Research Lab - Florian Rudroff
General research vision:
The rich repository of biologically active and intricate molecules found in nature has been a wellspring of inspiration for synthetic chemists over the centuries. Their persistent endeavor has been to replicate natural chemical processes and fabricate biomaterials through sophisticated methodologies such as biomimetic synthesis, asymmetric catalysis, and natural product synthesis. Biocatalysis, serving as the biological counterpart to organo- and metal catalysis, stands as an integral component within the chemists’ arsenal, facilitating the synthesis of both refined and large-scale chemical compounds. Our focal point lies in the adept exploration, amplification, and refinement of this extensive chemists’ toolbox, fostering its application in sustainable synthetic practices. In doing so, we aim to contribute to the advancement of environmentally conscious and resource-efficient synthetic methodologies.
Current research:
All of these research topics are addressed in my research group. We focus on the following content: The development of assays tailored to specific functional groups (e.g., R-OH, RCHO, RCOOH) is underway, coupled with the integration of a microfluidic device to enable ultra-high throughput screening of both novel and enhanced biocatalysts. These assays are meticulously designed for implementation in a fluorescence-activated droplet sorting (FADS) system.-
- Our research is fundamentally focused on unraveling and engineering novel enzyme classes, delving into the intricacies of their mechanisms. This encompasses the study of, for example, enoate reductases, BVMOs, alkene-cleaving enzymes known as ‘ozonylases,’ amin dehydrogenases, and hydrogenases.
- Drawing inspiration from nature, we have expanded upon the concept, rooted in the retro(bio)synthesis approach, of single-step (bio)transformations. This expansion involves transitioning towards cascade-type reactions, achieved by seamlessly combining either chemo/bio- or bio/biocatalysts, both in vitro and in vivo. These efforts aim to facilitate the production of fragrances, bioactive compounds, and chiral building blocks.
- Investigating the biodegradation of polyolefin-based plastics through a biocatalytic Fenton-type reaction in the presence of light and oxygen is a focal point of our research.
- We are actively engaged in exploring the capture and valorization of CO2 by leveraging the capabilities of phototrophic bacteria, marking a significant stride towards sustainable and environmentally conscious practices.
- We are actively engaged in exploring the capture and valorization of CO2 by leveraging the capabilities of phototrophic bacteria, marking a significant stride towards sustainable and environmentally conscious practices.
Chemo-Enzymatic Cascade for the Generation of Fragrance Aldehydes
Publication Title: Chemo-Enzymatic Cascade for the Generation of Fragrance Aldehydes by Daniel Schwendenwein, Anna K. Ressmann, Marcello Entner, Viktor Savic, Margit Winkler,* and Florian Rudroff*
Enzyme Cascade Design and Modelling
In our book “Enzyme Cascade Design and Modelling” we have joined our forces and brought a team of experts in multi-enzymatic and chemo-enzymatic cascades! Editors: Kara,
Cell‐free in vitro reduction of carboxylates to aldehydes: With crude enzyme preparations to a key pharmaceutical building block
The scarcity of practical methods for aldehyde synthesis in chemistry necessitates the development of mild, selective procedures. Carboxylic acid reductases catalyze aldehyde formation from stable
Biocatalysis in Green and Blue: Cyanobacteria
Recently, several studies have proven the potential of cyanobacteria as whole-cell biocatalysts for biotransformation. Compared to heterotrophic hosts, cyanobacteria show unique advantages thanks to their
Pyrazines: Synthesis and Industrial Application of these Valuable Flavor and Fragrance Compounds
Alkyl pyrazines—other than being extracted from various natural sources such as coffee beans, cocoa beans, and vegetables—can be synthesized by chemical methods or by certain
Boosting photobioredox catalysis by morpholine electron donors under aerobic conditions
Light-driven reduction of flavins, e.g. FAD or FMN, by sacrificial electron donors emerged as a convenient method to promote biocatalytic transformations. However, flavin activation has been restricted
Florian Rudroff, born in Vienna, studied Technical Chemistry, with specialization in Organic Chemistry at the TU Wien. He conducted his diploma and PhD thesis in the group of Prof. Marko Mihovilovic and obtained his PhD in 2007 from TU Wien. The topic of the PhD thesis was located at the interface between Biology and Chemistry in the field of Biocatalysis. Afterwards he was awarded with an ’Erwin Schrödinger fellowship’ and went for a postdoctoral stay to the group of Prof. Uwe Sauer at ETH Zurich. His research was mainly conducted to Molecular Systems Biology (metabolomics and fluxomics) and specifically to the rapid metabolic and TOR signalling responses in Saccharomyces cerevisiae upon different nitrogen input signals.
In 2011, he returned to the TU Wien and started his independent scientific career in the field of Systems Biocatalysis. Since December 2017, he became Assistant Professor and finished his Habilitation in the field of Bioorganic Chemistry at the Institute of Applied Synthetic Chemistry, TU Wien. In 2020, he was appointed as Assoc. Professor and group leader of the Bioorganic Synthetic Chemistry Group (BSC) at the TU Wien. Since 2018 he is leader of the local branch (Vienna, lower Austria, Burgenland) of the Austrian Chemical Society (GÖCH).
His main research interests are located in the field of enzyme cascade catalysis, photoredox biocatalysis (e.g, CO2 utilization by cyanobacteria), protein engineering, the development of high throughput platforms (e.g. fluorescence adsorption droplet sorting, FADS) and organic synthesis.
González Rodríguez, Jorge (Post Doc)
Jorge González Rodríguez studied his PhD under the supervision of Prof. Humberto Rodríguez Solla and Prof. Raquel María González Soengas at the University of Oviedo (Spain). His research during this period was focused on organometallic reactivity in combination with different biocatalytic approaches to access valuable building blocks for further synthetic and/or industrial uses. Currently, he is working on the development of new methodologies for the sustainable and efficient synthesis of pyrazines.
Keywords:
- sustainable synthesis
- pyrazines
Fried, Richard (PhD Student)
Keywords:
- metabolic pathways
- secondary metabolites
- purification strategies
- structure elucidation
Giparakis, Stefan (PhD Student)
Keywords:
- wacker-oxidation
- biotransformation
- green chemistry
Mortzfeld, Frederik (PhD Student)
ORCID: 0000-0003-2857-9504
Keywords:
- flow chemistry methodologies
- high energetic / ultrafast reactions
- biocatalysis
- directed evolution (FADS)
- API & fragrance synthesis
- process development
Jodlbauer, Julia (PhD Student)
Keywords:
- cyanobacteria
- redox biocatalysis
- RBS library
- light-driven whole-cell biotransformations
- synthetic biology
Schiefer, Astrid (PhD Student)
In the context of my Master’s thesis I took a closer look at ABAO (2-amino-benzamidoxime) assays for the detection of aldehydes with the subsequent aim of applying it for screening mutant libraries of aldehyde producing enzymes with FADS. I did my bachelor thesis “BIOCAR-Bioorthogonal capture and release” at the Institute of Applied Synthetic Chemistry in the research group Molecular Chemistry & Chemical Biology at TU Wien. After finishing my Master’s thesis in the Rudroff group, I am excited to continue on exploring the challenges towards enzyme assisted methods to cleave C=C double bonds.
Keywords:
- 2-amino-benzamidoxime
- FADS
- carboxylic acid reductase enzymes
- alkene cleavage
Rohr, Thomas (PhD Student)
I started my PhD-studies at this group in November 2019. My project focuses on establishing Cyanobacteria as host-platform for Whole-cell-catalysis (Biocatalysis). After finishing my Bachelor of Science in Applied Biology from the University of Applied Sciences Bonn-Rhein-Sieg, I started a Master in Biotechnology and Process-engineering before switching to the Heinrich-Heine-University Düsseldorf. There, I completed my Master of Science in Biology with focus on Synthetic Biology and Systems-Biotechnology with my Master-Thesis at the Instute of Synthetic Microbiology of Prof. Dr. Ilka Axmann. In parallel to my studies I worked for different Biotech-Start-ups, such as the NUMAFERM GmbH, Düsseldorf.
Keywords:
- cyanobacteria
- synthetic biology
- whole-cell-catalysis
- biocatalysis
Wutscher, Maximilian (PhD Student)
During my Master´s thesis, which I completed at TU Wien in 2023, my research was focused on an enzymatic cascade towards fragrance aldehydes.
The objective of my PhD is to develop a versatile biocatalytic approach employing redox-active enzymes for O-dealkylation. The enzymes applied facilitate the transformation under mild conditions, utilizing molecular oxygen as the oxidant. The final goal is to establish a comprehensive toolbox for organic chemists, enabling the selective and regio-specific removal of O-alkyl side chains on various substrates.
ORCID: 0009-0006-5200-2018
Keywords:
- biocatalysis
- protein engineering
- dealkylation
Suchy, Lydia (PhD Student)
I started my academic education at the University of Vienna in Chemistry. During my master studies in Chemistry I developed a growing interest in Biology. So, after completing my master thesis (conducted at the research institution CINVESTAV, in Mexico City) I started a second master program in Biology at the University of Vienna. I found an opportunity to combine my chemical and biological interests in the BSC group at the TU Wien, where I am completing my master thesis in Biology and will continue with my PhD. In my project I am trying to combine different enzymes for the realization of in vivo enzymatic cascade reactions in Escherichia Coli.
Keywords:
- whole-cell biocatalysis
- in vivo enzymatic cascade reactions
Scheibelreiter, Verena (PhD Student)
I completed my Master’s degree at TU Wien in 2023. My master thesis explored a catalytic method for starch oxidation.
My PhD research revolves around developing enzymatic deprotection strategies for alkyl ethers of different substrates including carbohydrates, utilizing heme-based proteins. Our goal is to expand the chemical space for this type of transformation, making existing synthesis pathways more sustainable and enabling new synthetic strategies.
ORCID: 0000-0002-8236-9658
Keywords:
- enzymatic oxidation
- sugar chemistry
- green chemistry
- dealkylases
Waltl, Christian (Master Student)
Christian did his bachelor`s thesis in the lab of Prof. Verena Jantsch at the Max Perutz Labs, where he concerned himself with rmh-1 which is a scaffolding protein in the RTR-complex of C. elegans, which is important in meiosis. He was part of a pilot project, were he tried to generate a new non-null allele of rmh-1 to gain further insight into the function of rmh-1. He obtained his bachelor`s degree in biology, with an emphasis on microbiology and genetics, in October 2020.
He started his master’s thesis in March 2022 under the supervision of Julia Jodlbauer and Prof. Florian Rudroff. The goal of his thesis is to establish a cascade in Synechocystis sp. PCC6803, converting carveol, which can be produced from limonene, which can be sourced from waste product orange peels, to carvolactone, which is a promising molecule for the generation of thermoplastic polymers. Synechocystis sp. PCC6803 is a cyanobacterium which, due to its photoautotrophic lifestyle, could present a greener alternative to the heterotrophic E. coli which is currently a widespread platform for the generation of desired compounds.
