubria (via Dunant 3, Varese), Italy

E-mail: loredano.pollegioni@uninsubria.it

D-Amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxygen-dependent oxida- tive deamination of amino acids D-isomers resulting in α-keto acids, ammonia and hydrogen peroxide.

Owing to the absolute stereoselectivity (L-amino acids are neither substrates nor inhibitors) and broad sub- strate specificity, in past years DAAO has been investigated for use as an industrial biocatalyst - the most important application being the two-step conversion of cephalosporin C into 7-amino cephalosporanic acid.

[2]

Most recently has light been shed on the extraordinary functional plasticity of this enzyme, mainly be- cause of detailed investigations into structure-function relationships on yeast DAAO (i.e. elucidating the 3D structure and performing site-directed mutagenesis) and by identifying novel DAAOs. These investigations showed that the active site residues of DAAO are not directly involved in catalysis, but operate by maintain- ing the proper conformation and a favorable microenvironment for optimal catalysis.

The extraordinary functional plasticity of DAAO makes this flavoenzyme a suitable scaffold for developing new properties.

These findings have boosted research on DAAO: by applying the most advanced techniques in protein en- gineering, substrate specificity, oxygen affinity, cofactor binding, and oligomeric state of the enzyme have been redesigned.

Recent developments devoted to produce improved DAAO variants for established ap- plications, including as a biocatalyst for resolving racemic amino acid mixtures (even unnatural ones) and as a tool for biosensing (i.e. for the rapid and reliable detection of the neurotransmitter D-serine in the brain or the total D-amino acid content in food specimen). Furthermore, DAAOs were also developed for novel areas of research, including within medical (an enzyme variant most active at low oxygen concentration al- lows a higher toxic effect on cancer cells), environmental and agricultural fields (i.e. as a new mechanism of herbicide resistance).

In the next future, new concepts and fields of research/application of DAAO will further develop mainly by synthesizing “creative thinking” and “classical scientific analysis” based on the solid foundation of previous studies.

[1] Pollegioni, L. et al. (2007) Physiological functions of D-amino acid oxidases: from yeast to humans. Cell. Mol. Life Sci. 64, 1373-94

[2] Pilone, M.S. and Pollegioni, L. (2010) Enzymes, D-amino acid oxidases. In Encyclopedia of Industrial Biotechnology:

Bioprocess, Bioseparation, and Cell Technology (Flickinger, M.C., ed.), pp. 1-11, John Wiley & Sons

[3] Pollegioni, L. and Molla, G. (2011) New biotech applications from evolved D-amino acid oxidases. Trends Biotechnol., in

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Laccases in biocatalytic cascade reactions: regeneration of cofactors coenzymes

Dietmar Haltrich, Roman Kittl, Christoph Sygmund, Roland Ludwig

Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU – University of Natu- ral Resources and Life Sciences, 1190 Vienna, Austria

E-mail: dietmar.haltrich@boku.ac.at

Laccases belong to a diverse superfamily of multi-copper oxidases (MCOs) that catalyse one-electron trans- fer oxidations of various phenolic compounds, complexed metal ions and dye molecules, with transfer of these electrons to the four copper centers of laccase. These electrons are then passed on to dioxygen, result- ing in its reduction to water rather than hydrogen peroxide. Laccases are widespread in nature, and – depend- ing on their origin – they can show widely varying properties including redox potential, substrate specificity, stability and catalytic optima. Laccases have found increased interest and application in organic synthesis in recent years.

We have shown that laccases can be used to efficiently regenerate flavin-containing oxidoreductases, both dehydrogenases and oxidases, by reoxidising artificial redox mediator used by these oxidoreductases as electron acceptors in reactions that are of biocatalytic interest. This can enable the use of flavin-dependent dehydrogenases in biocatalysis since then the electron acceptor can be added in minute, stoichiometric amounts. When using flavin-containing oxidases, this approach of laccase regeneration can contribute to the stabilisation of the enzyme during turnover, since the formation of highly reactive hydrogen peroxide can be greatly reduced when using alternative electron acceptors that are continuously regenerated by laccase.

This concept employing laccase can be even further expanded to continuous in situ regeneration of NAD+

and NADP+ from NADH and NADPH, respectively, in coenzyme-dependent, dehydrogenase-catalyzed reactions. Here, the enzymatic regeneration system uses laccase in combination with redox mediators to reoxidise NADH or NADPH by concomitant reduction of oxygen to water. We tested several redox media- tors under different conditions, and Meldola’s blue appeared to be the most promising candidate as a redox mediator for these processes. Laccases from the Japanese lacquer tree Rhus vernificera, the basidiomycete Trametes pubescens, and the ascomycete Melanocarpus albomyces were tested with Meldola’s blue as redox mediator in different dehydrogenase-catalyzed conversions to corroborate the application potential. One of these reactions employed NAD(P)-dependent glucose dehydrogenase for the oxidation of glucose, and the redox mediator Meldolas’s blue, laccase and oxygen as regenerative system. For both coenzymes turnover numbers of >1000 were obtained in 0.5-L batch biocatalytic experiments.

[1] Riva, S. Laccases: blue enzymes for green chemistry. Trends Biotechnol. (2006) 24:219–226

[2] Kunamneni, A., Camarero, S., García-Burgos, C., Plou, F.J., Ballesteros, A., Alcalde, M. Engineering and applications of fungal laccases for organic synthesis. Microb. Cell Fact. (2008) 7:32

Monday, October 3 Lectures

[1] Wang, Z.; Lie, F.; Lim, E.; Li, K.; Li, Z. Adv. Synth. Catal. 2009, 351, 1849 –1856.

[2] Wang, Z.; Wu, J.; Li, Z. Manuscript in preparation.

[3] Chen, Y., Lie, F.; Li. Z. Adv. Synth. Catal. 2009, 351, 2107 – 2112.

[4] Lie, F.; Chen, Y.; Wang, Z.; Li, Z. Tetrahedron: Asymmetry 2009, 20, 1206–1211.

[5] Zhang, W.; Tang, W. L.; Wang, Z.; Li, Z. Adv. Synth. Catal. 2010, 352, 3380-3390.

