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Biology subjects

Warden, A. C.

Publications and source records attributed to Warden, A. C..

4 recordsLinked to original sources

Data-Efficient Exploration of Enzyme Function Using Family-Specific Machine Learning

Enzymes are essential biocatalysts across diverse industries, driving demand for high-performing variants. Foundation models are attractive for guiding enzyme discovery, but often lack the resolution to model subtle variations driving function within homologous families. Navigating these rugged functional landscapes to identify elite variants remains challenging and experimentally costly, even when guided by such models. Here we show that coupling dense, family-specific experimental screening with targeted, sequence-based deep learning provides a data-efficient discovery strategy. We experimentally screened 1,513 natural homologues from an esterase superfamily (>7,500 assays) and used this functional landscape to train task-specific models that predict activity, thermostability, and substrate specificity from sequence alone. Prospective experimental validation of previously untested sequences demonstrated that these task-specific models significantly outperformed generalist pre-trained and physics-based models in enriching for target traits. Residue-level attribution further indicated that the models captured sequence patterns consistent with underlying structural features. Finally, retrospective simulations showed that iterative retraining compresses the search space, discovering 60% of top-tier hits using nearly half the samples required by pre-trained baseline models. Together, these results highlight that machine learning can provide mechanistic insight, and that integrating targeted data acquisition with iterative machine learning provides a more data-efficient discovery strategy than relying on generic model scale.

bioengineering↗

MMINT: a Metabolic Model Interactive Network Tool for the exploration and comparative visualisation of metabolic networks

Genome-scale metabolic models (GEMs) are essential tools in systems and synthetic biology, enabling the mathematical simulation of metabolic pathways encoded in genomes to predict phenotypes. The complexity of GEMs, however, can often limit the interpretation and comparison of their outputs. Here, we present MMINT (Metabolic Modelling Interactive Network Tool), designed to facilitate the exploration and comparison of metabolic networks. MMINT employs GEM networks and flux solutions derived from Constraint Based Analysis (e.g. Flux Balance Analysis) to create interactive visualizations. This tool allows for seamless toggling of source and target metabolites, network decluttering, enabling exploration and comparison of flux solutions by highlighting similarities and differences between metabolic states, which enhances the identification of mechanistic drivers of phenotypes. We demonstrate MMINTs capabilities using the Pyrococcus furiosus GEM, showcasing its application in distinguishing the metabolic drivers of acetate- and ethanol-producing phenotypes. By providing an intuitive and responsive model-exploration experience, MMINT addresses the need for a tool that simplifies the interpretation of GEM outputs and supports the discovery of novel metabolic engineering strategies. MMINT is available at https://doi.org/10.6084/m9.figshare.26409328 Graphical abstractMMINT functionalities provide an intuitive and responsive model-exploration experience, enabling flux solution comparison and the identification of metabolic drivers of phenotypes O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/606923v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@163d42eorg.highwire.dtl.DTLVardef@ff2922org.highwire.dtl.DTLVardef@1e5881aorg.highwire.dtl.DTLVardef@4a8bf0_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Engineering a functional NifDK polyprotein resistant to mitochondrial degradation

To engineer Mo dependent nitrogenase function in plants expression of proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in E. coli identified a subset that retained function, including a single amino acid (Y100Q) variant. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in E. coli based nitrogenase assays, demonstrating this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

synthetic biology↗

Engineered Enzymes that Retain and Regenerate their Cofactors Enable Continuous-Flow Biocatalysis

Introduction Introduction Results Conclusions Author Contributions Competing Interests Statement General DNA manipulation G3PDEc NoxCa GlpKTk AceKMs GlpKTk-AceKMs G3PDEc-NOXCa GlpKTk-AceKMsE2Aa G3PDEc-NOXCa-Est2Aa FruASc-Est2Aa Engineered Fusion Protein... Expression of individual enzymes... Expression of GlpKTk-AceKMs-E2Aa Expression and purification of... Expression and purification of... Protein Expression and... Synthesis of N6-2AE-ADP ...

synthetic biology↗