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RESEARCH

Research in the Schmidt Group

Our research harnesses the powerful reactivity and selectivity of enzymes for greener, more sustainable and resource-efficient production of pharmaceutical building blocks and active ingredients. We are an interdisciplinary research group working at the intersection of synthetic biology, enzymology, organic chemistry and biocatalysis. Our research is driven by the desire to understand the complex, molecular processes underlying enzyme catalysis and exploit them for sustainable synthesis to address scientific and societal challenges with innovative enzymatic and microbial approaches. For example, we seek to elucidate the tightly controlled mechanisms in redox enzymes and to engineer these processes to increase their applicability. We also aim to contribute to the necessary transition from fossil resources to the recycling of chemical elements, with carbon (compounds) as the central resource. In this context, we are particularly fascinated by the metabolic capabilities of autotrophic microorganisms to utilize C1 compounds for the synthesis of complex molecules. We envision that these autotrophic microbes can be developed into efficient cell factories for CO2-based biotechnology.

Rieske oxygenases

Electron transfer pathways in enzymes

Enzymatic nitrogen-nitrogen bond formation

Photobiocatalysis

Autotrophic chassis strain development

Our funding sources

Rieske Oxygenases

A group of enzymes called Rieske non-heme iron oxygenases (ROs) catalyzes reactions that are among the most challenging in organic syntheses. ROs are the only enzymes known to catalyze the stereoselective formation of vicinal cis-diols in one step leading to important building blocks for pharmaceuticals. These enzymes are soluble multicomponent systems that harness the reductive power of NAD(P)H for oxygen activation. Due to their versatility, ROs are considered as the non-heme analogue of cytochrome P450 monooxygenases and, in addition to their relaxed substrate specificity, these enzymes can catalyze various oxidation reactions, resulting in an enormous potential of these enzymes for manifold synthetically useful transformations. In contrast to many flavin-dependent monooxygenases, ROs depend on electron transfer from a reductase or the interplay of a reductase (FdR) and a ferredoxin (Fd). In the latter case, the cofactor-derived electrons are transferred via FdR and Fd to the terminal oxygenase (Oxy) component containing the active site.

In the last decade, Rieske oxygenases have also been revealed to play important roles in natural product biosynthesis. Often, the pharmaceutical potential of natural product scaffolds is underexplored due to the difficulty in accessing analogs to complex secondary metabolites using traditional synthetic methods. Recent advances in sequencing technology have facilitated the identification of secondary metabolite gene clusters from genomic and metagenomic samples, which has led to an explosion of newly identified and annotated secondary metabolite pathways.

 

We strive to leverage the powerful reactivity and selectivity of ROs from natural product pathways in concise approaches to natural products and their analogs. We are particularly interested in the biosynthesis of hapalindole-type alkaloids that have gained increasing attention as highly potential leads in recent drug discovery due to their diverse bioactivities. We strive to identify putative ROs from the hapalindole-type alkaloids biosynthetic gene clusters and to elucidate their catalytic role in the synthesis and diversification of these natural products. We employ mechanistic studies with biochemical characterization along with the determination of the substrate scope for their application in synthesizing biologically active molecules. Thereby, synthetic efforts feed biological studies on activity, which inform the subsequent selection of synthetic targets.

 

 

Electron transfer pathways in enzymes

Rieske oxygenases depend on electron transfer from a reductase or the interplay of a reductase and a ferredoxin. These complex redox machines require multistep electron tunneling architectures that can transfer electrons rapidly with only a small loss of free energy to the surface of the oxygenase, where the actual catalysis takes place. 

The applicability of enzymes depending on an electron transfer chain involving ferredoxins remains challenging, mainly due to their low catalytic activity, low stability, and the dependence on a complex electron transfer system. Especially in case of P450 monooxygenases, many efforts have been made to surmount these limitations by simplifying the electron transfer chain. This has led to an increased knowledge on the electron transfer pathways in oxidoreductases, however, in Rieske oxygenases the underlying molecular mechanisms that determine the interactions between ferredoxins and their oxygenases are much less understood.

 

We strive to elucidate the mechanisms that determine the redox partner specificity in Rieske oxygenases and in turn to increase the understanding of the essential aspects determining an efficient electron transfer based on specific protein-protein interactions between the respective partners. We investigate to which extend the redox partner specificity in Rieske oxygneases can be altered toward non-natural electron donors, and whether electron transfer chains in these complex redox machines can be simplified and/or engineered to increase the applicability of ROs.

Enzymatic nitrogen-nitrogen bond formation

Nitrogen-nitrogen (N-N) bonds have fascinating structural and functional properties, are ubiquitous in various pharmaceuticals and fine chemicals, and represent important key structural motifs in various natural products, endowing them with potent biological activities. However, traditional chemical methods for synthesizing N-N bond-containing compounds are often difficult to implement, costly, and involve harsh reaction conditions. Nitrogen-nitrogen bond forming enzymes (NNzymes) from natural product biosynthetic gene clusters catalyze unique chemical reactions, but are still mainly studied in the context of their native reactions/reactivities and often lack structural insights to support experimental findings. Thus, the lack of knowledge on how these enzymes perform the challenging N-N bond formation hinders a systematic exploration of their reactivity towards non-natural substrates and transformations for biocatalytic applications.