[6] Tang, W.; Li, Z.; Zhao, H. Chem. Commun. 2010, 46, 5461–5463.

[7] Chen, Y.; Tang W. L.; Mou, J.; Li, Z. Angew. Chem. Int. Ed. 2010, 49, 5278 –5283.

Regio- and stereo-selective biohydroxylations for organic synthesis: biocatalyst discovery and engineering

Zhi Li

Department of Chemical & Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576

E-mail: chelz@nus.edu.sg

Regio- and stereo-selective biohydroxylations are important reactions for the syntheses of enantiopure alco- hols that are useful synthons, aroma materials, and pharmaceutical intermediates. One of the key challenges for the practical application of this type of transformation in organic synthesis is the development of efficient biocatalysts. We recently identified Cellulosimicrobium cellulans EB-8-4 for the allylic hydroxylation of D- limonene with >99% regio- and stereo-selectivity to give (+)-trans-carveol, a useful and valuable fragrance and flavor compound.

This biotransformation afforded 42-times higher product concentration

than the best known result. Similarly, Pseudomonas monteilii TA-5 was developed as a powerful biocatalyst for the enantioselective benzylic hydroxylation to give several (R)-benzylic alcohols containing reactive functional groups in 93–99% ee as the only products with 56-66% isolated yield.

This strain catalyzed also the hy- droxylation of indan and tetralin, giving (R)-1-indanol and (R)-1-tetralol in 99% ee and 62–67% yields.

We also engineered a recombinant Escherichia coli expressing the P450pyr monooxygenase from Sphingo- monas sp. HXN-200 for the regio- and stereo-selective hydroxylation.

Biohydroxylation of N-benzylpyr- rolidin-2-one with the E. coli cells gave (S)-N-benzyl-4-hydroxypyrrolidin-2-one (a useful intermediate for preparing oral carbapenem antibiotic CS-834) in >99% ee, with 2.6-fold increase of product concentration in comparison with the wild type strain. The recombinant biocatalyst demonstrated also excellent regio- and stereo-selectivity for the hydroxylation of (-)-β-pinene giving the valuable (1R)-trans-pinocarveol in 82%

yield, with 200-fold increase of the product concentration compared with the best reported biosystem for the same transformation.

The enantioselectivity of P450pyr monooxygenase for the biohydroxylation of N-benzyl pyrrolidine was improved by directed evolution.

After three generations of evolution, the best mutant 1AF4A was able to produce the product in 83% (R) ee compared to the wild type’s 43% ee (S). This demonstrated the first ex- ample of evolution of a P450 monooxygenase with inverted enantioselectivity. To facilitate the evolution of enantioselective enzyme, a generally useful high-throughput method for determining the enantioselectivity of biohydroxylations was developed by the use of isotopically labeled enantiopure substrates and MS analy- sis.

Based on this assay and the structure information of the P450pyr hydroxylase, directed evolution led to the discovery of a triple mutant with excellent enantioselectivity to produce (S)-N-benzyl 3-hydroxypyr- rolidine (an useful pharmaceutical intermediate) in >98% ee.

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[1] Food Chem. 116:114 (2009)

[2] J. Mol. Catal. B:Enzymatic. 66:72 (2010) [3] Appl. Environ. Microbiol. 76:6397 (2010)

Combining protein engineering with statistical modeling for the novel synthesis of hydroxytyrosol by toluene 4-monooxygenase

Moran Brouk

, Yuval Nov

, Ayelet Fishman

a

Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, 32000, Israel;

Department of Statistics, University of Haifa, Haifa 31905, Israel

E-mail: afishman@tx.technion.ac.il

Hydroxytyrosol (HTyr), one of the most important phenols present in olives, stands out as a compound of high added value due to its exceptional antioxidant, antimicrobial and anti-carcinogenic activities. It is be- lieved to be the antioxidant with the highest free radical scavenging capacity: double that of quercetin and more than three times that of epicatechin. It has been demonstrated that HTyr inhibits human low-density li- poprotein (LDL) oxidation, scavenges free radicals, inhibits platelet aggregation and confers cell protection.

The vast amount of data accumulated regarding the benefits of HTyr, together with its high bioavailability in human, make it a good candidate to serve as an antioxidant for either pharmaceutical or food preparations (i.e. functional foods). Despite the great potential of HTyr, its commercial availablity is limited, therefore a biotechnological process is highly desired.

The goal of this research is to engineer toluene monooxygenases (TMOs) for the biosynthesis of commer- cially-valuable HTyr, from a cheap and abundant substrate, 2-phenylethanol (PEA). This enzymatic hy- droxylation is a novel and promising method to synthesize HTyr in a low cost single-step reaction, with high selectivity while utilizing an environmentally friendly process.

Escherichia coli cells manipulated to express TMOs are capable of oxidizing a wide range of substituted aromatic and phenolic compounds with high regiospecificity. Despite the resemblance of PEA to the natu- ral substrate, toluene, it was found to be a very poor substrate for the wild-type enzymes. In this research, by employing several protein engineering approaches, the substrate specificity and oxidation activity were dramatically improved. The non-rational approach of directed evolution, led to the discovery of a distant residue from the active site, S395, which affects the enzyme’s activity.

Another valuable residue, D285, located at the entrance of the channel leading to the active site, was found based on rational design.

Fur- thermore, a statistical model was developed to give predictions to which mutations should be combined to give further rise in activity. One triple mutant suggested by this model, had a 200-fold improvement in activ- ity compared to the wild-type enzyme.

It was concluded that increasing the size of the active site pocket and the channel entrance, enables for the first time HTyr formation, which the wild-type enzyme was not capable of producing.

[1] Labinger, J. A.; Bercaw, J. E. Nature 2002, 417, 507-514.

[2] Punniyamurthy, T.; Velusamy, S.; Iqbal, J. Chem. Rev. 2005, 105, 2329-2364.

[3] Kamata, K.; Yonehara, K.; Nakagawa, Y.; Uehara, K.; Mizuno, N. Nat Chem 2010, 2, 478-483.

[4] Beilen, J. B. v.; Funhoff, E. G. Curr. Opin.Biotechnol. 2005, 16, 308-314.

