IN VIVO CASCADES

Catalytic cascades for selective aldehyde syntheses in vivo

The main goal of this project is to understand the requirements and limitations of microbial cells for aldehyde synthesis. The synthesis of target products– chiral fragrance aldehydes – will be tackled by a combination of chemo-catalysis with a multi-enzymatic in vivo cascade reaction.

THE PROJECT

Many fragrances that we know from daily life share the same chemical functional group – the aldehyde. The most prominent example is probably vanillin, which is used in a huge number of food products, cosmetics and perfumes but there are also others like cinnamaldehyde (which gives the smell to cinnamon) and derivatives thereof. As many of these fragrances are difficult to obtain in sufficient amounts from natural sources, there is a lot of interest to find ways how to prepare them. Carboxylic acids could be promising precursors, but it is very difficult to reduce them selectively to aldehydes. Chemistry has some possibilities to do that, but they all suffer from drawbacks that enzymatic methods can circumvent. Enzymes are non-toxic and typically operate at ecologically benign conditions. So called carboxylate reductase enzymes can selectively make an aldehyde from an acid at the expense of cofactors.

These cofactors, however, are too expensive to be added to a reaction and therefore, we will use microorganisms, which produce these cofactors from sugar and air. The microorganism E. coli will be engineered such that they are able to catalyze a cascade reaction to fragrances like for example TropionalTM, which has an ‘ocean – like’ smell, from precursors that were synthesized with catalytic chemical methods. Ultimately, the aim is to integrate the chemical step into the biotransformation step and run the reaction in a chemoenzymatic one pot fashion.

One of the main outcomes from this project will be a better understanding of the complex interplay between the foreign enzymes and chemicals with the microorganism’s metabolism. Only with this knowledge it will be possible to find bottlenecks and to develop ideas how to alleviate them. This will not only be for the benefit of fragrance synthesis but also generally for other cascade reactions with aldehyde intermediates.

This project received funding from the Austrian Science Fund FWF (P 28477-B21). 