Keywords:
- biocatalysis
- green chemistry
- cyanobacteria
Pyrazine synthesis – A greener way for our daily flavor and smell
Project description:
- Biocatalytic retrosynthesis for the production of heterocycles
- Biocatalytic synthesis of flavour and fragrance compounds via different enzymatic cascades
- Comparison of chemical & enzymatic routes
- FADS-based (fluorscence activated droplet sorting) enzyme-assay development
- Biomimicking of chemical flavour production starting from sugar beet molasses
Bioinspired alkene cleavage – BioZone
The aim of this project is to develop a biological (enzyme-assisted) method for splitting carbon / carbon double bonds without the use of the dangerous ‘ozone’. The so-called ozonolysis is a widely used method for the production of so-called “carbonyl compounds”. Such carbon / carbon double bond containing compounds can be starting materials to make such as flavor compounds like vanillin or fragrances like Lilial. Ozonolyis is mainly used on a laboratory scale. The reason for this is the toxicity of ozone and the danger of the intermediate products, which are not safe: Some of them have already caused industrial reactors to explode. We want to find and investigate a safe alternative with enzymes that can split C = C double bonds at room temperature, in water and by means of oxygen. We use a high-throughput detection method that is specifically designed for the detection of aldehydes (carbonyl compounds) in order to examine different protein sequences for their ability to split C = C double bonds. This detection method is highly sensitive, completely independent of structure, easy to use and extremely specific for aldehydes. With this detection method in combination with a special technique, it is possible to examine up to a million protein sequences in a day. It is thus possible to examine known enzymes for their substrate diversity and new enzymes for their activity in a very short time, as quantitatively as possible. After suitable enzymes have been found, we will use them for the production of potential new active pharmaceutical ingredients or flavorings.
Cyanobacteria – Photo-Biocatalysis goes green and blue
Recently, several studies have proven the potential of cyanobacteria as whole-cell biocatalysts for biotransformations. Compared to heterotrophic hosts, cyanobacteria show unique advantages thanks to their photoautotrophic metabolism. Their ability to use light as energy and CO2 as carbon source promises a truly sustainable production platform. Moreover, their photoautotrophic metabolism offers an encouraging source of reducing power, which makes them attractive for redox-based biotechnological purposes. To exploit the full potential of these whole-cell biocatalysts, cyanobacterial cells must be considered in their entirety.
Project description:
- Expression of redox enzymes in cyanobacteria
- Comparison of different cyanobacteria
- Biocatalysis – single step and in a cascade fashion
- Systems biology of cyanobacteria
Enzyme cascades & Chemo/Biocatalysis
Project description:
- Development of novel strategies for the synthesis of high-value compounds by retrosynthesis
- Combination of chemo and biocatalysis
- Multi-step catalysis in vivo & in vitro
- Flux optimization – on transcriptional and translational level
- Protein engineering
Enzymatic reactions towards aldehydes: An overview
Lukas Schober, Hana Dobiašová, Valentina Jurkaš, Fabio Parmeggiani, Florian Rudroff, Margit Winkler*
Publications in Scientific Journals
Enlightening the Path to Protein Engineering: Chemoselective Turn-On Probes for High-Throughput Screening of Enzymatic Activity
Sebastian Hecko, Astrid Schiefer, Christoffel P. S. Badenhorst, Michael J. Fink, Marko D. Mihovilovic, Uwe T. Bornscheuer, and Florian Rudroff*
Chem. Rev. 2023, XXXX, XXX, XXX-XXX
Many successful stories in enzyme engineering are based on the creation of randomized diversity in large mutant libraries, containing millions to billions of enzyme variants. Methods that enabled their evaluation with high throughput are dominated by spectroscopic techniques due to their high speed and sensitivity. A large proportion of studies relies on fluorogenic substrates that mimic the chemical properties of the target or coupled enzymatic assays with an optical read-out that assesses the desired catalytic efficiency indirectly. The most reliable hits, however, are achieved by screening for conversions of the starting material to the desired product. For this purpose, functional group assays offer a general approach to achieve a fast, optical read-out. They use the chemoselectivity, differences in electronic and steric properties of various functional groups, to reduce the number of false-positive results and the analytical noise stemming from enzymatic background activities. This review summarizes the developments and use of functional group probes for chemoselective derivatizations, with a clear focus on screening for enzymatic activity in protein engineering.
Organic Acid to Nitrile: A Chemoenzymatic Three-Step Route
Margit Winkler*, Melissa Horvat, Astrid Schiefer, Victoria Weilch, Florian Rudroff, Miroslav Pátek, Ludmila Martínková*
Various widely applied compounds contain cyano-groups, and this functional group serves as a chemical handle for a whole range of different reactions. We report a cyanide free chemoenzymatic cascade for nitrile synthesis. The reaction pathway starts with a reduction of carboxylic acid to aldehyde by carboxylate reductase enzymes (CARs) applied as living cell biocatalysts. The second – chemical – step includes in situ oxime formation with hydroxylamine. The final direct step from oxime to nitrile is catalyzed by aldoxime dehydratases (Oxds). With compatible combinations of a CAR and an Oxd, applied in one-pot two-step reactions, several aliphatic and aryl-aliphatic target nitriles were obtained in more than 80% conversion. Phenylacetonitrile, for example, was prepared in 78% isolated yield. This chemoenzymatic route does not require cyanide salts, toxic metals, or undesired oxidants in contrast to entirely chemical procedures.
Publications in Scientific Journals
Biogenic colourants in the textile industry – a promising and sustainable alternative to synthetic dyes
Richard Fried, Ilinca Oprea, Karin Fleck and Florian Rudroff*
Green Chemistry 2021
Chemo-Enzymatic Cascade for the Generation of Fragrance Aldehydes
Catalysts 2021, 11(8), 932
In this study, we present the synthesis of chiral fragrance aldehydes, which was tackled by a combination of chemo-catalysis and a multi-enzymatic in vivo cascade reaction and the development of a highly versatile high-throughput assay for the enzymatic reduction of carboxylic acids. We investigated a biocompatible metal-catalyzed synthesis for the preparation of α or β substituted cinnamic acid derivatives which were fed directly into the biocatalytic system. Subsequently, the target molecules were synthesized by an enzymatic cascade consisting of a carboxylate reduction, followed by the selective C-C double bond reduction catalyzed by appropriate enoate reductases. We investigated a biocompatible oxidative Heck protocol and combined it with cells expressing a carboxylic acid reductase from Neurospora crassa (NcCAR) and an ene reductase from Saccharomyces pastorianus for the production fragrance aldehydes.
Biocatalysis in Green and Blue: Cyanobacteria
Jodlbauer, Julia; Rohr, Thomas; Spadiut, Oliver; Mihovilovic, Marko D.; Rudroff, Florian*
Trends in biotechnology (2021)
A major challenge for the enzymatic synthesis of sugar esters is the low solubility of sugars in anhydrous, often toxic, organic solvents. We overcame this limitation by using acyltransferases for efficient acetylation of sugars in water. Selective 6-O-acetylation of glucose, maltose, and maltotriose with conversions of up to 78% was achieved within 15 min using engineered acyltransferases (4 μM). Moreover, we identified EstA as a promiscuous acyltransferase preferentially acetylating sugars instead of hydrophobic acyl acceptors. This expands the applicability of promiscuous acyltransferases to sugar modifications and contributes to the understanding of how to adapt acyltransferases to hydrophilic substrates.
Publications in Scientific Journals
Biogenic colourants in the textile industry – a promising and sustainable alternative to synthetic dyes
Richard Fried, Ilinca Oprea, Karin Fleck and Florian Rudroff*
Green Chemistry 2021
Chemo-Enzymatic Cascade for the Generation of Fragrance Aldehydes
Catalysts 2021, 11(8), 932
In this study, we present the synthesis of chiral fragrance aldehydes, which was tackled by a combination of chemo-catalysis and a multi-enzymatic in vivo cascade reaction and the development of a highly versatile high-throughput assay for the enzymatic reduction of carboxylic acids. We investigated a biocompatible metal-catalyzed synthesis for the preparation of α or β substituted cinnamic acid derivatives which were fed directly into the biocatalytic system. Subsequently, the target molecules were synthesized by an enzymatic cascade consisting of a carboxylate reduction, followed by the selective C-C double bond reduction catalyzed by appropriate enoate reductases. We investigated a biocompatible oxidative Heck protocol and combined it with cells expressing a carboxylic acid reductase from Neurospora crassa (NcCAR) and an ene reductase from Saccharomyces pastorianus for the production fragrance aldehydes.
Biocatalysis in Green and Blue: Cyanobacteria
Jodlbauer, Julia; Rohr, Thomas; Spadiut, Oliver; Mihovilovic, Marko D.; Rudroff, Florian*
Trends in biotechnology (2021)
A major challenge for the enzymatic synthesis of sugar esters is the low solubility of sugars in anhydrous, often toxic, organic solvents. We overcame this limitation by using acyltransferases for efficient acetylation of sugars in water. Selective 6-O-acetylation of glucose, maltose, and maltotriose with conversions of up to 78% was achieved within 15 min using engineered acyltransferases (4 μM). Moreover, we identified EstA as a promiscuous acyltransferase preferentially acetylating sugars instead of hydrophobic acyl acceptors. This expands the applicability of promiscuous acyltransferases to sugar modifications and contributes to the understanding of how to adapt acyltransferases to hydrophilic substrates.
Publications in Scientific Journals
An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation
ChemCatChem, 12 (2020), 7; 2032 – 2039.
Immobilized cell physiology imaging and stabilization of enzyme cascade reaction using recombinant cells escherichia coli entrapped in polyelectrolyte complex beads by jet break-up encapsulator
M. Bucko*, P. Gemeiner, T. Krajcovic, M. Hakarova, D. Chorvat, A. Chorvatova, I. Lacik, F. Rudroff, M.D. Mihovilovic
A novel, high performance, and scalable immobilization protocol using a laminar jet break-up technique was developed for the production of polyelectrolyte complex beads with entrapped viable Escherichia coli cells expressing an enzyme cascade of alcohol dehydrogenase, enoate reductase, and cyclohexanone monooxygenase. A significant improvement of operational stability was achieved by cell immobilization, which was manifested as an almost two-fold higher summative product yield of 63% after five cascade reaction cycles as compared to the yield using free cells of 36% after the maximum achievable number of three cycles. Correspondingly, increased metabolic activity was observed by multimodal optical imaging in entrapped cells, which was in contrast to a complete suppression of cell metabolism in free cells after five reaction cycles. Additionally, a high density of cells entrapped in beads had a negligible effect on bead permeability for low molecular weight substrates and products of cascade reaction.
Keywords:
enzyme cascade reaction; immobilization; polyelectrolyte; multimodal optical imaging; biocatalysis; whole-cell biocatalyst
Amino Benzamidoxime (ABAO)-Based Assay to Identify Efficient Aldehyde-Producing Pichia pastoris Clones
Investigation of a New Type I Baeyer-Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability
Pyrazines: Synthesis and Industrial Application of these Valuable Flavor and Fragrance Compounds
Frederik B. Mortzfeld, Chiam Hashem, Kvetoslava Vranková, Margit Winkler, Florian Rudroff*
Contributions to Books
Book Title: Applied Biocatalysis : The Chemist’s Enzyme Toolbox
Synthesis of Six out of Eight Carvo-Lactone Stereoisomers via a Novel Concurrent Redox Cascade Starting from (R)- and (S)-Carvones
Pages 426 – 434.