To address this knowledge gap, we are leveraging our expertise in metal-dependent biocatalysis to develop novel chemistries from the under-explored family of NNzymes. Among others, these enzymes utilize different iron cofactors and reaction types for N-N couplings to form unique natural products, providing an opportunity to complement existing chemical methods for selective N-N bond formation. To elucidate their catalytic potential, we systematically explore their biotechnological potential for (non)-natural transformations to discover and engineer first-in-class promiscuous NNzymes as a novel addition to the biocatalytic toolbox.

For example, we are exploiting biocatalytic N-N bond formation with piperazate synthases by combining experimental and in silico approaches to elucidate the catalytic potential of these enzymes. In addition, we are exploiting the catalytic capabilities of piperazate synthases in a biocatalytic cascade to synthesize various cyclic N-N bonded compounds of high value as pharmaceutical building blocks. In doing so, we are able to exploit the substrate promiscuity of this enzyme family, a feature that is critical to continuing to access a wide variety of N-N bonded products through protein engineering.

Building on these successes, we aim to establish a versatile NNzyme platform by deciphering novel reaction chemistries from this under-explored enzyme family. In doing so, we will address reactions that are difficult to realize using traditional chemical approaches and that are currently not feasible using enzyme catalysis. Thus, we will develop an enzymatic toolbox for versatile N-N bond forming reaction chemistry by exploring naturally promiscuous and engineered NNzymes for diverse intra- and intermolecular as well as non-natural N-N bond formation. To achieve this, we are combining methods from enzyme catalysis and evolution, genome mining, and the latest advances in bioinformatics. In this way, we will not only determine the chemistry performed by the enzymes, but also elucidate enzyme structures and catalytic mechanisms, and design biocatalytic cascades to demonstrate the potential of tailor-made N-N enzymes.

Photobiocatalysis

Photo-biocatalytic Cascades:

The combination of several catalytic steps to conduct a precisely arranged sequence of chemical transformations in a single reaction vessel exhibits an enormous potential for more economically and ecologically efficient synthetic routes, and thus, the development of one-pot (cascade) reactions is a growing research field. Combining catalysts from different catalysis fields (“worlds”), however, is sometimes more challenging for compatibility reasons. Especially photocatalysts have been combined with various types of other catalysts yielding synergistic dual catalytic systems, where two types of catalysts participate in one catalytic cycle. The combination of photocatalytic and biocatalytic steps for organic synthesis, however, has not been systematically explored until now. Most examples of combining photo- and biocatalysis focus on photocatalytic in situ regeneration of redox enzymes.

We strive to couple photocatalytic reactions that use light energy directly to drive small molecule conversions with further enzymatic functionalization to develop photobiocatalytic concurrent tandem or sequential reactions. We investigate limitations associated to photochemoenzymatic cascades, and develop strategies to overcome the often observed incompatibilities between enzymes and photocatalysts.

Autotrophic chassis strain development

Chemists already strive for more than 100 years for possibilities to produce valuable chemicals from CO2. With the growing world population, depletion of fossil resources and the on-going climate change, the last years’ research strongly focuses on innovative and applicable solutions via chemical as well as biotechnological routes. Thereby, biotechnological routes provide several advantages over chemical strategies. With their capacity to utilize renewable energy for the accumulation of biomass from CO2, autotrophic microorganisms have an enormous potential for the selective production of a broad palette of future materials for our society.

However, the biotechnological utilization of CO2 is associated to several challenges, mainly related to the enzymatic fixation of CO2 to enter metabolism as well as the supply of energy, which is at the same time the greatest chance for microbial biotechnology in this context. In order to overcome these challenges, synthetic biology and process engineering must be integrated to demonstrate the successful production of commodities from CO2.

 

We will explore the potential of the autotrophic bacterium Cupriavidus necator for the production of biochemicals. Particular interest lies thereby on the combination of synthetic biology approaches with metabolic engineering for an adaptive laboratory evolution to create an efficient route from CO2 fixation to the production of commodities. The native machinery for H2/CO2-utilization in the CO2-fixing C. necator is highly optimized from evolution, and thus represents a tremendous space to connect the metabolism with additional electron sinks and to increase space-time yields. Based on this, we strive to develop robust C. necator production strains and investigate metabolic routes using the strengths of C. necator. In order to provide optimal metabolic supply for the envisioned products, we will develop molecular genetic tools for an efficient chassis strain engineering to produce several commodities from CO2.

Our funding sources

Start-up fund S. Schmidt
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ERC Starting Grant – ReCNNSTRCT (GA no. 101075934)
NWO Talent Programme – Vidi (VI.VIDI.213.025)
NWO ENW – XS (OCENW.XS22.1.044 and OCENW.XS23.3.002)
Funding by the European Union
H2020 MSCA ITN-EJD 764920 (PhotoBioCat)
H2020 MSCA ITN-EJD 955740 (ConCO2rde)
Horizon Europe MSCA DN 101073065 (BiodeCCodiNNg)
China Scholarship Council (Personal fellowships Yongxin Li and Hui Miao)