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RESEARCH
Research in the Schmidt Group
Our research exploits the powerful reactivity and selectivity of enzymes from secondary metabolite pathways for the production of natural products and their analogs for pharmaceutical applications. One of the main areas of interest is the identification, characterization and the engineering of Rieske non-heme iron oxygenases for their application in biocatalytic reactions towards pharmaceuticals and/or precursors thereof. Along this line, the outstanding reactivities of these biocatalysts are exploited in synthetic metabolic pathways and (chemo)enzymatic cascade reactions for the production of complex compounds from simple precursors. The Schmidt group is thereby using state-of-the-art synthetic biology tools for the design and assembly of artificial metabolic pathways, and is developing new methodologies for the genetic engineering of several chassis strains, including autotrophic bacteria such as Cupriavidus necator. Finally, the Schmidt group aims at the development of new concepts for electron transfer pathways in microorganisms, and is developing concepts to optimize electron transfer chains in multi-component oxidoreductases. By expanding our knowledge on the parameters determining efficient electron transfer and thus catalysis, it is expected that this will increase the broad applicability of multi-component oxidoreductases relying on complex electron transfer mechanisms.
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. Nature developed several strategies to transfer electrons over long distances along chains of closely spaced redox relays: iron-sulfur clusters, copper centers and hemes in respiratory enzymes. Biological electron-transfer proteins can be placed into several categories according to the complexity of the reaction and the structural architecture. The simplest ones are the ferredoxins. Ferredoxins are one-domain one-electron carrier proteins in which the electron resides transiently on an iron-sulfur cluster. These electron carriers are small proteins and act in diverse biochemical processes as universal electron carrier.
The applicability of enzymes depending on an electron transfer chain involving ferredoxins remains to be 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 to increase the applicability of ROs.
Photobiocatalysis
Whole-cell photobiocatalysis:
Due to their dependency on complex electron transport chains as well as to provide an efficient in situ cofactor regeneration, the majority of synthetic applications of Rieske oxygenases relies on recombinant whole-cell catalysts. In synthetic applications, the nicotinamide cofactors are recycled by using energy-rich organic molecules as electron donors. In most cases, only a small fraction of the electrons provided by these sacrificial co-substrates is utilized, resulting in a poor atom efficiency. In order to solve this challenge, many alternative solutions are currently under consideration. Linking photochemistry to enzymes in vitro for cofactor regeneration is one of the possible approaches. On the other hand, introducing artificial photosynthesis in heterotrophic bacteria such as E. coli offers the advantage of utilizing a genetically easy-to-manipulate organism along with the capability of producing high amounts of soluble protein within the cells. Additionally, these systems are less prone to the inhibiting effects of self-shading at high cell densities compared to photoautotrophic organisms.
We are interested in coupling a well-established whole-cell system based on E. coli via light-harvesting complexes to RO-catalyzed hydroxylations in vivo. We explore the boundaries of E. coli as chassis for artificial photosynthesis, and due to the complex electron transfer chain in ROs, we strive to simplify these electron supply chains by this light-driven approach. The simplification of the overall electron transfer system could potentially improve the overall yields, thus paving the way for developing cost-effective, efficient and sustainable processes for RO-catalyzed reactions.
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.
Synthetic metabolic pathways
Synthetic biology applies the principles of engineering to biology in order to create biological functionalities not seen before in nature. One of the most exciting applications of synthetic biology is the design of new organisms with the ability to produce valuable chemicals, including pharmaceuticals and biomaterials in a greener, sustainable fashion. Nature has developed a very effective system of cascade reactions to enable metabolic pathways and thus life. The living cell can basically be regarded as a single-vessel system in which the interconnection of various multi-stage reactions with enzymes in the aqueous medium is made possible. The cellular metabolism clearly demonstrates the benefit of multi-stage cascade reactions: although no separation and processing of the different intermediates takes place, selective product formation occurs. When the concept is transferred to organic or biotechnological syntheses, time, costs and waste are saved compared to a step-by-step process by eliminating the need to isolate the intermediate products.
We strive to exploit the outstanding reactivities of Rieske oxygenases and other biocatalysts in synthetic metabolic pathways for the production of pharmaceuticals and/or precursors thereof. In order to shorten development times of in vivo cascade reactions, we aim to develop efficient synthetic biology tools for the rapid modular assembly of synthetic enzyme cascades and the finetuning of expression on different molecular levels to pave the way for industrial applications of designer microorganisms. We are thereby using state-of-the-art synthetic biology tools for the design and assembly of artificial metabolic pathways, and are developing new methodologies for the genetic engineering of several chassis strains.
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
ERC Starting Grant – ReCNNSTRCT
(GA no. 101075934)
NWO Talent Programme – Vidi
(VI.VIDI.213.025)
NWO ENW – XS
(OCENW.XS22.1.044)
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 fellowship Yongxin Li)