Publications in Scientific Journals
Boosting photobioredox catalysis by morpholine electron donors under aerobic conditions
C. P. Goncalves, H. Mansouri Khosravi, S. Pourmehdi, M. Abdellah, B. Fadiga, E. Bastos, J. Sá, M.D. Mihovilovic, F. Rudroff*
Morpholine-based buffers activate aerobic photobiocatalysis via spin correlated ion pair formation
L. Gonçalves*, H. Mansouri Khosravi, E. Bastos, M. Abdellah, B. Fadiga, F. Rudroff, M.D. Mihovilovic
Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes
Advanced Synthesis & Catalysis, 361 (2019), 11; 2538 – 2543.
Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime-Mediated High-Throughput Assay
Publications in Scientific Journals
Novel concurrent redox cascades of (R)- and (S)-carvones enables access to carvo-lactones with distinct regio- and enantioselectivity
N. Iqbal, J.D. Stewart, P. Macheroux, F. Rudroff*, M.D. Mihovilovic
Tetrahedron, 52 (2018), 74; 7389 – 7394.
Whole-cell based synthetic enzyme cascades – Light and Shadow of a promising technology
F. Rudroff*
Current Opinion in Chemical Biology, 49 (2018), 84 – 90.
Combining chemo- and biocatalysis, opportunities and challenges
F. Rudroff, M.D. Mihovilovic, H. Gröger, R. Snajdrova, H. Iding, U. Bornscheuer*
Nature Catalysis, 1 (2018), 1; 12 – 33.
Biocompatible metal-assisted C-C cross-coupling combined with biocatalytic chiral reductions in a concurrent tandem cascade
P. Schaaf, T. Bayer, M. Koley, M. Schnürch, U. Bornscheuer, F. Rudroff*, M.D. Mihovilovic
Chemical Communications, 54 (2018), 12978 – 12981.
In this study, we present a concurrent chemo/biocatalytic one pot reaction cascade by combining a metal (Pd/Cu) assisted Liebeskind–Srogl (L–S) coupling with an enantioselective enzymatic reduction for the production of chiral amines and alcohols. The latter transformation was realized by applying enantiocomplementary alcohol dehydrogenases from Lactobacillus kefir (R-selective) and Rhodococcus ruber (S-selective). Compatibility issues were solved by investigating first the L–S-coupling protocol in water at room temperature. Subsequently, we investigated two different biphasic systems and applied a biomimicking approach to separate enzyme-deactivating components. By using a lipophilic membrane in a smart reactor design, we were able to perform concurrent catalytic cascades with overall concentrations up to 100 mM substrate and to produce 1-phenylethylamine and several chiral alcohols in high yields (up to 81% over 2 steps) and enantiomeric purity ((+) and (−)-enantiomers both with 99% ee).
Easy Access to Enantiopure (S)- and (R)-Aryl Alkyl Alcohols by a Combination of Gold(III)-Catalyzed Alkyne Hydration and Enzymatic Reduction
P. Schaaf, V. Gojic, T. Bayer, F. Rudroff, M. Schnürch, M.D. Mihovilovic*
Chemical Communications, 54 (2018), 12978 – 12981.
Publications in Scientific Journals
In vivo synthesis of polyhydroxylated compounds from a ‘hidden reservoir’ of toxic aldehyde species
T. Bayer, T. Wiesinger, S. Milker, M. Winkler, M.D. Mihovilovic, F. Rudroff*
ChemCatChem, 9 (2017), 2919 – 2923.
Synthetic enzyme cascades in living cells often lack efficiency owing to the formation of byproducts by endogenous enzymes or toxicity of the cascade intermediates. Highly reactive aldehyde species can trigger a metabolic stress response, and this leads to undesired side reactions and decreased yields. Owing to the metabolic background of Escherichia coli (E. coli), aldehydes may be irreversibly oxidized to carboxylic acids or reduced to the corresponding alcohols. Herein, we applied an approach to equilibrate the aldehyde concentration in vivo. We oxidized primary alcohols to the corresponding aldehydes by AlkJ, an alcohol dehydrogenase from Pseudomonas putida. Introduction of a carboxylic acid reductase from Nocardia iowensis allowed the target compound to be retrieved from the carboxylate sink. Further reduction of the aldehydes to alcohols by endogenous E. coli enzymes completed the equilibration between alcohols, aldehydes, and carboxylic acids. Thus, the aldehyde concentrations remained below nonviable concentrations. We demonstrated the concept on several primary alcohols, which reached the redox equilibrium within 6 h and persisted up to 24 h. Subsequent combination with a dihydroxyacetone‐dependent aldolase (Fsa1‐A129S, E. coli) demonstrated that the reactive aldehyde species were freely available and gave the aldol product, (3S,4R)‐1,3,4‐trihydroxy‐5‐phenylpentan‐2‐one, in 70 % yield within short reaction times.
Mutagenesis‐Independent Stabilization of Class B Flavin Monooxygenases in Operation
C. P. Goncalves, D. Kracher, S. Milker, M. Fink*, F. Rudroff*, R. Ludwig*, A. Bommarius, M.D. Mihovilovic
Advanced Synthesis & Catalysis, 359 (2017), 2121 – 2131.
Nicotinamide adenine dinucleotide-dependent redox-neutral convergent cascade for lactonizations with Type II flavin-containing monooxygenase
L. Hunag, E. Romero, A. Ressmann, F. Rudroff, F. Hollmann, M.W. Fraaije, S. Kara*
Advanced Synthesis & Catalysis, 12 (2017), 2142 – 2148.
Manipulating the stereoselectivity of the thermostable Baeyer–Villiger monooxygenase TmCHMO by directed evolution
G. Li, M. Fürst, H. Mansouri Khosravi, A. Ressmann, A. Ilie, F. Rudroff, M.D. Mihovilovic*, M.W. Fraaije*, M.T Reetz*
Organic & Biomolecular Chemistry, 15 (2017), 9824 – 9829.
Kinetic modeling of an enzymatic redox cascade in vivo reveals cofactor-caused bottlenecks
S. Milker, M. Fink, N. Oberleitner, A. Ressmann, U. Bornscheuer, M.D. Mihovilovic, F. Rudroff*
ChemCatChem, 9 (2017), 3420 – 3427.
Non-hazardous biocatalytic oxidation in Nylon-9 monomer synthesis on a 40-gram scale with efficient downstream processing
S. Milker, M. Fink*, F. Rudroff, M.D. Mihovilovic
This paper describes the development of a biocatalytic process on the multi‐dozen gram scale for the synthesis of a precursor to Nylon‐9, a specialty polyamide. Such materials are growing in demand, but their corresponding monomers are often difficult to synthesize, giving rise to biocatalytic approaches. Here, we implemented cyclopentadecanone monooxygenase as an Escherichia coli whole‐cell biocatalyst in a defined medium, together with a substrate feeding–product removal concept, and an optimized downstream processing (DSP). A previously described hazardous peracid‐mediated oxidation was thus replaced with a safe and scalable protocol, using aerial oxygen as oxidant, and water as reaction solvent. The engineered process converted 42 g (0.28 mol) starting material ketone to the corresponding lactone with an isolated yield of 70% (33 g), after highly efficient DSP with 95% recovery of the converted material, translating to a volumetric yield of 8 g pure product per liter. Biotechnol. Bioeng. 2017;114: 1670–1678. © 2017 Wiley Periodicals, Inc.
Escherichia coli Fails to Efficiently Maintain the Activity of an Important Flavin Monooxygenase in Recombinant Overexpression
S. Milker, C. P. Goncalves, M. Fink*, F. Rudroff*
Frontiers in Microbiology, 8 (2017).
This paper describes the measurement and analysis of in vivo activity and stability of cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO), a model Baeyer–Villiger monooxygenase, in the recombinant host Escherichia coli. This enzyme was often described as poorly stable in vitro, and has recently been found to deactivate rapidly in the absence of its essential cofactors and antioxidants. Its stability in vivo was scarcely studied, so far. Under conditions common for the overexpression of CHMO we investigated the ability of the host to support these properties using metabolomics. Our results showed that E. coli failed to provide the intracellular levels of cofactors required to functionally stabilize the enzyme, although the biocatalyst was produced in high concentration, and was invariably detected after protein synthesis had stopped. We thus infer that biotechnological applications of CHMO with this host relied on a residual activity of approximately 5-10%. Other microorganisms might offer a more efficient solution for recombinant production of CHMO and related enzymes.
From waste to value – Direct utilization of limonene from orange peel in a biocatalytic cascade reaction towards chiral carvolactone
N. Oberleitner, A. Ressmann, K. Schröder, P. Gärtner, M.W. Fraaije, U. Bornscheuer, M.D. Mihovilovic, F. Rudroff*:
A fusion protein of an enoate reductase and a Baeyer-Villiger monoxygenase facilitates synthesis of chiral lactones
C. Peters, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer*
Biological Chemistry, 1 (2017), 398; 31 – 37.
First chemo-enzymatic synthesis of (R)-Taniguchi lactone and substrate profiles of CAMO and OTEMO, two new Baeyer-Villiger monooxygenases
F. Rudroff*, M. Fink, R. Pydi, U. Bornscheuer, M.D. Mihovilovic
Monatshefte für Chemie, 148 (2017), 157 – 165.
Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes
H. Stolterfohta, D. Schwendenwein, W. Sensen, F. Rudroff, M. Winkler
Journal of Biotechnology, 257 (2017), 222 – 232.
Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds
T. Wiesinger, T. Bayer, S. Milker, M.D. Mihovilovic, F. Rudroff*
ChemBioChem, 18 (2017), 1 – 9.
Baeyer-Villiger oxidations: biotechnological approach
M. Bucko, P. Gemeiner, A. Schenkmayerova, T. Krajcovic, F. Rudroff*, M.D. Mihovilovic
Applied Microbiology and Biotechnology, 100 (2016), 15; 6585 – 6599.
Synthesis of tetrahydrofuran-based natural products and their carba analogs via stereoselective enzyme mediated Baeyer-Villiger oxidation
F. Rudroff, D. Bianchi, R. Moran-Ramallal, N. Iqbal, D. Dreier, M.D. Mihovilovic*
Tetrahedron, 72 (2016), 7212 – 7221.
Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa
D. Schwendenwein, G. Fiume, H. Weber, F. Rudroff, M. Winkler*
Advanced Synthesis & Catalysis, 21 (2016), 358; 3414 – 3421.
The enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. We have increased the spectrum of carboxylate‐reducing enzymes (CARs) with the sequence of a fungal CAR from Neurospora crassa OR74A (NcCAR). NcCAR was efficiently expressed in E. coli using an autoinduction protocol at low temperature. It was purified and characterized in vitro, revealing a broad substrate acceptance, a pH optimum at pH 5.5–6.0, a Tm of 45 °C and inhibition by the co‐product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of NcCAR was demonstrated in a whole‐cell biotransformation using the Escherichia coli K‐12 MG1655 RARE strain in order to suppress overreduction to undesired alcohol. The fragrance compound piperonal was prepared from piperonylic acid (30 mM) on gram scale in 92 % isolated yield in >98% purity. This corresponds to a productivity of 1.5 g/L/h.
Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives
T. Bayer, S. Milker, T. Wiesinger, M.D. Mihovilovic, F. Rudroff*
Advanced Synthesis & Catalysis, 357 (2015), 1587 – 1618.
An immense number of chemical reactions are carried out simultaneously in living cells. Nature’s optimization approach encompasses the assembly of reactions in cascades and to embed them in finely tuned metabolic networks. With the vast progress in the field of biocatalysis, man‐made cascades, especially redox cascades, have reached a degree of complexity that needs tools for improved control and optimization. Combined strategies from biocatalysis, metabolic engineering and synthetic biology lead to the establishment of artificial metabolic pathways with minimized interference with the cellular host environment. This review will focus on genetic and metabolic engineering tools for the assembly and introduction of de novo redox pathways into the host Escherichia coli and will present state of the art redox cascades performed by tailor‐made microbial cell factories.
First Total Synthesis of Piperenol B and Configuration Revision of the Enantiomers Piperenol B and Uvarirufol A
T. Fischer, B. Cerra, M. Fink, F. Rudroff, E. Horkel, M.D. Mihovilovic*
European Journal of Organic Chemistry, 2015 (2015), 1464 – 1471.
The first total synthesis of piperenol B, a polyoxygenated cyclohexene natural product from Piper cubeb, has been developed starting with the multi‐ten‐gram scale enzymatic dihydroxylation of sodium benzoate. The originally predicted absolute configurations of piperenol B and its enantiomer uvarirufol A were eventually revised based on this synthesis and NMR analysis of late‐stage Mosher’s esters.
Cascade catalysis – strategies and challenges en route to preparative synthetic biology
J. Muschiol, C. Peters, N. Oberleitner, M.D. Mihovilovic, U. Bornscheuer, F. Rudroff*
Chemical Communications, 51 (2015), 5798 – 5811.
Nature’s smartness and efficient assembling cascade type reactions inspired biologists and chemists all around the world. Tremendous effort has been directed towards the understanding and mimicking of such networks. In recent years considerable progress has been made in developing multistep one-pot reactions combining either advantage of chemo-, regio-, and stereoselectivity of biocatalysts or promiscuity and productivity of chemocatalysts. In this context several concepts, inspired by different disciplines (biocatalysis, metabolic engineering, synthetic chemistry, and material science), have been evolved. This review will focus on major contributions in the field of cascade reactions over the last three years.
Inferring causal metabolic signals that regulate the dynamic TORC1-dependent transcriptom
A. Oliveira, S. Dimopoulos, A. Busetto, S. Christen, R. Dechant, L. Falter, M. Chehreghani, S. Jozefczuk, C. Ludwig, F. Rudroff, J. Schulz, A. González, A. Soulard, D. Stracka, R. Aebersold, J. Buhmann, N. Hall, M. Peter, U. Sauer*, J. Stelling*
Molecular Systems Biology, 4 (2015), 11; 802.
Cells react to nutritional cues in changing environments via the integrated action of signaling, transcriptional, and metabolic networks. Mechanistic insight into signaling processes is often complicated because ubiquitous feedback loops obscure causal relationships. Consequently, the endogenous inputs of many nutrient signaling pathways remain unknown. Recent advances for system‐wide experimental data generation have facilitated the quantification of signaling systems, but the integration of multi‐level dynamic data remains challenging. Here, we co‐designed dynamic experiments and a probabilistic, model‐based method to infer causal relationships between metabolism, signaling, and gene regulation. We analyzed the dynamic regulation of nitrogen metabolism by the target of rapamycin complex 1 (TORC1) pathway in budding yeast. Dynamic transcriptomic, proteomic, and metabolomic measurements along shifts in nitrogen quality yielded a consistent dataset that demonstrated extensive re‐wiring of cellular networks during adaptation. Our inference method identified putative downstream targets of TORC1 and putative metabolic inputs of TORC1, including the hypothesized glutamine signal. The work provides a basis for further mechanistic studies of nitrogen metabolism and a general computational framework to study cellular processes.
In vitro characterization of an enzymatic redox cascade composed of an alcohol dehydrogenase, an enoate reductases and a Baeyer-Villiger monooxygenase
N. Oberleitner, C. Peters, F. Rudroff, U. Bornscheuer, M.D. Mihovilovic*
Journal of Biotechnology, 192 (2014), 393 – 399.
- C. Peters, R. Koelzsch, M. Kadow, L. Skalden, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer:
“Identification, Characterization, and Application of Three Enoate Reductases from Pseudomonas putida in In Vitro Enzyme Cascade Reactions“;
ChemCatChem, 6 (2014), 1021 – 1027.
- J. Santos-Aberturas, J. Engel, J. Dickerhoff, M. Dorr, F. Rudroff, K. Weisz, U. Bornscheuer:
“Exploration of the substrate promiscuity of biosynthetic tailoring enzymes as a new source of structural diversity for polyene macrolide antifungals“;
ChemCatChem, 7 (2014), 3; 490 – 500.
- D. Stracka, S. Jozefczuk, F. Rudroff, U. Sauer, N. Hall:
“Nitrogen source activates TOR (target of rapamycin) complex 1 via glutamine and independently of Gtr/Rag proteins“;
Journal of Biological Chemistry, 289 (2014), 25010 – 25020.
- M. Sunnaker, E. Zamora-Sillero, A. Garcia de Lomana, F. Rudroff, U. Sauer, J. Stelling, A. Wagner:
“Topological augmentation to infer hidden processes in biological systems“;
Bioinformatics, 2 (2014), 30; 221 – 227.
- D. Bianchi, R. Moran-Ramallal, N. Iqbal, F. Rudroff, M.D. Mihovilovic:
“Enantiocomplementary access to carba-analogs of C-nucleoside derivatives by recombinant Baeyer-Villiger monooxygenases“;
Bioorganic & Medicinal Chemistry Letters, 23 (2013), 2718 – 2720.
- M. Fink, M. Schön, F. Rudroff, M. Schnürch, M.D. Mihovilovic:
“Single Operation Stereoselective Synthesis of Aerangis Lactones: Combining Continuous Flow Hydrogenation and Biocatalysts in a Chemoenzymatic Sequence“;
ChemCatChem, 5 (2013), 724 – 727.
- M. Geier, A. Braun, P. Fladischer, P. Stepniak, F. Rudroff, C. Hametner, M.D. Mihovilovic, A. Glieder:
“Double site saturation mutagenesis of the human cytochrome P450 2D6 for steroid hydroxylation“;
FEBS Journal, 280 (2013), 3094 – 3108.
- F. Leipold, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer:
“The Steroid Monooxygenase from Rhodococcus rhodochrous – a Versatile Biocatalyst“;
Tetrahedron – Asymmetry, 24 (2013), 1620 – 1624.
- N. Oberleitner, C. Peters, J. Muschiol, M. Kadow, S. Saß, T. Bayer, P. Schaaf, N. Iqbal, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer:
“An Enzymatic Toolbox for Cascade Reactions: A Showcase for an In Vivo Redox Sequence in Asymmetric Synthesis“;
ChemCatChem, 5 (2013), 3524 – 3528.
- M. Fink, D.V. Rial, P. Kapitanova, A. Lengar, J. Rehdorf, Q. Cheng, F. Rudroff, M.D. Mihovilovic:
“Quantitative Comparison of Chiral Catalysts Selectivity and Performance: A Generic Concept Illustrated with Cyclododecanone Monooxygenase as Baeyer-Villiger Biocatalyst“;
Advanced Synthesis & Catalysis, 18 (2012), 354; 3491 – 3500.
- N. Iqbal, F. Rudroff, A. Brige, J. van Beeumen, M.D. Mihovilovic:
“Asymmetric Bioreduction of Activated Carbon-Carbon Double Bonds Using Shewanella Yellow Enzyme (SYE-4) as Novel Enoate Reductase“;
Tetrahedron, 68 (2012), 37; 7619 – 7623.
- M. Fink, T. Fischer, F. Rudroff, H. Dudek, M.W. Fraaije, M.D. Mihovilovic:
“Extensive substrate profiling of cyclopentadecanone monooxygenase as Baeyer-Villiger biocatalyst reveals novel regiodivergent oxidations“;
Journal of Molecular Catalysis B Enzymatic, 73 (2011), 9 – 16.
- M. Fink, F. Rudroff, M.D. Mihovilovic:
“Baeyer-Villiger monooxygenases in aroma compound synthesis“;
Bioorganic & Medicinal Chemistry Letters, 21 (2011), 6135 – 6138.
- M. Hucik, M. Bucko, P. Gemeiner, V. Stefuca, A. Vikartovska, M.D. Mihovilovic, F. Rudroff, N. Iqbal, D. Chorvat, I. Lacik:
“Encapsulation of recombinant E. coli expressing cyclopentanone monooxygenase in polyelectrolyte complex capsules for Baeyer-Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one“;
Biotechnology Letters, 32 (2010), 675 – 680.
- D. Pazmino, A. Riebel, J. de Lange, F. Rudroff, M.D. Mihovilovic, M.W. Fraaije:
“Efficient Biooxidations Catalyzed by a new Generation of Self-Sufficient Baeyer-Villiger Monooxygenases“;
ChemBioChem, 10 (2009), 2595 – 2598.
- M.D. Mihovilovic, B. Grötzl, W. Kandioller, A. Muskotal, R. Snajdrova, F. Rudroff, H. Spreitzer:
“Recombinant Whole-Cell Mediated Baeyer-Villiger Oxidation of Perhydropyran-Type Ketones“;
Chemistry & Biodiversity, 5 (2008), 490 – 498.
- I. Braun, F. Rudroff, M.D. Mihovilovic, T. Bach:
“Ring Opening and Rearrangement Reactions of Tricyclo[4.2.1.02,5]nonan-9-one“;
Synthesis, 24 (2007), 3896 – 3906.
- F. Rudroff, J. Rydz, F.H. Ogink, M. Fink, M.D. Mihovilovic:
“Comparing the Stereoselective Biooxidation of Cyclobutanones byRecombinant Strains Expressing Bacterial Baeyer-Villiger Monooxygenases“;
Advanced Synthesis & Catalysis, 349 (2007), 1436 – 1444.
- I. Braun, F. Rudroff, M.D. Mihovilovic, T. Bach:
“Synthesis of Enantiomerically Pure Bicyclo[4.2.0]octanes by Cu-Catalyzed [2+2] Photocycloaddition and Enantiotopos-Differentiating Ring Opening“;
Angewandte Chemie – International Edition, 45 (2006), 5541 – 5543.
- M.D. Mihovilovic, D. Bianchi, F. Rudroff:
“Accessing tetrahydrofuran-based natural products by microbial Baeyer-Villiger biooxidation“;
Chemical Communications, 2006 (2006), 3214 – 3216.
