Biology subjects

Madsen, N. G.

Publications and source records attributed to Madsen, N. G..

5 recordsLinked to original sources

bioRxiv2026

Biocatalytic Production of Galantamine in Yeast through Cytochrome P450 Optimization

Galantamine is a pharmaceutically relevant Amaryllidaceae alkaloid used for the treatment of Alzheimer's disease. Its structural complexity and low abundance in plants motivate the development of alternative manufacturing routes. Here, we established the first engineered yeast platform for the biocatalytic production of galantamine from 4OMe-norbelladine. Heterologous expression of the downstream pathway enzymes NtCYP96T6, NtNMT1, and NtAKR1 from Narcissus cv. Tete-a-Tete in Saccharomyces cerevisiae was complemented by optimization of cultivation temperature, carbon source, and medium pH enabling the first demonstration of galantamine production in yeast. Systematic screening of cytochrome P450 reductase and cytochrome b partners to boost NtCYP96T6 activity led to the identification of a novel reductase mined from the Narcissus pseudonarcissus transcriptome, NpCPR, which supported the highest pathway flux and yielded 7.0 {+/-} 0.6 mg/L galantamine, corresponding to a 7.6 % molar yield from 250 M 4OMe-norbelladine and an approximately 173-fold improvement over the parental strain. We further exploited this yeast cell factory for the precursor-directed biosynthesis of 7F-galantamine, highlighting the potential of pathway enzyme promiscuity to access new-to-nature GAL analogues that may be challenging to produce through conventional chemical synthesis. Together, this work establishes a foundation for microbial galantamine production and biosynthetic diversification of its pharmaceutically relevant scaffold.

synthetic biology↗

bioRxiv2026

Engineering Biosensors to Enhance Monoterpene Indole Alkaloid Production in Yeast

Monoterpene Indole Alkaloids (MIAs) are a diverse family of plant natural products with various medicinal applications. Although MIAs, such as vinblastine and reserpine, are clinically validated, sourcing of MIAs for clinical use or drug discovery from natural resources or via chemical synthesis is hampered due to their scarcity and chemical complexity. Refactoring MIA biosynthesis pathways in microbial cell factories could offer an alternative, more stable and potentially sustainable manufacturing route for alkaloid medicines and novel therapies. However, reaching commercially attractive titers, rates and yields remains challenging owing to the length and complexity of these metabolic pathways. One critical bottleneck is the low screening throughput and very high cost of the analytical methods used to quantify MIA for optimizing production. In this study, we evolved RamR, a promiscuous bacterial transcription factor to respond to five different MIAs, resulting in highly sensitive and selective sensor variants (EC50<10 M). X-ray crystallography and computational modeling provided insight into the MIA binding of the evolved biosensor variants. The RamR biosensing platform was functionalized in yeast and subsequently applied in a cost-effective semi-throughput screening campaign of a 188-gene overexpression library to identify high-performing cell factory designs for strictosidine, the common precursor for all MIAs. The fluorescent biosensor signal correlated with HPLC quantification (r2 = 0.932) allowing identification of single metabolic engineering hits which when combined yielded a maximum titer of >220 mg/L strictosidine, 3-fold higher than the parental reference strain. This study demonstrates the development of selective biosensors for MIAs and the cost-effective identification of novel metabolic engineering hits for optimizing MIA production in microbial cell factories.

synthetic biology↗

bioRxiv2024

ProteusAI: An Open-Source and User-Friendly Platform for Machine Learning-Guided Protein Design and Engineering

AO_SCPLOWBSTRACTC_SCPLOWProtein design and engineering are crucial for advancements in biotechnology, medicine, and sustainability. Machine learning (ML) models are used to design or enhance protein properties such as stability, catalytic activity, and selectivity. However, many existing ML tools require specialized expertise or lack open-source availability, limiting broader use and further development. To address this, we developed ProteusAI, a user-friendly and open-source ML platform to streamline protein engineering and design tasks. ProteusAI offers modules to support researchers in various stages of the design-build-test-learn (DBTL) cycle, including protein discovery, structure-based design, zero-shot predictions, and ML-guided directed evolution (MLDE). Our benchmarking results demonstrate ProteusAIs efficiency in improving proteins and enyzmes within a few DBTL-cycle iterations. ProteusAI democratizes access to ML-guided protein engineering and is freely available for academic and commercial use. Future work aims to expand and integrate novel methods in computational protein and enzyme design to further develop ProteusAI.

bioinformatics↗

bioRxiv2024

Harnessing Chemical Space Neural Networks to Systematically Annotate GPCR ligands

Machine learning (ML) has revolutionized drug discovery by enabling the exploration of vast, uncharted chemical spaces essential for discovering novel patentable drugs. Despite the critical role of human G protein-coupled receptors (hGPCRs) in FDA-approved drugs, exhaustive in-distribution drug-target interaction (DTI) testing across all pairs of hGPCRs and known drugs is rare due to significant economic and technical challenges. This often leaves off-target effects unexplored, which poses a considerable risk to drug safety. In contrast to the traditional focus on out-of-distribution (OOD) exploration (drug discovery), we introduce a neighborhood-to-prediction model termed Chemical Space Neural Networks (CSNN) that leverages network homophily and training-free graph neural networks (GNNs) with Labels as Features (LaF). We show that CSNNs ability to make accurate predictions strongly correlates with network homophily. Thus, LaFs strongly increase a ML models capacity to enhance in-distribution prediction accuracy, which we show by integrating labeled data during inference. We validate these advancements in a high-throughput yeast biosensing system (3773 DTIs, 539 compounds, 7 hGPCRs) to discover novel DTIs for FDA-approved drugs and to expand the general understanding of how to build reliable predictors to guide experimental verification.

bioinformatics↗

bioRxiv2023

De novo Design of a Polycarbonate Hydrolase

Enzymatic degradation of plastics is currently limited to the use of engineered natural enzymes. As of yet, all engineering approaches applied to plastic degrading enzymes retain the natural /{beta} -fold. While mutations can be used to increase thermostability, an inherent maximum likely exists for the /{beta} -fold. It is thus of interest to introduce catalytic activity toward plastics in a different protein fold to escape the sequence space of plastic degrading enzymes. Here, a method for designing highly thermostable enzymes that can degrade plastics is described. This has been used to design an enzyme that can catalyze the hydrolysis of polycarbonate, which no known natural enzymes can degrade. Rosetta enzyme design is used to introduce a catalytic triad into a set of thermostable scaffolds. Through computational evaluation, a potential PCase was selected and produced recombinantly in E. coli. CD spectroscopy suggests that the design has a melting temperature of >95{degrees}C. Activity towards a commercially used polycarbonate (Makrolon 2808) was confirmed using AFM, which showed that a PCase had been designed successfully. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/532063v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1369436org.highwire.dtl.DTLVardef@3c87b3org.highwire.dtl.DTLVardef@1f1138forg.highwire.dtl.DTLVardef@3b4bba_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