[5] Funhoff, E. G.; Salzmann, J.; Bauer, U.; Witholt, B.; van Beilen, J. B. Enz. Microbiol. Technol. 2007, 40, 806-812.

Enzymatic selective oxidation of alkanes under mild conditions

Mélanie Bordeaux

, Nakry Pen

, Anne Galarneau

, François Fajula

, Jullien Drone

a

Institut Charles Gerhardt Montpellier UMR 5253 CNRS/ENSCM/UM2/UM1,8 rue de l’Ecole Normale 34296 Montpellier, France

E-mail: jullien.drone@enscm.fr

The selective catalytic oxidation of alkanes under mild conditions remains a major challenge in industrial and synthetic chemistry because these routes are generally energy intensive and environmentally unfriendly.

[1,2]

As a result, much research is conducted on finding and engineering new catalysts to approach the ideal

catalyst for alkane hydroxylation.

Biocatalysts that oxidize alkanes allow organisms to use hydrocarbons as a source of energy and cellular building blocks are theorically approaching the features of the ideal biocatalyst.

The recent discovery of a new and promising P450 alkane hydroxylase family (the CYP153 family) has attracted attention.

We have used molecular engineering to successfully convert a low-activity octane hydroxylase (CY- P153A13a from Alcanivorax borkumensis) into a fast and regioselective medium-chain terminal alkane hydroxylase we named P450 A13-red

(Figure 1).

Figure 1. The regioselective medium-chain terminal alkane hydroxylation by P450 A13-red.

We will present our results concerning the construction and the characterization of A13-red as well as our efforts to develop a sustainable, continous-flow process based on this potent biocatalyst.

Monday, October 3 Lectures

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[1] Cremonesi, P., Carrea, G., et al. 1973, Arch. Biochem. Biophys., 159, 1, 7-10.

[2] Donova, M.V., Egorova, O.V. et al. 2005, Process Biochem., 40, 7, 2253-2262.

[3] Rheault, P. and Charbonneau, A. 1999, Biochim. Biophys. Acta, 1447, 17-24.

[4] Fogal S., Motterle R. et al. 2010. Chem. Eng. Transaction, 20, 61-66.

From literature to industrial scale.

Testosterone synthesis: a case history

Riccardo Motterle

, Stefano Fogal

, Giancarlo Arvotti

, Chiara Bezze

, Elisabetta Bergantino

, Andrea Castellin

.

a

F.I.S. Fabbrica Italiana Sintetici S.p.A., Viale Milano 26, Alte di Montecchio Maggiore, Vicenza, Italy;

Dipartimento di Biologia, Università degli Studi di Padova, Viale G. Colombo 3, Padova, Italy

E-mail: riccardo.motterle@fisvi.com

A chemo-enzymatic process for testosterone (TS) synthesis from commercially available androstendione (AD) is presented (see fig. 1). Stereo- and regio-selective reduction of AD cheto group in 17 position af- forded the corresponding alcohol (TS).

Figure 1. Testosterone synthesis from androstendione.

With the purpose of developping a sustainable process to be implemented at industrial level, we selected the chemo-enzymatic approach as the most appropriate.

According to literature, no commercial enzymes were available to efficiently catalyze this transformation. Crossing bibliographic and bioinformatics data, we assumed that murine 17βHSD (17β-hydroxysteroid dehydrogenase) type 5

could represent a good candidate. The enzyme was genetically modified and then cloned for recombinant expression in E. coli

(KRD-FIS 001). After verification of the ability of the enzyme to specifically catalyze the desired transfor- mation, fermentation and isolation processes were optimized and scaled-up to few m3 bioreactor level. At the same time, the development of the chemo-enzymatic process was carried out. Since the enzyme KRD- FIS 001 can use both NADH and NADPH as cofactors, a glucose dehydrogenase (GDH) was coupled for cofactor regeneration. Moreover, due to the extremely low water solubility of both AD and TS, the stability of the enzyme to organic co-solvent presence was investigated and methanol was found to be the best choice.

Optimal pH and temperature for the biotransformation were set up and kinetic parameters were measured.

From an initial dilution of more than 30.000 L/Kg, unfeasible and useless for industrial implementation, the process was further optimized by stabilizing the enzyme to work at highly concentrated conditions. The re- sulting engineered chemo-enzymatic process was claimed in a patent application and verified at Kilo level.

Optimization is still ongoing, with the aim to scale-up at industrial level for multiton production campaign.

Engineering of oxidase enzymes for large scale production of APIs and intermediates

Ee Lui Ang

Codexis Laboratories Singapore Pte Ltd, 61 Science Park Rd #03-15/24, The Galen, Singapore Science Park II, Singapore 117525, Singapore

E-mail:eelui.ang@codexis.com

This presentation focuses on the recent work by Codexis to develop oxidase enzymes, namely mono-oxyge- nases and amine oxidases, to accept a range of substrates with high enantioselectivity for large scale produc- tion of specific APIs and intermediates. To engineer mono-oxygenase enzymes to work as biocatalysts for scalable and economically feasible industrial sulfoxidation processes, significant changes had to be made to various properties of the wild type enzyme. Using directed evolution, we were able to reverse the enanati- oselectivity of the mono-oxgenase enzyme, and suppress the formation of over-oxidation by-products. We also made significant improvements in multiple parameters such as activity and thermostability, as well as substrate and product tolerance to deliver a high productivity enzyme suitable for large scale biocatalysis.

The development of amine oxidases for large-scale chiral synthesis of important drug intermediates with marked advantages over current chemical process routes will also be discussed.