- M.D. Mihovilovic, F. Rudroff, A. Winninger, T. Schneider, F. Schulz, M.T Reetz:
“Microbial Baeyer-Villiger Oxidation: Stereopreference and Substrate Acceptance of Cyclohexanone Monooxygenase Mutants Prepared by Directed Evolution“;
Organic Letters, 8 (2006), 1221 – 1224.
- F. Rudroff, V. Alphand, R. Furstoss, M.D. Mihovilovic:
“Optimizing Fermentation Conditions of Recombinant Escherichia coli Expressing Cyclopentanone Monooxygenase“;
Organic Process Research & Development, 10 (2006), 599 – 604.
- M.D. Mihovilovic, P. Kapitan, J. Rydz, F. Rudroff, F.H. Ogink, M.W. Fraaije:
“Biooxidation of ketones with a cyclobutanone structural motif by recombinant whole-cells expressing 4-hydroxyacetophenone monooxygenase“;
Journal of Molecular Catalysis B Enzymatic, 32 (2005), 135 – 140.
- M.D. Mihovilovic, F. Rudroff, B. Grötzl, P. Kapitan, J. Rydz, R.L. Mach:
“Family Clustering of Baeyer-Villiger Monooxygenase Based on Protein Sequences and Stereopreference“;
Angewandte Chemie – International Edition, 44 (2005), 3609 – 3613.
- M.D. Mihovilovic, F. Rudroff, B. Grötzl, P. Stanetty:
“Microbial Baeyer-Villiger Oxidation of Prochiral Polysubstituted Cyclohaxanones by Recombinant Whole-Cells Expressing Two Bacterial Monooxygenases“;
European Journal of Organic Chemistry, 2005 (2005), 809 – 816.
- M.D. Mihovilovic, R. Snajdrova, A. Winninger, F. Rudroff:
“Baeyer-Villiger Oxidations of Bridged endo-Tricyclic Ketones with Engineered Escherichia coli Expressing Monooxygenase of Bacterial Origin“;
Synlett, 18 (2005), 2751 – 2754.
Contributions to Books
- N. Oberleitner, C. Peters, J. Muschiol, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer:
“Three Enzyme-Catalyzed Redox Cascade for the Production of a Carvo-Lactone“;
in: “Practical Methods for Biocatalysis and Biotransformations 3“, 3; W. Kroutil, W. Sutton, J. Whittall (ed.); John Wiley & Sons, Ltd., London, 2016, (invited), ISBN: 978-1-118-60525-7, 222 – 225. - F. Rudroff, M. Fink, M.D. Mihovilovic:
“Miscellaneous key non C-C bond forming enzyme reactions“;
in: “Organic Synthesis Using Biocatalysis“, A. Goswami, J.D. Stewart (ed.); Elsevier Science Ltd., 2015, ISBN: 978-0-12-411518-7, 243 – 283. - F. Rudroff, M.D. Mihovilovic:
“Monooxygenase-Catalyzed Redox Cascade Biotransformations“;
in: “Cascade Biocatalysis“, S. Riva, W.-D. Fessner (ed.); Wiley-VCH, 2014, ISBN: 978-3-527-33522-0, 43 – 63.
- N. Oberleitner, C. Peters, J. Muschiol, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer:
Florian Rudroff
Connect
- +43 (1) 58801 163618
- +43 664 605 887 137
- florian.rudroff@tuwien.ac.at
General research vision:
The rich repository of biologically active and intricate molecules found in nature has been a wellspring of inspiration for synthetic chemists over the centuries. Their persistent endeavor has been to replicate natural chemical processes and fabricate biomaterials through sophisticated methodologies such as biomimetic synthesis, asymmetric catalysis, and natural product synthesis. Biocatalysis, serving as the biological counterpart to organo- and metal catalysis, stands as an integral component within the chemists’ arsenal, facilitating the synthesis of both refined and large-scale chemical compounds. Our focal point lies in the adept exploration, amplification, and refinement of this extensive chemists’ toolbox, fostering its application in sustainable synthetic practices. In doing so, we aim to contribute to the advancement of environmentally conscious and resource-efficient synthetic methodologies.
Current research:
All of these research topics are addressed in my research group. We focus on the following content:
The development of assays tailored to specific functional groups (e.g., R-OH, RCHO, RCOOH) is underway, coupled with the integration of a microfluidic device to enable ultra-high throughput screening of both novel and enhanced biocatalysts. These assays are meticulously designed for implementation in a fluorescence-activated droplet sorting (FADS) system.
- Our research is fundamentally focused on unraveling and engineering novel enzyme classes, delving into the intricacies of their mechanisms. This encompasses the study of, for example, enoate reductases, BVMOs, alkene-cleaving enzymes known as ‘ozonylases,’ amin dehydrogenases, and hydrogenases.
- Drawing inspiration from nature, we have expanded upon the concept, rooted in the retro(bio)synthesis approach, of single-step (bio)transformations. This expansion involves transitioning towards cascade-type reactions, achieved by seamlessly combining either chemo/bio- or bio/biocatalysts, both in vitro and in vivo. These efforts aim to facilitate the production of fragrances, bioactive compounds, and chiral building blocks.
- Investigating the biodegradation of polyolefin-based plastics through a biocatalytic Fenton-type reaction in the presence of light and oxygen is a focal point of our research.
- We are actively engaged in exploring the capture and valorization of CO2 by leveraging the capabilities of phototrophic bacteria, marking a significant stride towards sustainable and environmentally conscious practices.
- We are actively engaged in exploring the capture and valorization of CO2 by leveraging the capabilities of phototrophic bacteria, marking a significant stride towards sustainable and environmentally conscious practices.
Chemo-Enzymatic Cascade for the Generation of Fragrance Aldehydes
Publication Title: Chemo-Enzymatic Cascade for the Generation of Fragrance Aldehydes by Daniel Schwendenwein, Anna K. Ressmann, Marcello Entner, Viktor Savic, Margit Winkler,* and Florian Rudroff*
Enzyme Cascade Design and Modelling
In our book “Enzyme Cascade Design and Modelling” we have joined our forces and brought a team of experts in multi-enzymatic and chemo-enzymatic cascades! Editors: Kara,
Cell‐free in vitro reduction of carboxylates to aldehydes: With crude enzyme preparations to a key pharmaceutical building block
The scarcity of practical methods for aldehyde synthesis in chemistry necessitates the development of mild, selective procedures. Carboxylic acid reductases catalyze aldehyde formation from stable
Biocatalysis in Green and Blue: Cyanobacteria
Recently, several studies have proven the potential of cyanobacteria as whole-cell biocatalysts for biotransformation. Compared to heterotrophic hosts, cyanobacteria show unique advantages thanks to their
Pyrazines: Synthesis and Industrial Application of these Valuable Flavor and Fragrance Compounds
Alkyl pyrazines—other than being extracted from various natural sources such as coffee beans, cocoa beans, and vegetables—can be synthesized by chemical methods or by certain
Boosting photobioredox catalysis by morpholine electron donors under aerobic conditions
Light-driven reduction of flavins, e.g. FAD or FMN, by sacrificial electron donors emerged as a convenient method to promote biocatalytic transformations. However, flavin activation has been restricted
Florian Rudroff, born in Vienna, studied Technical Chemistry, with specialization in Organic Chemistry at the TU Wien. He conducted his diploma and PhD thesis in the group of Prof. Marko Mihovilovic and obtained his PhD in 2007 from TU Wien. The topic of the PhD thesis was located at the interface between Biology and Chemistry in the field of Biocatalysis. Afterwards he was awarded with an ’Erwin Schrödinger fellowship’ and went for a postdoctoral stay to the group of Prof. Uwe Sauer at ETH Zurich. His research was mainly conducted to Molecular Systems Biology (metabolomics and fluxomics) and specifically to the rapid metabolic and TOR signalling responses in Saccharomyces cerevisiae upon different nitrogen input signals.
In 2011, he returned to the TU Wien and started his independent scientific career in the field of Systems Biocatalysis. Since December 2017, he became Assistant Professor and finished his Habilitation in the field of Bioorganic Chemistry at the Institute of Applied Synthetic Chemistry, TU Wien. In 2020, he was appointed as Assoc. Professor and group leader of the Bioorganic Synthetic Chemistry Group (BSC) at the TU Wien. Since 2018 he is leader of the local branch (Vienna, lower Austria, Burgenland) of the Austrian Chemical Society (GÖCH).
His main research interests are located in the field of enzyme cascade catalysis, photoredox biocatalysis (e.g, CO2 utilization by cyanobacteria), protein engineering, the development of high throughput platforms (e.g. fluorescence adsorption droplet sorting, FADS) and organic synthesis.
González Rodríguez, Jorge (Post Doc)
Jorge González Rodríguez studied his PhD under the supervision of Prof. Humberto Rodríguez Solla and Prof. Raquel María González Soengas at the University of Oviedo (Spain). His research during this period was focused on organometallic reactivity in combination with different biocatalytic approaches to access valuable building blocks for further synthetic and/or industrial uses. Currently, he is working on the development of new methodologies for the sustainable and efficient synthesis of pyrazines.
Keywords:
- sustainable synthesis
- pyrazines
Fried, Richard (PhD Student)
Keywords:
- metabolic pathways
- secondary metabolites
- purification strategies
- structure elucidation
Giparakis, Stefan (PhD Student)
Keywords:
- wacker-oxidation
- biotransformation
- green chemistry
Mortzfeld, Frederik (PhD Student)
ORCID: 0000-0003-2857-9504
Keywords:
- flow chemistry methodologies
- high energetic / ultrafast reactions
- biocatalysis
- directed evolution (FADS)
- API & fragrance synthesis
- process development
Jodlbauer, Julia (PhD Student)
Keywords:
- cyanobacteria
- redox biocatalysis
- RBS library
- light-driven whole-cell biotransformations
- synthetic biology
Schiefer, Astrid (PhD Student)
In the context of my Master’s thesis I took a closer look at ABAO (2-amino-benzamidoxime) assays for the detection of aldehydes with the subsequent aim of applying it for screening mutant libraries of aldehyde producing enzymes with FADS. I did my bachelor thesis “BIOCAR-Bioorthogonal capture and release” at the Institute of Applied Synthetic Chemistry in the research group Molecular Chemistry & Chemical Biology at TU Wien. After finishing my Master’s thesis in the Rudroff group, I am excited to continue on exploring the challenges towards enzyme assisted methods to cleave C=C double bonds.
Keywords:
- 2-amino-benzamidoxime
- FADS
- carboxylic acid reductase enzymes
- alkene cleavage
Rohr, Thomas (PhD Student)
I started my PhD-studies at this group in November 2019. My project focuses on establishing Cyanobacteria as host-platform for Whole-cell-catalysis (Biocatalysis). After finishing my Bachelor of Science in Applied Biology from the University of Applied Sciences Bonn-Rhein-Sieg, I started a Master in Biotechnology and Process-engineering before switching to the Heinrich-Heine-University Düsseldorf. There, I completed my Master of Science in Biology with focus on Synthetic Biology and Systems-Biotechnology with my Master-Thesis at the Instute of Synthetic Microbiology of Prof. Dr. Ilka Axmann. In parallel to my studies I worked for different Biotech-Start-ups, such as the NUMAFERM GmbH, Düsseldorf.