Our team

Dr. Margit Winkler
Project Coordination

Dr. Florian Rudroff
Project Coordination

Anna Ressmann
PostDoc

Daniel Schwendenwein
PhD Student

Holly Stolterfoht
Master Student

David Gradischnig
Student

Kinga Rudnicka
Erasmus trainee

Weronika Chojnacka
Erasmus trainee

Magdalena Górecka
Erasmus trainee

PUBLICATIONS

Publication Tabs

 Margit Winkler (2017) Invited Keynote Lecture Discovery of new enzymes by homology and beyond International Congress of the Malaysian Society for Microbiology 2017 (ICMSM2017), Dec.6, Bandar Baru Bangi, Malaysia
 Daniel Schwendenwein (2017) Heliotropin preparation by a carboxylate reductase in resting cells School of Molecular and Cell Biology, University of the Witwatersrand, Nov.23, Johannesburg, South Africa
 Margit Winkler (2017) Enzymatic Functional Group Transformations for Industrial Biotechnology CATSA 2017, Nov.21, Sun City, South Africa
Florian Rudroff (2017) Science Flash Presentation Catalytic Cascades – ‘En Route’ to Applied Biochemical Cell Factories, European Summit of Industrial Biotechnology, Nov.14-16, Graz, Austria
 Daniel Schwendenwein (2017) Heliotropin preparation by a carboxylate reductase in resting cells 15th Weurman Flavour Research Symposium, Sep.18-22, Graz, Austria
Margit Winkler (2017) Speed Talk Carboxylic acid reductase (CAR) mediated Biotransformations Biotrans 2017, Jul.9-13, Budapest, Hungary
 Margit Winkler (2016) Science Flash Presentation Selective preparation of Aldehydes using carboxylate reductases, European Summit of Industrial Biotechnology, Nov.14-16, Graz, Austria.
 Margit Winkler (2016) Aldehyde preparation with new fungal carboxylate reductases 25. Nachwuchswissenschaftlersymposiums Bioorganische Chemie, Sep 21-23, Jena, Germany
 Margit Winkler (2016) New enzymes for the selective enzymatic reduction of carboxylic acids to aldehydes ICC05-AEM2016, Sep 4-8, Toyama, Japan
 Florian Rudroff (2016) Biocat2016, Aug 28-Sep 1, Hamburg, Germany
 Margit Winkler (2016) Carboxylate reductases from fungal Origin for the preparation of aldehyde fragrances, 17th European Congress on Biotechnology, Jul 3-6, Krakow, Poland
 Florian Rudroff (2016) Oxyzymes, Jul 3-6, Wageningen, The Netherlands
 Margit Winkler (2016) New Enzymes for Biocatalytic Syntheses, Symposium Molecular Biotechnology – from the Origins to New Horizons, April 14-15, Graz, Austria
 Margit Winkler (2016) Five Examples of Enzymatic Functional Group Transformations, Mikrobiologisches Seminar, Universität Stuttgart, Feb 3, Stuttgart, Germany.
 Margit Winkler (2016) Formation and transformation of carboxylic acid derivatives, Seminar of the Institute of Applied Synthetic Chemistry, Vienna University of Technology, Jan 14, Vienna, Austria.
 Margit Winkler (2016) Enzymatic Carboxylate Reduction, Institute Seminar of the Institute for Molecular Biotechnology, Graz University of Technology, Jan 13, Graz, Austria
 Margit Winkler Carboxylic acid Reductse Enzymes (CARs) Current Opinion in Chemical Biology, 2018, 43, 23-29. DOI: 10.1016/j.cbpa.2017.10.006
 Thomas Bayer, Thomas Wiesinger, Sofia Milker, Margit Winkler, Marko D. Mihovilovic, Florian Rudroff In vivo synthesis of polyhydroxylated compounds from a 'hidden reservoir' of toxic aldehyde species, ChemCatChem, 2017, 9, 2919-2923. DOI: 10.1002/cctc.201700469
Holly Stolterfoht, Daniel Schwendenwein, Christoph W. Sensen, Florian Rudroff, Margit Winkler Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes, J Biotechnol, 2017, 257, 222-232. DOI:10.1016/j.jbiotec.2017.02.014
Daniel Schwendenwein, Giuseppe Fiume, Hansjörg Weber, Florian Rudroff, Margit Winkler Selective enzymatic transformation to aldehydes in vivo by fungal carboxylate reductase from Neurospora crassa, Adv. Syn. Catal., 2016, 358, 3414-3421. DOI: 10.1002/adsc.201600914
Daniel Schwendenwein, Florian Rudroff, Margit Winkler (2017) Selective preparation of Aldehydes using carboxylate reductases, European Summit of Industrial Biotechnology, Nov.14-16, Graz, Austria
Florian Rudroff Anna K. Ressmann, Daniel Schwendenwein, Simon Leonhartsberger, Mark Dörr, Marko D. Mihovilovic, Uwe T. Bornscheuer, Margit Winkler, Michael J. Fink, Sofia Milker, Thomas Bayer, Thomas Wiesinger, Nikolin Oberleitner, (2017) Catalytic Cascades – ‘En Route’ to Applied Biochemical Cell Factories, European Summit of Industrial Biotechnology, Nov.14-16, Graz, Austria
Anna K. Ressmann, Simon Leonhartsberger, Holly Stolterfoht, Daniel Schwendenwein, Marko D. Mihovilovic, Margit Winkler, Florian Rudroff (2017) Whole Cell Substrate Screening of Carboxylic Acid Reductases, Biotrans 2017, Jul.9-13, Budapest, Hungary
Daniel Schwendenwein, Giuseppe Fiume, Harald Pichler, Margit Winkler (2017) Carboxylic Acid Reductase (CAR) mediated Biotransformations, Biotrans 2017, Jul.9-13, Budapest, Hungary
Daniel Schwendenwein, Holly Stolterfoht, Florian Rudroff, Margit Winkler (2017) Modulation of NcCAR and PPTase Expression levels for Aldehyde Preparation with whole cells, Biotrans 2017, Jul.9-13, Budapest, Hungary
Holly Stolterfoht, Daniel Schwendenwein, Florian Rudroff, Kinga Rudnicka, Margit Winkler (2017) Identification of Key-Residues for Enzymatic Carboxylate Reduction, Biotrans 2017, Jul.9-13, Budapest, Hungary
Thomas Bayer, Sofia Milker, Thomas Wiesinger, Margit Winkler, Marko D. Mihovilovic, Florian Rudroff (2017) Substrate funnelling complements flux optimization tools to maximize product titers of in vivo cascades, Biotrans 2017, Jul.9-13, Budapest, Hungary
Daniel Schwendenwein, Phillipe Oberhemm, Giuseppe Fiume, Florian Rudroff, Kerstin Steiner, Kinga Rudnicka, Margit Winkler (2016) Selective preparation of Aldehydes using carboxylate reductases, European Summit of Industrial Biotechnology, Nov.14-16, Graz, Austria
Giuseppe Fiume, Philippe Oberhemm, Daniel Schwendenwein, Margit Winkler (2016) A new fungal carboxylic acid reductase for the preparation of aldehydes from acids, 8th ÖGMBT Annual Meeting, Sep 12-14, Graz, Austria
Daniel Schwendenwein, Florian Rudroff, Margit Winkler (2016) Tuning the expression of Neurospora crassa carboxylic acid reductase towards an efficient whole cell catalyst for aldehyde preparation, 8th ÖGMBT Annual Meeting, Sep 12-14, Graz, Austria.
Christoph K. Winkler, Markus Plank, Margit Winkler, Kurt Faber (2016) Chemo-, regio- and stereoselectivity of carboxylic acid reductases, BIOCAT 2016– 8th International Congress on Biocatalysis, Aug 28-Sep 1, Hamburg, Germany
Daniel Schwendenwein, Florian Rudroff, Kerstin Steiner, Philippe Oberhemm, Giuseppe Fiume, Margit Winkler (2016) Aldehyde preparation with new fungal carboxylate reductases, Gordon Research Conference Biocatalysis, Jul 10-15, Biddeford, ME, USA
Thomas Bayer, Thomas Wiesinger, Sofia Milker, Margit Winkler, Florian Rudroff, Marko D. Mihovilovic (2016) Convergent Carbon Flux via Substrate Funelling for the In Vivo Production of Reactive Aldehyde Species, Gordon Research Conference Biocatalysis, Jul 10-15, Biddeford, ME, USA This Poster was outstanding and selected for a Talk
 Margit Winkler Blog article: How to prepare flavors and fragrances by enzymes https://www.acib.at/how-flavors-and-fragrances-are-produced-by-enzymes/, May 18th 2017 
 Martin Walpot Press release: Von der Säure zum Aldehyd, May 12th 2017 

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This project is carried out in collaboration with