Monday, October 3 Lectures

OH

O

CH₃ H CH₃ O

O

CH₃ H CH₃

NAD(P) NAD(P)H

KRD-FIS 001

GDH Glucose Gluconic Acid

Androstendione Testosterone

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[1] K. Goldberg, K. Schroer, S. Lütz, A. Liese Applied Microbiology & Biotechnology 76 (2007), 237-248, DOI: 10.1007/

s00253-007-1002-0

[2] K. Goldberg, K. Schroer, S. Lütz, A. Liese, Applied Microbiology & Biotechnology 76 (2007), 249-255, DOI: 10.1007/

s00253-007-1005-x

[3] K. Schroer, M. Kittelmann, S. Lütz, Biotechnology&Bioengineering 106 (2010), 699-706, DOI: 10.1002/bit.22775

Recent developments in the application of oxidoreductases

Falk Hildebrand

, Christina Kohlmann

, Kirsten Schroer

, Matthias Kittelmann

, Stephan Lütz

a

Institute of Biotechnology 2, Reserach Centre Jülich, Germany

b

Novartis Institutes for BioMedical Research, Basel, Switzerland E-mail: stephan.luetz@novartis.com

Oxidoreductases are valuable tools for the synthesis of chiral builduing blocks as well as drug metabolites on preparative scale, e.g.

They are especially appealing biocatalysts for several reasons, e.g. their ability to be used in asymmetric synthesis reactions (100% yield) rather than kinetic resolutions or their ability to catalyze reaction which are difficult to achieve chemically, e.g. selective C-H-bond oxidation.

Several recent developments in the application of Oxidoreductases will be given, including the use of al- ternative reaction media (e.g. ionic liquids) to solubilize poorly water-soluble substrates

as well as the engineering of whole cell catalysts for redox reactions will be presented.

Limitations and opportunities will be discussed from an application point of view.

Monday, October 3 Lectures

[1] E. Burda, W. Hummel, H. Gröger, Angew. Chem. 2008, 120, 9693; Angew. Chem. Int. Ed. 2008, 47, 9551.

[2] E. Burda, W. Bauer, W. Hummel, H. Gröger, ChemCatChem 2010, 2, 67-72.

[3] K. Baer, M. Kraußer, E. Burda, W. Hummel, A. Berkessel, H. Gröger, Angew. Chem. 2009, 121, 9519; Angew. Chem. Int.

Ed. 2009, 48, 9355.

[4] G. Rulli, N. Duangdee, K. Baer, W. Hummel, A. Berkessel, H. Gröger, Angew. Chem. 2011, in press; Angew. Chem. Int. Ed.

Combination of chemo- and biocatalysis in multi-step one-pot processes in aqueous reaction media

Harald Gröger

Faculty of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany E-mail: harald.groeger@uni-bielefeld.de

Multi-step one-pot processes represent an attractive synthetic concept for the improvement of overall pro- cess efficiency by decreasing the required number of work up and purification steps. By avoiding such time-, capacity- and solvent-intensive process steps, multi-step one-pot syntheses contribute to a signifi- cantly improved process economy as well as to more sustainable synthetic routes. A key criterion for multi- step one-pot processes is the compatibility of the individual reaction steps with each other. Accordingly, most of today´s known multi-step one-pot processes are based on either chemocatalytic multi-step reactions or “pure” biotechnological processes such as, e.g., fermentation. In contrast, successful combinations of chemo- and biocatalytic reactions, in particular in aqueous reaction media, are much less widely known.

In this contribution strategies for the combination of chemo- and biocatalysts towards the development of multi-step one-pot processes in aqueous reaction media are presented. Since palladium-catalyzed cross-cou- pling reactions are of particular importance in the field of metal catalysis, as enzymatic reductions are in the field of biocatalysis, we were interested in the investigation of the compatibility of these types of reactions with each other in water. As an example for such a one-pot process the synthesis of chiral biaryl-containing alcohols via Suzuki-cross-coupling reaction and subsequent asymmetric enzymatic reduction is discussed.

[1,2]

Very recently we could also demonstrate the compatibility of a metal-catalyzed cross-metathesis reaction

with a biotransformation.

A further research focus has been on the combination of an organo- and biocatalytic reaction sequence. It turned out that the reaction mixture resulting from an asymmetric organocatalytic aldol reaction is compat- ible with a direct subsequent enzymatic reduction without the need for a work-up step of the aldol reaction.

[3,4]

Furthermore, a combination of non-catalyzed Wittig reactions with biocatalytic reductions of C=O and

C=C double bonds, respectively, in aqueous reaction media has been achieved.

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Novel applications of ene-reductases in dynamic kinetic resolutions and redox-cascade biotransformations

Marko D. Mihovilovic

a

Vienna University of Technology, Institute of Applied Synthetic Chemistry, Getreidemarkt 9/163-OC, A-1060 Vienna, Austria

E-mail: mmihovil@pop.tuwien.ac.at

Novel applications of ene-reductases for the preparation of chiral compounds will be discussed. The biore- duction of Baylis-Hillman reaction products was studied; in this context, a partial dynamic kinetic resolu- tion process was observed yielding syn-products, predominantly (X = OH). The conversion of substrates containing protected amines (X = NHCOOR) was found to proceed to anti-products, exclusively. This rep- resents a novel approach to non-natural β-amino acids.

Recently, we discovered regiodivergent Baeyer-Villiger oxygenations of various enantio- and regioisomeric terpenones. By combining this biotransformation with a preceeding ene-reductase reaction, troublesome chemical hydrogenation of easily available enone precursors can be avoided. This three enzyme redox cas- cade process (including a suitable dehydrogenase for cofactor regeneration) represents a novel combination of redox biocatalysts in a modular fashion aiming at the formation of potential fragrance compounds.

Monday, October 3 Lectures

[1] Van Rantwijk, F. and Sheldon, R.A., Curr. Opin. Biotechnol. 11 (2000) 554

[2] Perez, D.I., Mifsud Grau M., Arends I.W.C.E. and Hollmann F., Chem. Commun. 44 (2009) 6848

Enantiospecific photobiocatalytic oxyfunctionalization reactions

E. Churakova, I.W.C.E. Arends, F. Hollmann

Department of Biotechnology, Delft University of Technology, Julianalaan 136, Delft, The Netherlands

E-mail: e.churakova@tudelft.nl

Heme peroxidases widely considered as versatile biocatalysts. Unlike the P450 monooxygenases, heme- peroxidases do not rely on expensive NADH cofactor and catalyze a broad range of oxidation/oxyfunc- tionalization reactions simply utilizing hydrogen peroxide (H2O2) as oxidant with high degrees of chemo-, regio- and stereo-specificity. But the prosthetic heme group can be easily inactivated even by low H2O2 concentrations.