Keywords:
- cyanobacteria
- synthetic biology
- whole-cell-catalysis
- biocatalysis
Wutscher, Maximilian (PhD Student)
During my Master´s thesis, which I completed at TU Wien in 2023, my research was focused on an enzymatic cascade towards fragrance aldehydes.
The objective of my PhD is to develop a versatile biocatalytic approach employing redox-active enzymes for O-dealkylation. The enzymes applied facilitate the transformation under mild conditions, utilizing molecular oxygen as the oxidant. The final goal is to establish a comprehensive toolbox for organic chemists, enabling the selective and regio-specific removal of O-alkyl side chains on various substrates.
ORCID: 0009-0006-5200-2018
Keywords:
- biocatalysis
- protein engineering
- dealkylation
Suchy, Lydia (PhD Student)
I started my academic education at the University of Vienna in Chemistry. During my master studies in Chemistry I developed a growing interest in Biology. So, after completing my master thesis (conducted at the research institution CINVESTAV, in Mexico City) I started a second master program in Biology at the University of Vienna. I found an opportunity to combine my chemical and biological interests in the BSC group at the TU Wien, where I am completing my master thesis in Biology and will continue with my PhD. In my project I am trying to combine different enzymes for the realization of in vivo enzymatic cascade reactions in Escherichia Coli.
Keywords:
- whole-cell biocatalysis
- in vivo enzymatic cascade reactions
Scheibelreiter, Verena (PhD Student)
I completed my Master’s degree at TU Wien in 2023. My master thesis explored a catalytic method for starch oxidation.
My PhD research revolves around developing enzymatic deprotection strategies for alkyl ethers of different substrates including carbohydrates, utilizing heme-based proteins. Our goal is to expand the chemical space for this type of transformation, making existing synthesis pathways more sustainable and enabling new synthetic strategies.
ORCID: 0000-0002-8236-9658
Keywords:
- enzymatic oxidation
- sugar chemistry
- green chemistry
- dealkylases
Waltl, Christian (Master Student)
Christian did his bachelor`s thesis in the lab of Prof. Verena Jantsch at the Max Perutz Labs, where he concerned himself with rmh-1 which is a scaffolding protein in the RTR-complex of C. elegans, which is important in meiosis. He was part of a pilot project, were he tried to generate a new non-null allele of rmh-1 to gain further insight into the function of rmh-1. He obtained his bachelor`s degree in biology, with an emphasis on microbiology and genetics, in October 2020.
He started his master’s thesis in March 2022 under the supervision of Julia Jodlbauer and Prof. Florian Rudroff. The goal of his thesis is to establish a cascade in Synechocystis sp. PCC6803, converting carveol, which can be produced from limonene, which can be sourced from waste product orange peels, to carvolactone, which is a promising molecule for the generation of thermoplastic polymers. Synechocystis sp. PCC6803 is a cyanobacterium which, due to its photoautotrophic lifestyle, could present a greener alternative to the heterotrophic E. coli which is currently a widespread platform for the generation of desired compounds.
Keywords:
- biocatalysis
- green chemistry
- cyanobacteria
Pyrazine synthesis – A greener way for our daily flavor and smell
Project description:
- Biocatalytic retrosynthesis for the production of heterocycles
- Biocatalytic synthesis of flavour and fragrance compounds via different enzymatic cascades
- Comparison of chemical & enzymatic routes
- FADS-based (fluorscence activated droplet sorting) enzyme-assay development
- Biomimicking of chemical flavour production starting from sugar beet molasses
Bioinspired alkene cleavage – BioZone
The aim of this project is to develop a biological (enzyme-assisted) method for splitting carbon / carbon double bonds without the use of the dangerous ‘ozone’. The so-called ozonolysis is a widely used method for the production of so-called “carbonyl compounds”. Such carbon / carbon double bond containing compounds can be starting materials to make such as flavor compounds like vanillin or fragrances like Lilial. Ozonolyis is mainly used on a laboratory scale. The reason for this is the toxicity of ozone and the danger of the intermediate products, which are not safe: Some of them have already caused industrial reactors to explode. We want to find and investigate a safe alternative with enzymes that can split C = C double bonds at room temperature, in water and by means of oxygen. We use a high-throughput detection method that is specifically designed for the detection of aldehydes (carbonyl compounds) in order to examine different protein sequences for their ability to split C = C double bonds. This detection method is highly sensitive, completely independent of structure, easy to use and extremely specific for aldehydes. With this detection method in combination with a special technique, it is possible to examine up to a million protein sequences in a day. It is thus possible to examine known enzymes for their substrate diversity and new enzymes for their activity in a very short time, as quantitatively as possible. After suitable enzymes have been found, we will use them for the production of potential new active pharmaceutical ingredients or flavorings.
Cyanobacteria – Photo-Biocatalysis goes green and blue
Recently, several studies have proven the potential of cyanobacteria as whole-cell biocatalysts for biotransformations. Compared to heterotrophic hosts, cyanobacteria show unique advantages thanks to their photoautotrophic metabolism. Their ability to use light as energy and CO2 as carbon source promises a truly sustainable production platform. Moreover, their photoautotrophic metabolism offers an encouraging source of reducing power, which makes them attractive for redox-based biotechnological purposes. To exploit the full potential of these whole-cell biocatalysts, cyanobacterial cells must be considered in their entirety.
Project description:
- Expression of redox enzymes in cyanobacteria
- Comparison of different cyanobacteria
- Biocatalysis – single step and in a cascade fashion
- Systems biology of cyanobacteria
Enzyme cascades & Chemo/Biocatalysis
Project description:
- Development of novel strategies for the synthesis of high-value compounds by retrosynthesis
- Combination of chemo and biocatalysis
- Multi-step catalysis in vivo & in vitro
- Flux optimization – on transcriptional and translational level
- Protein engineering
Publications in Scientific Journals
An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation
ChemCatChem, 12 (2020), 7; 2032 – 2039.
Immobilized cell physiology imaging and stabilization of enzyme cascade reaction using recombinant cells escherichia coli entrapped in polyelectrolyte complex beads by jet break-up encapsulator
M. Bucko*, P. Gemeiner, T. Krajcovic, M. Hakarova, D. Chorvat, A. Chorvatova, I. Lacik, F. Rudroff, M.D. Mihovilovic
A novel, high performance, and scalable immobilization protocol using a laminar jet break-up technique was developed for the production of polyelectrolyte complex beads with entrapped viable Escherichia coli cells expressing an enzyme cascade of alcohol dehydrogenase, enoate reductase, and cyclohexanone monooxygenase. A significant improvement of operational stability was achieved by cell immobilization, which was manifested as an almost two-fold higher summative product yield of 63% after five cascade reaction cycles as compared to the yield using free cells of 36% after the maximum achievable number of three cycles. Correspondingly, increased metabolic activity was observed by multimodal optical imaging in entrapped cells, which was in contrast to a complete suppression of cell metabolism in free cells after five reaction cycles. Additionally, a high density of cells entrapped in beads had a negligible effect on bead permeability for low molecular weight substrates and products of cascade reaction.
Keywords:
enzyme cascade reaction; immobilization; polyelectrolyte; multimodal optical imaging; biocatalysis; whole-cell biocatalyst
Amino Benzamidoxime (ABAO)-Based Assay to Identify Efficient Aldehyde-Producing Pichia pastoris Clones
Investigation of a New Type I Baeyer-Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability
Pyrazines: Synthesis and Industrial Application of these Valuable Flavor and Fragrance Compounds
Frederik B. Mortzfeld, Chiam Hashem, Kvetoslava Vranková, Margit Winkler, Florian Rudroff*
Contributions to Books
Book Title: Applied Biocatalysis : The Chemist’s Enzyme Toolbox
Synthesis of Six out of Eight Carvo-Lactone Stereoisomers via a Novel Concurrent Redox Cascade Starting from (R)- and (S)-Carvones
Pages 426 – 434.
Publications in Scientific Journals
Boosting photobioredox catalysis by morpholine electron donors under aerobic conditions
C. P. Goncalves, H. Mansouri Khosravi, S. Pourmehdi, M. Abdellah, B. Fadiga, E. Bastos, J. Sá, M.D. Mihovilovic, F. Rudroff*
Morpholine-based buffers activate aerobic photobiocatalysis via spin correlated ion pair formation
L. Gonçalves*, H. Mansouri Khosravi, E. Bastos, M. Abdellah, B. Fadiga, F. Rudroff, M.D. Mihovilovic
Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes
Advanced Synthesis & Catalysis, 361 (2019), 11; 2538 – 2543.
Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime-Mediated High-Throughput Assay
Publications in Scientific Journals
Novel concurrent redox cascades of (R)- and (S)-carvones enables access to carvo-lactones with distinct regio- and enantioselectivity
N. Iqbal, J.D. Stewart, P. Macheroux, F. Rudroff*, M.D. Mihovilovic
Tetrahedron, 52 (2018), 74; 7389 – 7394.
Whole-cell based synthetic enzyme cascades – Light and Shadow of a promising technology
F. Rudroff*
Current Opinion in Chemical Biology, 49 (2018), 84 – 90.
Combining chemo- and biocatalysis, opportunities and challenges
F. Rudroff, M.D. Mihovilovic, H. Gröger, R. Snajdrova, H. Iding, U. Bornscheuer*
Nature Catalysis, 1 (2018), 1; 12 – 33.
Biocompatible metal-assisted C-C cross-coupling combined with biocatalytic chiral reductions in a concurrent tandem cascade
P. Schaaf, T. Bayer, M. Koley, M. Schnürch, U. Bornscheuer, F. Rudroff*, M.D. Mihovilovic
Chemical Communications, 54 (2018), 12978 – 12981.
In this study, we present a concurrent chemo/biocatalytic one pot reaction cascade by combining a metal (Pd/Cu) assisted Liebeskind–Srogl (L–S) coupling with an enantioselective enzymatic reduction for the production of chiral amines and alcohols. The latter transformation was realized by applying enantiocomplementary alcohol dehydrogenases from Lactobacillus kefir (R-selective) and Rhodococcus ruber (S-selective). Compatibility issues were solved by investigating first the L–S-coupling protocol in water at room temperature. Subsequently, we investigated two different biphasic systems and applied a biomimicking approach to separate enzyme-deactivating components. By using a lipophilic membrane in a smart reactor design, we were able to perform concurrent catalytic cascades with overall concentrations up to 100 mM substrate and to produce 1-phenylethylamine and several chiral alcohols in high yields (up to 81% over 2 steps) and enantiomeric purity ((+) and (−)-enantiomers both with 99% ee).
Easy Access to Enantiopure (S)- and (R)-Aryl Alkyl Alcohols by a Combination of Gold(III)-Catalyzed Alkyne Hydration and Enzymatic Reduction
P. Schaaf, V. Gojic, T. Bayer, F. Rudroff, M. Schnürch, M.D. Mihovilovic*
Chemical Communications, 54 (2018), 12978 – 12981.