To avoid this chemical degradation of enzyme active site we have developed an alterna- tive, light-driven approach for in situ H2O2 generation. Visible-light exited flavins such as flavin adenine mononucleotide (FMN) can oxidize simple and abundant electron donors such as ascorbic acid. The result- ing reduced flavins (FMNH2) quickly react with molecular oxygen to eventually yield the re-oxidized fla- vins and hydrogen peroxide. Promising preliminary results for photoenzymatic approach have been obtained with chloroperoxidase-catalyzed sulfoxidation reactions.

Here, we report a practical and simple photoenzymatic oxyfunctionalization method by combining the pho- tocatalytic approach for the controlled in situ H

O

generation method from O

to a novel and highly promis- ing haloperoxidase from Agrocybe aegerita (AaP) (Figure 1). We have investigated the applicability of AaP as catalyst for photobiocatalytic epoxidation and hydroxylation reactions. The proposed in situ generation of H2O2 proved to be a suitable approach to achieve stable and robust oxyfunctionalization activity (more than 40-fold increase of productivity time as compared to the stoichiometric use of H

O

). High productivities and excellent enantiomeric excesses (>97%) were obtained for the majority of studied substrates.

Figure 1. Photoenzymatic oxyfunctionalization by combining in situ H

O

generation with AaP-catalyzed hydroxylation/epoxidation

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[1] R. D. Norcross and I. Paterson, Chem. Rev. 1995, 95, 2041

[2] K. Nakamura, T. Matsuda, in “Enzyme Catalysis in Organic Synthesis”, 2002, vol.3, Edited by Drauz K, Waldmann H.

Weinheim: Wiley-VCH, pp.991-1047.

[3] D. Kalaitzakis, J. D. Rozzell, S. Kambourakis, I. Smonou, Org. Lett. 2005, 7, 4799-4801.

[4] D. Kalaitzakis, J. D. Rozzell, S. Kambourakis, I. Smonou, Adv. Synth. Catal. 2006, 348, 1958-1969.

A two-step, one-pot enzymatic synthesis of 2-substituted-1,3-diols

Ioulia Smonou*, Kalaitzakis Dimitris

Department of Chemistry, University of Crete, 71003, Heraklio, Crete, Greece smonou@chemistry.uoc.gr

Optically active 1,3-diols are important targets for many synthetic methodologies, since they are high value chiral synthons or building blocks for the synthesis of many natural products and pharmaceuticals. They have frequently been used as valuable intermediates in the synthesis of drugs and natural products with important biological activity.

Stereoselective ketoreductase-catalysed reductions

of a-substituted-b- hydroxy ketones will be described. The stereoisomeric syn or anti α-substituted 1,3-diols were prepared in high optical purities (>99% de, >99% ee) and chemical yields. This is a simple, highly stereoselective and quantitative method for the synthesis of different diastereomers of chiral diols.

Furthermore, we will present a biocatalytic cascade reaction, which was designed for the stereoselective synthesis of optically pure 2-alkyl-1,3-diols employing two enzymes.

The cascade process consists of two consecutive steps: a stereoselective diketone reduction and a hydroxy ketone reduction. Chiral diols were formed by the addition of ketoreductases in the same vessel, in high stereoselectivity and chemical yield, without the isolation of the intermediate β-hydroxy ketones.

Monday, October 3 Lectures

[1] S. Mukherjee, J. W. Yang, S. Hoffmann, B. List, Chem. Rev. 2007, 107, 5471-5569.

[2] P. Clapés, W.-D. Fessner, in Stereoselective Reactions of Carbonyl and Imino Groups, Vol. 2 (Ed.: G. A. Molander), Georg Thieme Verlag KG, Suttgart (Germany), 2011, pp. 677-734.

[3] M. Schürmann, G. A. Sprenger, J. Biol. Chem. 2001, 276, 11055-11061.

[4] J. A. Castillo, J. Calveras, J. Casas, M. Mitjans, M. P. Vinardell, T. Parella, T. Inoue, G. A. Sprenger, J. Joglar, P. Clapés, Org.

Aldolases in cascade reactions for asymmetric synthesis

Pere Clapés

Instituto de Química Avanzada de Cataluña-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain.

E-mail: pere.clapes@iqac.csic.es

Carbon-carbon bond formation is a cornerstone reaction in synthetic organic chemistry and a pivotal process in the construction of the skeletal framework of complex molecular targets. Among the methods available, the aldol addition reaction is a powerful strategy that enables the concomitant funcionalization and creation of stereogenic centers.

Asymmetric direct aldol additions mediated by aldolases are finding increasing ac- ceptance in chemical research and production of asymmetric compounds due to the high selectivity and cata- lytic efficacy.

Enzymatic cascade reactions as the consecutive series of biocatalytic reactions, including sequential process in combination with organic reactions, have undeniable benefits namely atom economy, time labor, resource management and waste generation. In this paper, the application of biocatalytic cascade reactions involving the use of aldolases will be discussed. Particularly, D-fructose-6-phosphate aldolase from E. coli (FSA)

was found to catalyze stereoselectively aldol additions of dihydroxyacetone, hydroxy- acetone and hydroxybutanone to a variety of aldehyde acceptors.

Recently it was uncovered that FSA has the ability to catalyze the homo- and cross-aldol additions of glycolaldehyde.

This property has expanded its synthetic potential in cascade reactions by the virtue to generate an aldehyde, which may be use as accep- tor in another consecutive aldol addition. Different redesigns of FSA wild-type may allow to control its se- lectivity towards donors and acceptors and therefore to modulate its reactivity in front of different substrates.

Figure 1. Example of in situ product utilization of FSA-catalyzed cross-aldol addition of glycolaldehyde.

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[1] B. Sauerzapfe et al., Glycoconjugate J. 2009, 26, 141.

One-pot enzymatic synthesis of

poly-n-acetyllactosamine oligosaccharides

Claudia Rech, Ruben P. Rosencrantz, and Lothar Elling

Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineer- ing, RWTH Aachen University, Aachen, Germany

E-Mail: l.elling@biotec.rwth-aachen.de

The glycan poly-N-acetyllactosamine (poly-LacNAc) is a major constituent of glycoproteins and glycolip- ids of the outer cellular glycocalix and plays an essential role in galectin-mediated cell-cell and cell-matrix recognition. Poly-LacNAc is therefore a target molecule for the biofunctionalisation of biomaterial sur- faces. It consists of a repeating unit of Gal(β1-4)GlcNAc (type 2 LacNAc). Multi-step sequential enzymatic synthesis combining human β1-4galactosyltransferase (β4GalT) and β1-3N-acetylglucosaminyltransferase (β3GlcNAc) from Helicobacter pylori was previously established.

Poly-LacNAc oligomers with up to three LacNAc units were obtained. We here demonstrate for the first time the combination of two glycosyl- transferases in a one-pot synthesis resulting in the synthesis of defined mixtures of poly-LacNAc oligomers (Figure 1). Parameters such as reaction time, enzyme ratios, length and concentration of the starting acceptor substrate were optimized. Mixtures of poly-LacNAc oligomers ranging from tri- to trideca-saccharides were obtained as analysed by MALDI-TOF. After a final galactosylation step single LacNAc oligomers ([Lac- NAc]1-6) could be isolated by preparative HPLC.

Work is in progress to analyse the binding specificity of galectins to defined mixtures poly-LacNAc oligom- ers and to explore their potential use for the biofunctionalization of biomaterial surfaces.

Figure 1. One-pot enzymatic synthesis of poly-LacNAc oligomers.

We thank Prof. Dr. V. Křen (Academy of Sciences of the Czech Republic) for the synthesis of linker-modified GlcNAc and NMR analysis, Prof.

B. Ernst (University of Basel) for the gift of UDP-Gal., Dr. W.W. Wakarchuck (National Research Council of Canada) for β3GlcNAcT. We also thank Prof. Dr. F.-G. Hanisch (Cologne University) for MALDI-TOF analysis. Financial support by the DFG within the Research Training Group 1035 “Biointerface”, by the EU-COST action CM07010, and by the excellence initiative of the German federal and state governments through ERS@RWTH Aachen University is gratefully acknowledged.

O HO

HO OH O

OH

O HO AcHN OH

O HO

OH O OH

O HO AcHN

OH

O O

HO AcHN OH O O

HO OH OH HO

O HO

OH O OH

O HO AcHN

OH

HO O

HO AcHN OH

O

N O H

HN N O

H S

N O H

HN N O

H S

N O H

HN N O

H S

1-5

1-5

β3GlcNAc-T

UDP-GlcNAc UDP-Glc 4´-Epimerase UDP-Gal

AP UDP Uridine + 2 P_i

UDP-Glc

UDP AP Uridine + 2 P_i β4Gal-T1

Monday, October 3 Lectures

[1] (a) Falus P., Boros Z., Hornyánszky G., Nagy J., Darvas F., Ürge L., Poppe L. Tetrahedron Lett., 2011, 52, 1310-1312; (b) Poppe L., Tomin A., Boros Z., Varga E., Ürge L., Darvas F. Novel dinamic kinetic resolution process, Hung. Pat. P0900720, 2009.

Continuous-flow systems for synthesis, kinetic resolution and dynamic kinetic resolution of amines

Zoltán Boros

, Péter Falus

, Gábor Hornyánszky

, József Nagy

, László Ürge

, Ferenc Darvas

, László Poppe

a

Department of Organic Chemistry and Technology, and Research Group for Alkaloid Chemistry of the Hungarian Academy of Sciences; Budapest University of Technology and Economics, Műegyetem rkp. 3., H-1111 Budapest, Hungary;

SynBiocat Ltd, Lázár deák u 4/1, H-1173 Budapest, Hungary;

ThalesNano Inc., Graphisoft Park, Záhony u. 7., H-1031 Budapest, Hungary.

E-mail: poppe@mail.bme.hu

Amines are indispensable building blocks in numerous drugs, pesticides and color pigments. Thus, devel- opment of general and efficient methods to prepare enantiopure amino compounds is still required. First, novel reductive amination of ketones with ammonium formate and various metals was developed in one- pot and one-step reactions using batch and continuous-flow methods (Fig. 1a).

Next, lipase-catalyzed kinetic resolutions in continuous-flow reactors were studied in between 0-70 °C (Fig. 1b). Depending on the substrate, maximum of enantiomer selectivity (E) at certain temperatures or even continuous increase of E with increasing temperature has been found (Fig. 1c). A chemoenzymatic cascade system was used for continuous-flow dynamic kinetic resolutions.

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[1] Bruggink, A., Schoevaart, R. and Kieboom, T. Org. Process Res. Dev. 2003, 4, 622

[2] Santacoloma, P.A., Sin, G., Gernaey, K.V. and Woodley, J.M. Org. Process Res. Dev. 2011, 15, 203

[3] Koszelewski, D., Lavandera, I., Clay, I., Guebitz, G.M., Rozzell, D. and Kroutil, W. Adv. Synth. Catal. 2008, 350 (17), 2761 [4] Truppo, M.D., Rozzell, J.D. and Turner, N.J. Org. Process Res. Dev. 2010, 14, 234

Modeling framework for multi-enzyme in-pot processes applied to chiral amine production

Paloma A. Santacoloma

, Kresimir Janesa, Pär Tufvesson

, Gürkan Sinb, Krist V. Gernaey

, John M.

Woodley

a

PROCESS; bCAPEC; Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229,DK-2800 Kgs. Lyngby

E-mail: psa@kt.dtu.dk

Nowadays multi-enzyme processes are seen as an alternative to assist in the synthesis of complex com- pounds of industrial interest.

In general, a multi-enzyme in-pot process is characterized by the mixture of enzymes that catalyze several reactions in a single pot. In this manner, purification steps of intermediate products may be eliminated. Consequently, it potentially leads to considerable process improvements like increases in the process yield, and reduction in downstream processing and operating costs. In the analysis of these types of processes, mathematical models and computational tools enable a systematic development of multi-enzyme in-pot processes.