Publications in Scientific Journals
In vivo synthesis of polyhydroxylated compounds from a ‘hidden reservoir’ of toxic aldehyde species
T. Bayer, T. Wiesinger, S. Milker, M. Winkler, M.D. Mihovilovic, F. Rudroff*
ChemCatChem, 9 (2017), 2919 – 2923.
Synthetic enzyme cascades in living cells often lack efficiency owing to the formation of byproducts by endogenous enzymes or toxicity of the cascade intermediates. Highly reactive aldehyde species can trigger a metabolic stress response, and this leads to undesired side reactions and decreased yields. Owing to the metabolic background of Escherichia coli (E. coli), aldehydes may be irreversibly oxidized to carboxylic acids or reduced to the corresponding alcohols. Herein, we applied an approach to equilibrate the aldehyde concentration in vivo. We oxidized primary alcohols to the corresponding aldehydes by AlkJ, an alcohol dehydrogenase from Pseudomonas putida. Introduction of a carboxylic acid reductase from Nocardia iowensis allowed the target compound to be retrieved from the carboxylate sink. Further reduction of the aldehydes to alcohols by endogenous E. coli enzymes completed the equilibration between alcohols, aldehydes, and carboxylic acids. Thus, the aldehyde concentrations remained below nonviable concentrations. We demonstrated the concept on several primary alcohols, which reached the redox equilibrium within 6 h and persisted up to 24 h. Subsequent combination with a dihydroxyacetone‐dependent aldolase (Fsa1‐A129S, E. coli) demonstrated that the reactive aldehyde species were freely available and gave the aldol product, (3S,4R)‐1,3,4‐trihydroxy‐5‐phenylpentan‐2‐one, in 70 % yield within short reaction times.
Mutagenesis‐Independent Stabilization of Class B Flavin Monooxygenases in Operation
C. P. Goncalves, D. Kracher, S. Milker, M. Fink*, F. Rudroff*, R. Ludwig*, A. Bommarius, M.D. Mihovilovic
Advanced Synthesis & Catalysis, 359 (2017), 2121 – 2131.
Nicotinamide adenine dinucleotide-dependent redox-neutral convergent cascade for lactonizations with Type II flavin-containing monooxygenase
L. Hunag, E. Romero, A. Ressmann, F. Rudroff, F. Hollmann, M.W. Fraaije, S. Kara*
Advanced Synthesis & Catalysis, 12 (2017), 2142 – 2148.
Manipulating the stereoselectivity of the thermostable Baeyer–Villiger monooxygenase TmCHMO by directed evolution
G. Li, M. Fürst, H. Mansouri Khosravi, A. Ressmann, A. Ilie, F. Rudroff, M.D. Mihovilovic*, M.W. Fraaije*, M.T Reetz*
Organic & Biomolecular Chemistry, 15 (2017), 9824 – 9829.
Kinetic modeling of an enzymatic redox cascade in vivo reveals cofactor-caused bottlenecks
S. Milker, M. Fink, N. Oberleitner, A. Ressmann, U. Bornscheuer, M.D. Mihovilovic, F. Rudroff*
ChemCatChem, 9 (2017), 3420 – 3427.
Non-hazardous biocatalytic oxidation in Nylon-9 monomer synthesis on a 40-gram scale with efficient downstream processing
S. Milker, M. Fink*, F. Rudroff, M.D. Mihovilovic
This paper describes the development of a biocatalytic process on the multi‐dozen gram scale for the synthesis of a precursor to Nylon‐9, a specialty polyamide. Such materials are growing in demand, but their corresponding monomers are often difficult to synthesize, giving rise to biocatalytic approaches. Here, we implemented cyclopentadecanone monooxygenase as an Escherichia coli whole‐cell biocatalyst in a defined medium, together with a substrate feeding–product removal concept, and an optimized downstream processing (DSP). A previously described hazardous peracid‐mediated oxidation was thus replaced with a safe and scalable protocol, using aerial oxygen as oxidant, and water as reaction solvent. The engineered process converted 42 g (0.28 mol) starting material ketone to the corresponding lactone with an isolated yield of 70% (33 g), after highly efficient DSP with 95% recovery of the converted material, translating to a volumetric yield of 8 g pure product per liter. Biotechnol. Bioeng. 2017;114: 1670–1678. © 2017 Wiley Periodicals, Inc.
Escherichia coli Fails to Efficiently Maintain the Activity of an Important Flavin Monooxygenase in Recombinant Overexpression
S. Milker, C. P. Goncalves, M. Fink*, F. Rudroff*
Frontiers in Microbiology, 8 (2017).
This paper describes the measurement and analysis of in vivo activity and stability of cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO), a model Baeyer–Villiger monooxygenase, in the recombinant host Escherichia coli. This enzyme was often described as poorly stable in vitro, and has recently been found to deactivate rapidly in the absence of its essential cofactors and antioxidants. Its stability in vivo was scarcely studied, so far. Under conditions common for the overexpression of CHMO we investigated the ability of the host to support these properties using metabolomics. Our results showed that E. coli failed to provide the intracellular levels of cofactors required to functionally stabilize the enzyme, although the biocatalyst was produced in high concentration, and was invariably detected after protein synthesis had stopped. We thus infer that biotechnological applications of CHMO with this host relied on a residual activity of approximately 5-10%. Other microorganisms might offer a more efficient solution for recombinant production of CHMO and related enzymes.
From waste to value – Direct utilization of limonene from orange peel in a biocatalytic cascade reaction towards chiral carvolactone
N. Oberleitner, A. Ressmann, K. Schröder, P. Gärtner, M.W. Fraaije, U. Bornscheuer, M.D. Mihovilovic, F. Rudroff*:
A fusion protein of an enoate reductase and a Baeyer-Villiger monoxygenase facilitates synthesis of chiral lactones
C. Peters, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer*
Biological Chemistry, 1 (2017), 398; 31 – 37.
First chemo-enzymatic synthesis of (R)-Taniguchi lactone and substrate profiles of CAMO and OTEMO, two new Baeyer-Villiger monooxygenases
F. Rudroff*, M. Fink, R. Pydi, U. Bornscheuer, M.D. Mihovilovic
Monatshefte für Chemie, 148 (2017), 157 – 165.
Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes
H. Stolterfohta, D. Schwendenwein, W. Sensen, F. Rudroff, M. Winkler
Journal of Biotechnology, 257 (2017), 222 – 232.
Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds
T. Wiesinger, T. Bayer, S. Milker, M.D. Mihovilovic, F. Rudroff*
ChemBioChem, 18 (2017), 1 – 9.
Baeyer-Villiger oxidations: biotechnological approach
M. Bucko, P. Gemeiner, A. Schenkmayerova, T. Krajcovic, F. Rudroff*, M.D. Mihovilovic
Applied Microbiology and Biotechnology, 100 (2016), 15; 6585 – 6599.
Synthesis of tetrahydrofuran-based natural products and their carba analogs via stereoselective enzyme mediated Baeyer-Villiger oxidation
F. Rudroff, D. Bianchi, R. Moran-Ramallal, N. Iqbal, D. Dreier, M.D. Mihovilovic*
Tetrahedron, 72 (2016), 7212 – 7221.
Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa
D. Schwendenwein, G. Fiume, H. Weber, F. Rudroff, M. Winkler*
Advanced Synthesis & Catalysis, 21 (2016), 358; 3414 – 3421.
The enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. We have increased the spectrum of carboxylate‐reducing enzymes (CARs) with the sequence of a fungal CAR from Neurospora crassa OR74A (NcCAR). NcCAR was efficiently expressed in E. coli using an autoinduction protocol at low temperature. It was purified and characterized in vitro, revealing a broad substrate acceptance, a pH optimum at pH 5.5–6.0, a Tm of 45 °C and inhibition by the co‐product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of NcCAR was demonstrated in a whole‐cell biotransformation using the Escherichia coli K‐12 MG1655 RARE strain in order to suppress overreduction to undesired alcohol. The fragrance compound piperonal was prepared from piperonylic acid (30 mM) on gram scale in 92 % isolated yield in >98% purity. This corresponds to a productivity of 1.5 g/L/h.
Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives
T. Bayer, S. Milker, T. Wiesinger, M.D. Mihovilovic, F. Rudroff*
Advanced Synthesis & Catalysis, 357 (2015), 1587 – 1618.
An immense number of chemical reactions are carried out simultaneously in living cells. Nature’s optimization approach encompasses the assembly of reactions in cascades and to embed them in finely tuned metabolic networks. With the vast progress in the field of biocatalysis, man‐made cascades, especially redox cascades, have reached a degree of complexity that needs tools for improved control and optimization. Combined strategies from biocatalysis, metabolic engineering and synthetic biology lead to the establishment of artificial metabolic pathways with minimized interference with the cellular host environment. This review will focus on genetic and metabolic engineering tools for the assembly and introduction of de novo redox pathways into the host Escherichia coli and will present state of the art redox cascades performed by tailor‐made microbial cell factories.
First Total Synthesis of Piperenol B and Configuration Revision of the Enantiomers Piperenol B and Uvarirufol A
T. Fischer, B. Cerra, M. Fink, F. Rudroff, E. Horkel, M.D. Mihovilovic*
European Journal of Organic Chemistry, 2015 (2015), 1464 – 1471.
The first total synthesis of piperenol B, a polyoxygenated cyclohexene natural product from Piper cubeb, has been developed starting with the multi‐ten‐gram scale enzymatic dihydroxylation of sodium benzoate. The originally predicted absolute configurations of piperenol B and its enantiomer uvarirufol A were eventually revised based on this synthesis and NMR analysis of late‐stage Mosher’s esters.
Cascade catalysis – strategies and challenges en route to preparative synthetic biology
J. Muschiol, C. Peters, N. Oberleitner, M.D. Mihovilovic, U. Bornscheuer, F. Rudroff*
Chemical Communications, 51 (2015), 5798 – 5811.
Nature’s smartness and efficient assembling cascade type reactions inspired biologists and chemists all around the world. Tremendous effort has been directed towards the understanding and mimicking of such networks. In recent years considerable progress has been made in developing multistep one-pot reactions combining either advantage of chemo-, regio-, and stereoselectivity of biocatalysts or promiscuity and productivity of chemocatalysts. In this context several concepts, inspired by different disciplines (biocatalysis, metabolic engineering, synthetic chemistry, and material science), have been evolved. This review will focus on major contributions in the field of cascade reactions over the last three years.
Inferring causal metabolic signals that regulate the dynamic TORC1-dependent transcriptom
A. Oliveira, S. Dimopoulos, A. Busetto, S. Christen, R. Dechant, L. Falter, M. Chehreghani, S. Jozefczuk, C. Ludwig, F. Rudroff, J. Schulz, A. González, A. Soulard, D. Stracka, R. Aebersold, J. Buhmann, N. Hall, M. Peter, U. Sauer*, J. Stelling*
Molecular Systems Biology, 4 (2015), 11; 802.