A systematic methodology is exemplified stepwise through the production of chiral amines by combining the action of three enzymes in a single reactor.

In the applied method, chiral amines are synthesized from ketones by using the first enzyme (transaminase, E.C. 2.6.1.2) together with L-alanine which provides the amino donor (see figure 1). The generated pyruvate, from the first reaction, is reduced by the second enzyme (lactate dehydrogenase, LDH, E.C. 1.1.1.27) to L-lactate. Removing the pyruvate contributes to both driv- ing the reaction to completion and eliminating pyruvate inhibition of the transaminase. The third enzyme (glucose dehydrogenase, GDH, E.C. 1.1.1.47) is added to the system in order to recycle the NADH cofactor.

[4]

The aim of this contribution is to present a methodological framework for modeling multi-enzyme in-pot processes in order to formulate reliable models for further applications such as optimization, control and prediction.

Figure 1. Reaction scheme for the synthesis of chiral amines by applying a three-enzymatic in-pot process (transaminase, LDH and GDH)

Monday, October 4 Lectures

Microbial and enzymatic processes for the production of useful chemicals: screening and development of unique microbial

enzymes and their industrial applications

Sakayu Shimizu

New Frontiers Research Laboratories, Toray Industries, Inc., Kamakura, Kanagawa 248-8555; Department of Bioscience and Biotechnology, Kyotogakuen University, Kameoka, Kyoto, 621-8555, Japan.

E-mail: Sakayu_Shimizu@nts.toray.co.jp; sshimizu@kyotogakuen.ac.jp

Over the past decade, the industrial use of microbial functions, such as their unique enzyme systems, catalysis and so on, has developed rapidly and is gathering increasing attention, particularly their use in solving environmental problems. Here, several unique microbial enzymes or reactions recently discovered by us and now used industrially are introduced, through which I will emphasize importance of screening for potential microorganisms and mutual collaboration between academia and industries.

Screening is a key step in process development, because, in many cases, the substrates in industrial processes are artificial compounds, and, in many cases, enzymes to catalyze suitable reactions for such processes are still unknown. Therefore, screening for novel enzymes that are capable of catalyzing new reactions is constantly needed. In addition, the discovery of new enzymes sometimes provides clues for designing new enzymatic processes. One of the most efficient and successful means of finding new enzymes is to screen a large number of microorganisms, because of their characteristic diversity and versatility. However, it is obviously very difficult to propose rational method of screening for novel enzymes; it sometimes involves something like midnight-walk without moonlight. There are three important stages in a general strategy:

(1) designing the process and deciding the type of enzymatic activity desired; (2) deciding which groups of microorganisms are to be selected and screened; and (3) designing an appropriate, convenient and sensitive assay that will allow as many microorganisms as possible to be screened. It is also important, during the course of screening, to observe the functions of microorganisms carefully in order to obtain the desired (but serendipitous or random) results.

Here, several unique microbial enzymes or reactions recently discovered by us and now used industrially are introduced (single cell oil production by oleaginous microorganisms, reductive transformation of unsaturated fatty acids, lactonase process for the optical resolution of lactones, bioreduction system for chiral alcohol synthesis, hydroxylation of amino acids, novel membrane reactor system for the continuous fermentaion, etc.), through which I will emphasize importance of the followings: i) screening for potential microorganisms from our rich resources, ii) thoughtful use of new technologyies, iii) mutual collaborations between academia and industries, and iv) rational and strategic support by the government.

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32 Lectures Tuesday, October 4 33

[1] Bishnu Prasad Pandey, et. al, Enzyme and Microbial Technology, in press, 2011 [2] Bishnu Prasad Pandey, et. al , Biotechnology & Bioengineering, 105(4):697 - 704, 2010

Modification of natural products using oxygenases

Byung-Gee Kim

, Nahum Lee, Kwon-Young Choi, Bishnu Pandey, EunOk-Jung

1 School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-744, Korea 2 Bioengineering Institute and Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-744, Korea

Phone: +82-2-880-8945 Fax: +82-2-872-7528 E-mail: E-mail: byungkim@snu.ac.kr

Natural products are mainly synthesized through secondary metabolite pathways. After the synthesis, they are further modified using tailoring enzymes to generate their diversities. Among them, the most interesting and prevalent enzyme reaction systems are the ones for hydroxylation, methylation, and glycosylation. Here, we will talk on several examples of monooxygenase reactions for such products which can enhance and/or change their biological functions: various hydroxylations of i) (iso)flavonoids(daidzein, genistein, apigenin, etc), ii) lignan and iii) stilbenoids using P450s and tyrosinase from Actinomycetes(Streptomyces, Norcadia etc.), and how we can develop self-sufficient P450s of Streptomyces in E.coli system as a successful substi- tute for the P450 reactions.

Figure 1. Daidzein Hydroxylation

[1] T. Fischer, J. Pietruszka, Top. Cur. Chem. 2010, 297, 1-43.

[2] (a) M. Bischop, V. Doum, A. C. M. Nordschild (née Rieche), J. Pietruszka, D. Sandkuhl, Synthesis 2010, 527-537; (b) J.

Pietruszka, R. C. Simon, Eur. J. Org. Chem. 2009, 3628-3634; (c) M. Korpak, J. Pietruszka, Adv. Synth. Catal. 2011, in press.

[3] (a) R. H. Cichewicz, F. A. Valeriote, P. Crews, Org. Lett. 2004, 6, 1951–1954; (b) G. R. Pettit, J. Xu, J. Chapius, R. K. Pettit, L. P. Tackett, D. L. Doubek, J. N. A. Hooper, J. M. Schmidt, J. Med. Chem. 2004, 47, 1149–1152.