Cells react to nutritional cues in changing environments via the integrated action of signaling, transcriptional, and metabolic networks. Mechanistic insight into signaling processes is often complicated because ubiquitous feedback loops obscure causal relationships. Consequently, the endogenous inputs of many nutrient signaling pathways remain unknown. Recent advances for system‐wide experimental data generation have facilitated the quantification of signaling systems, but the integration of multi‐level dynamic data remains challenging. Here, we co‐designed dynamic experiments and a probabilistic, model‐based method to infer causal relationships between metabolism, signaling, and gene regulation. We analyzed the dynamic regulation of nitrogen metabolism by the target of rapamycin complex 1 (TORC1) pathway in budding yeast. Dynamic transcriptomic, proteomic, and metabolomic measurements along shifts in nitrogen quality yielded a consistent dataset that demonstrated extensive re‐wiring of cellular networks during adaptation. Our inference method identified putative downstream targets of TORC1 and putative metabolic inputs of TORC1, including the hypothesized glutamine signal. The work provides a basis for further mechanistic studies of nitrogen metabolism and a general computational framework to study cellular processes.
In vitro characterization of an enzymatic redox cascade composed of an alcohol dehydrogenase, an enoate reductases and a Baeyer-Villiger monooxygenase
N. Oberleitner, C. Peters, F. Rudroff, U. Bornscheuer, M.D. Mihovilovic*
Journal of Biotechnology, 192 (2014), 393 – 399.
- C. Peters, R. Koelzsch, M. Kadow, L. Skalden, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer:
“Identification, Characterization, and Application of Three Enoate Reductases from Pseudomonas putida in In Vitro Enzyme Cascade Reactions“;
ChemCatChem, 6 (2014), 1021 – 1027.
- J. Santos-Aberturas, J. Engel, J. Dickerhoff, M. Dorr, F. Rudroff, K. Weisz, U. Bornscheuer:
“Exploration of the substrate promiscuity of biosynthetic tailoring enzymes as a new source of structural diversity for polyene macrolide antifungals“;
ChemCatChem, 7 (2014), 3; 490 – 500.
- D. Stracka, S. Jozefczuk, F. Rudroff, U. Sauer, N. Hall:
“Nitrogen source activates TOR (target of rapamycin) complex 1 via glutamine and independently of Gtr/Rag proteins“;
Journal of Biological Chemistry, 289 (2014), 25010 – 25020.
- M. Sunnaker, E. Zamora-Sillero, A. Garcia de Lomana, F. Rudroff, U. Sauer, J. Stelling, A. Wagner:
“Topological augmentation to infer hidden processes in biological systems“;
Bioinformatics, 2 (2014), 30; 221 – 227.
- D. Bianchi, R. Moran-Ramallal, N. Iqbal, F. Rudroff, M.D. Mihovilovic:
“Enantiocomplementary access to carba-analogs of C-nucleoside derivatives by recombinant Baeyer-Villiger monooxygenases“;
Bioorganic & Medicinal Chemistry Letters, 23 (2013), 2718 – 2720.
- M. Fink, M. Schön, F. Rudroff, M. Schnürch, M.D. Mihovilovic:
“Single Operation Stereoselective Synthesis of Aerangis Lactones: Combining Continuous Flow Hydrogenation and Biocatalysts in a Chemoenzymatic Sequence“;
ChemCatChem, 5 (2013), 724 – 727.
- M. Geier, A. Braun, P. Fladischer, P. Stepniak, F. Rudroff, C. Hametner, M.D. Mihovilovic, A. Glieder:
“Double site saturation mutagenesis of the human cytochrome P450 2D6 for steroid hydroxylation“;
FEBS Journal, 280 (2013), 3094 – 3108.
- F. Leipold, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer:
“The Steroid Monooxygenase from Rhodococcus rhodochrous – a Versatile Biocatalyst“;
Tetrahedron – Asymmetry, 24 (2013), 1620 – 1624.
- N. Oberleitner, C. Peters, J. Muschiol, M. Kadow, S. Saß, T. Bayer, P. Schaaf, N. Iqbal, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer:
“An Enzymatic Toolbox for Cascade Reactions: A Showcase for an In Vivo Redox Sequence in Asymmetric Synthesis“;
ChemCatChem, 5 (2013), 3524 – 3528.
- M. Fink, D.V. Rial, P. Kapitanova, A. Lengar, J. Rehdorf, Q. Cheng, F. Rudroff, M.D. Mihovilovic:
“Quantitative Comparison of Chiral Catalysts Selectivity and Performance: A Generic Concept Illustrated with Cyclododecanone Monooxygenase as Baeyer-Villiger Biocatalyst“;
Advanced Synthesis & Catalysis, 18 (2012), 354; 3491 – 3500.
- N. Iqbal, F. Rudroff, A. Brige, J. van Beeumen, M.D. Mihovilovic:
“Asymmetric Bioreduction of Activated Carbon-Carbon Double Bonds Using Shewanella Yellow Enzyme (SYE-4) as Novel Enoate Reductase“;
Tetrahedron, 68 (2012), 37; 7619 – 7623.
- M. Fink, T. Fischer, F. Rudroff, H. Dudek, M.W. Fraaije, M.D. Mihovilovic:
“Extensive substrate profiling of cyclopentadecanone monooxygenase as Baeyer-Villiger biocatalyst reveals novel regiodivergent oxidations“;
Journal of Molecular Catalysis B Enzymatic, 73 (2011), 9 – 16.
- M. Fink, F. Rudroff, M.D. Mihovilovic:
“Baeyer-Villiger monooxygenases in aroma compound synthesis“;
Bioorganic & Medicinal Chemistry Letters, 21 (2011), 6135 – 6138.
- M. Hucik, M. Bucko, P. Gemeiner, V. Stefuca, A. Vikartovska, M.D. Mihovilovic, F. Rudroff, N. Iqbal, D. Chorvat, I. Lacik:
“Encapsulation of recombinant E. coli expressing cyclopentanone monooxygenase in polyelectrolyte complex capsules for Baeyer-Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one“;
Biotechnology Letters, 32 (2010), 675 – 680.
- D. Pazmino, A. Riebel, J. de Lange, F. Rudroff, M.D. Mihovilovic, M.W. Fraaije:
“Efficient Biooxidations Catalyzed by a new Generation of Self-Sufficient Baeyer-Villiger Monooxygenases“;
ChemBioChem, 10 (2009), 2595 – 2598.
- M.D. Mihovilovic, B. Grötzl, W. Kandioller, A. Muskotal, R. Snajdrova, F. Rudroff, H. Spreitzer:
“Recombinant Whole-Cell Mediated Baeyer-Villiger Oxidation of Perhydropyran-Type Ketones“;
Chemistry & Biodiversity, 5 (2008), 490 – 498.
- I. Braun, F. Rudroff, M.D. Mihovilovic, T. Bach:
“Ring Opening and Rearrangement Reactions of Tricyclo[4.2.1.02,5]nonan-9-one“;
Synthesis, 24 (2007), 3896 – 3906.
- F. Rudroff, J. Rydz, F.H. Ogink, M. Fink, M.D. Mihovilovic:
“Comparing the Stereoselective Biooxidation of Cyclobutanones byRecombinant Strains Expressing Bacterial Baeyer-Villiger Monooxygenases“;
Advanced Synthesis & Catalysis, 349 (2007), 1436 – 1444.
- I. Braun, F. Rudroff, M.D. Mihovilovic, T. Bach:
“Synthesis of Enantiomerically Pure Bicyclo[4.2.0]octanes by Cu-Catalyzed [2+2] Photocycloaddition and Enantiotopos-Differentiating Ring Opening“;
Angewandte Chemie – International Edition, 45 (2006), 5541 – 5543.
- M.D. Mihovilovic, D. Bianchi, F. Rudroff:
“Accessing tetrahydrofuran-based natural products by microbial Baeyer-Villiger biooxidation“;
Chemical Communications, 2006 (2006), 3214 – 3216.
- M.D. Mihovilovic, F. Rudroff, A. Winninger, T. Schneider, F. Schulz, M.T Reetz:
“Microbial Baeyer-Villiger Oxidation: Stereopreference and Substrate Acceptance of Cyclohexanone Monooxygenase Mutants Prepared by Directed Evolution“;
Organic Letters, 8 (2006), 1221 – 1224.
- F. Rudroff, V. Alphand, R. Furstoss, M.D. Mihovilovic:
“Optimizing Fermentation Conditions of Recombinant Escherichia coli Expressing Cyclopentanone Monooxygenase“;
Organic Process Research & Development, 10 (2006), 599 – 604.
- M.D. Mihovilovic, P. Kapitan, J. Rydz, F. Rudroff, F.H. Ogink, M.W. Fraaije:
“Biooxidation of ketones with a cyclobutanone structural motif by recombinant whole-cells expressing 4-hydroxyacetophenone monooxygenase“;
Journal of Molecular Catalysis B Enzymatic, 32 (2005), 135 – 140.
- M.D. Mihovilovic, F. Rudroff, B. Grötzl, P. Kapitan, J. Rydz, R.L. Mach:
“Family Clustering of Baeyer-Villiger Monooxygenase Based on Protein Sequences and Stereopreference“;
Angewandte Chemie – International Edition, 44 (2005), 3609 – 3613.
- M.D. Mihovilovic, F. Rudroff, B. Grötzl, P. Stanetty:
“Microbial Baeyer-Villiger Oxidation of Prochiral Polysubstituted Cyclohaxanones by Recombinant Whole-Cells Expressing Two Bacterial Monooxygenases“;
European Journal of Organic Chemistry, 2005 (2005), 809 – 816.
- M.D. Mihovilovic, R. Snajdrova, A. Winninger, F. Rudroff:
“Baeyer-Villiger Oxidations of Bridged endo-Tricyclic Ketones with Engineered Escherichia coli Expressing Monooxygenase of Bacterial Origin“;
Synlett, 18 (2005), 2751 – 2754.
Contributions to Books
- N. Oberleitner, C. Peters, J. Muschiol, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer:
“Three Enzyme-Catalyzed Redox Cascade for the Production of a Carvo-Lactone“;
in: “Practical Methods for Biocatalysis and Biotransformations 3“, 3; W. Kroutil, W. Sutton, J. Whittall (ed.); John Wiley & Sons, Ltd., London, 2016, (invited), ISBN: 978-1-118-60525-7, 222 – 225. - F. Rudroff, M. Fink, M.D. Mihovilovic:
“Miscellaneous key non C-C bond forming enzyme reactions“;
in: “Organic Synthesis Using Biocatalysis“, A. Goswami, J.D. Stewart (ed.); Elsevier Science Ltd., 2015, ISBN: 978-0-12-411518-7, 243 – 283. - F. Rudroff, M.D. Mihovilovic:
“Monooxygenase-Catalyzed Redox Cascade Biotransformations“;
in: “Cascade Biocatalysis“, S. Riva, W.-D. Fessner (ed.); Wiley-VCH, 2014, ISBN: 978-3-527-33522-0, 43 – 63.
- N. Oberleitner, C. Peters, J. Muschiol, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer:
Florian Rudroff
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- florian.rudroff@tuwien.ac.at