Key building blocks for natural product synthesis via enzyme-mediated transformations

Jörg Pietruszka

, Martina Bischop

, Thomas Classen

, Nils Eichenauer

, Thomas Fischer

, Margarete Korpak

, Irene Kullartz

, Katharina Neufeld

, Anja C. M. Nordschild

, Melanie Schölzel

, and Diana Sandkuhl

a

Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, Jülich, Germany E-mail: j.pietruszka@fz-juelich.de

Biocatalytic approaches towards new building blocks in organic synthesis have increasingly emerged as an important tool in recent years. Nowadays, a number of biotransformations are primarily applied in the chemical and pharmaceutical industries delivering fine chemicals, e.g. for drugs. The mild reaction condi- tions - also triggering high stereo-, regio-, and chemoselectivity - and the often elegant short-cuts in syn- thetic endeavours lead to economic and ecological advantages of the enzymatic conversions.

The focus of our projects is on natural product syntheses and the development of new synthetic methods (see e.g. ref.

). The synthesis of the highly selective antitumor reagent psymberin (Figure 1), isolated from the sponge Psammocinia spp.

, is one of the more recent target molecules in our group, but also marine oxylipins such as the constanolactones caught our attention. The progress on a chemoenzymatic approach towards key building blocks for organic synthesis will be presented. Enzymes utilized range from hydrolases and (ene)reductases to monooxygenases.

Figure 1. Selected natural target compounds.

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[1] Hoshino, T.; Ohashi, S. Org. Lett. 2002, 4, 2553-2556.

[2] Wendt, K. U.; Lenhart, A.; Schulz, G. E. J Mol Biol 1999, 286, (1), 175-87.

[3] Bohlmann, J.; Meyer-Gauen, G.; Croteau, R. Proc Natl Acad Sci U S A 1998, 95, (8), 4126-33.

[4] Davis, E. M.; Croteau, R. Top. Curr. Chem. 2000, 209, 53-95.

Biocatalytic cyclization of citronellal

Gabriele Siedenburg

, Michael Breuer

, Bernhard Hauer

, Dieter Jendrossek

a

Inst. f. Microbiology, University Stuttgart,

BASF-SE, Ludwigshafen,

Inst. f. Technical Biochemistry, Uni- versity Stuttgart, Germany;

E-mail: imbgsi@imb.uni-stuttgart.de; imbdj@imb.uni-stuttgart.de

Key enzyme of hopanoid biosynthesis is squalene-hopene cyclase (SHC) which catalyzes the polycycliza- tion reaction of squalene to the pentacyclic triterpene hopene - the precursor of all hopanoids. Hopanoids stabilize the cytoplasm membrane of many bacteria similar to the function of sterols in eukarotes. The SHC- catalyzed reaction is one of the most complex biochemical reactions and involves the formation of 5 ring structures, the alteration of 13 covalent bonds, and the formation of 9 stereo centers. Zymomonas mobilis – an important ethanol producing bacterium - harbours two SHC-encoding genes (ZMO872, ZMO1548) that were cloned and over-expressed in E. coli. Hopene-forming activity was confirmed for both SHCs. In- terestingly, ZMO1548 was able to cyclise monoterpene citronellal to isopulegol (see figure). This finding is contrary to former results using the model SHC from Alicyclobacillus acidocaldarius

and several other SHCs cloned from different organisms in this study. Isopulegol is used as a flavor in different products and is an important inter-mediate in the production of menthol. Our finding is remarkable because cyclization of mono- sesqui- and diterpenes normally requires activation of the linear precursor by diphosphate.

De- pending on the stereo-configuration of the substrate [(S)- or (R)-citronellal] different isopulegol stereoiso- mers were formed. Cyclization of citronellal by SHC is the first example of an enzyme-catalyzed cyclization of a not-activated linear monoterpene.

[1] M. A. Gregory et al., Angew. Chem. Int. Ed. 2005, 44, 4757.

[2] N. L. Paiva, M. F. Roberts, A. L. Demain, J. Ind. Microbiol. 1993, 12, 423.

[3] J. Andexer et al., Proc. Natl. Acacd. Sci. USA 2011, 108, 4776.

[4] F. Rusnak et al., Biochemistry 1990, 29, 1425.

[5] Y. Hayashi et al., J. Antibiot. 2008, 61, 164.

Chorismatases in natural product biosynthesis

Jennifer N. Andexer

, Steven G. Kendrew

, Orestis Lazos

, Mohammed Nur-e-Alam

, Anna-Sophie Zim- mermann

, Steven Moss

, Barrie Wilkinson

, Peter F. Leadlay

a

Pharmaceutical and Medicinal Chemistry, University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany;

b

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, Unit- ed Kingdom;

Biotica Technology Ltd., Chesterford Research Park, Cambridge CB10 1XL, United Kingdom E-mail: jennifer.andexer@pharmazie.uni-freiburg.de

Ascomycin (FK520, 1) is a macrocyclic polyketide produced by Streptomyces hygroscopicus var. ascomy- ceticus with potent antifungal and immunosuppressive properties.

Figure 1. Structures of ascomycin (1)/ brasilicardin (2) and chorismatase-catalysed reactions.

As for the closely related rapamycin and FK506, the unusual 3,4- dihydroxycyclohexane carboxylic acid starter unit has been shown to be derived from shikimate metabolism.

However, the exact nature of the genes and enzymes involved in this feeder pathway has never been established. Here, we have examined the hypothesis that the FkbO gene product catalyses a key enzymatic step linking the shikimic pathway to the starter unit.

FkbO was expressed in Escherichia coli, purified, and tested for its ability to catalyse the direct hydrolysis of chorismate (4) (Figure 1). It was found to be an efficient catalyst for this reaction.

The only precedent for such a chorismatase activity is in the side reaction of known isochorismatases such as EntC from E. coli.

A homologous enzyme (Hyg5) is found in a less characterised cluster in the rapamycin-producing strain.

This enzyme was shown to carry out the hydrolysis of chorismate as well, but leads to 3-hydroxybenzoic acid (5) instead of 3.[3] Bra8, an enzyme from the brasilicardin cluster

exhibits very high similarity to Hyg5 and is suggested to be responsible for the biosynthesis of the 3-hydroxybenzoate moiety in brasilicar- din (4).

Our results show that FkbO and Hyg5 are genuine chorismatase and reveal another metabolic fate for choris- mate apart from the classic pathways leading to aromatic amino acids, isochorismate or ubiquinones.

Tuesday, October 4 Lectures

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