Jan Ježek

Enzyme cascades enable complex biochemical transformations, but optimizing them requires searching high-dimensional spaces of enzyme stoichiometry, reaction conditions, and buffer composition. Here we introduce CascadeMAP, an autonomous microfluidic platform for closed-loop optimization of enzyme cascades, integrating high-throughput microfluidics with Bayesian optimization and assisted by agentic analyses. We demonstrate the platform across two three-enzyme cascades: (i) a glycerol detection pathway monitored by fluorescence and (ii) a 1,2,3-trichloropropane degradation pathway monitored by label-free Raman spectroscopy, providing orthogonal detection modalities. Bayesian optimization reached comparable optima with threefold fewer experimental conditions than Design of Experiments. A multi-agent AI system automated processing 2.1 GB of experimental data, pattern recognition, and insight synthesis. Operating without human intervention for 7 days, CascadeMAP processed >295,000 reactions across ∼7,400 different conditions. This capability establishes a generalizable, resource-efficient framework for autonomously optimizing enzyme cascades and metabolic pathways and accelerates the development of biocatalytic and synthetic biological systems.

Reliable Raman spectroscopy (RS) and surface-enhanced Raman spectroscopy (SERS) of small, thin, transparent, or translucent samples requires careful selection of the substrate to minimize parasitic background contributions. In this study, we present a systematic comparison of some commonly used substrates under excitation at 532 nm and 785 nm. The evaluated substrates include standard glass and quartz microscope slides, lab grade aluminum (Al) foil, commercial Al-adhesive tape, custom-made Al-plate and polished monocrystalline calcium fluoride. Using a commercial Raman microspectroscope, we measured the raw background spectra of these substrates and compared them to the baseline spectrum of the instrument alone. We then quantified the differences using the sum of Raman signal across the complete spectral interval. We also isolated and compared the spectral contributions of these substrates while testing them with model analytes for RS and SERS. The calculations were performed by a custom baseline projection Python script for substrate evaluation using the Peakiness Index, and the useful Signal-to-Parasite Ratio. The results demonstrate that inexpensive Al-foil provides excellent performance, offering low background and stable optical behavior across both excitation wavelengths. Al-adhesive tapes, while comparable in performance, exhibited higher variability of background signals highly dependent on the brand, attributable mainly to the differences in reflectivity. This work provides a robust methodological framework for evaluating substrate-induced artifacts and offers experimentally validated recommendations for selecting cost-effective, low-background materials suitable for high-quality Raman spectroscopic analyses.

The creation of bioenergy based on the biomass wood pellet industry, which accounts for the majority of the global biomass supply, is one of the most common and important ways to utilize waste wood, wood dust, and other byproducts of wood manufacturing, known as forestry residues. Pellet production processes might greatly benefit from fast monitoring systems that may allow for at least a semi-quantitative measurement of crucial parameters such as lignin and cellulose. The determination of lignin and cellulose is complicated and time-consuming because it usually requires time-demanding and labor-intensive sample preparation. This, however, might be a crucial problem. In this context, the application of Raman spectroscopic techniques is considered a promising approach, as it enables rapid, reliable, and label-free analysis of wood pellets, providing information about the chemical composition of the biomass, specifically lignin and cellulose. The purpose of this article is to report on the application of Raman spectroscopy exemplified by the detection of the lignin/cellulose ratio. In our methodological approach, we integrated the area under the selected Raman bands to avoid a large scatter of data when only the intensities of the bands were used. Moreover, the acquired Raman spectra displayed very strong signals from both substances, which contributes to the feasibility of the analysis even with a portable instrument. This study is expected to be of assistance in situations when the monitoring of the chemical changes and the quick inspection of pellets are required in near real time, online, and in situ.

This pilot study investigated the metabolic responses of five selected bacteria to physiological stress. Surface-enhanced Raman spectroscopy was used to analyze spectral changes associated with the release of adenine, a key metabolite indicative of stress conditions. Laboratory-synthesized spherical silver and gold nanoparticles, which remained stable over an extended period, were employed as enhanced surfaces. Bacterial cultures were analyzed under standard conditions and in the presence of a selected stressor—demineralized water—inducing osmotic stress. The results showed that the adenine signal originated from metabolites released into the surrounding environment rather than directly from the bacterial cell wall. The study confirms the suitability of these cost-effective and easily synthesized stable nanoparticles for the qualitative detection of bacterial metabolites using a commercially available Raman instrument.

Correlative imaging, integrating diverse observational methods, has become increasingly vital for comprehensive sample analysis by linking distinct datasets that are accessible only through individual techniques. This study combines cryogenic scanning electron microscopy (cryo-SEM) and cryogenic Raman micro-spectroscopy (cryo-Raman), which together offer extensive insight into sample analysis. Cryo-SEM effectively investigates the surface, structure, and morphology, while cryo-Raman excels in detecting and identifying chemical composition. To integrate these techniques, we developed a novel assembly compatible with commercial cryo-SEM sample holders. The assembly is designed for precise sample positioning and observation at cryogenic temperatures of liquid nitrogen. This setup, controlled by LabVIEW software, includes a Dewar vessel containing liquid nitrogen, a 3D-printed stage for mounting sample holders, and a piezoelectric stage for XY motion, with z axis adjustments connected to the Raman spectrometer table. A plexiglass cover minimizes contamination and preserves sample integrity during analysis. The assembly was validated by first analyzing polystyrene at cryogenic temperatures to ensure its functionality. Subsequent tests on Cupriavidus necator H16—bacteria capable of producing polyhydroxyalkanoates (PHAs), a type of biodegradable polyester—demonstrated the system’s efficiency. PHAs, which are of interest as a sustainable alternative to petrochemical plastics, can be produced from industrial waste streams. Our results show that the combination of cryo-SEM and cryo-Raman is suitable for studying these microorganisms and permits a deeper understanding of the properties of polymer granules in microbial cells. This integrated approach represents a significant advance in semi-correlative imaging, providing more efficient and detailed analyses for future research in microbial physiology production and related fields.

Genetic screens have been used extensively to probe interactions between nuclear genes and their impact on phenotypes. Probing interactions between mitochondrial genes and their phenotypic outcome, however, has not been possible due to a lack of tools to map the responsible polymorphisms. Here, using a toolkit we previously established in Drosophila, we isolate over 300 recombinant mitochondrial genomes and map a naturally occurring polymorphism at the cytochrome c oxidase III residue 109 (CoIII¹⁰⁹) that fully rescues the lethality and other defects associated with a point mutation in cytochrome c oxidase I (CoI^T300I). Through lipidomics profiling, biochemical assays and phenotypic analyses, we show that the CoIII¹⁰⁹ polymorphism modulates cardiolipin binding to prevent complex IV instability caused by the CoI^T300I mutation. This study demonstrates the feasibility of genetic interaction screens in animal mitochondrial DNA. It unwraps the complex intra-genomic interplays underlying disorders linked to mitochondrial DNA and how they influence disease expression.

Mitochondria are dynamic organelles that undergo frequent remodeling to accommodate developmental needs. Here, we describe a striking organization of mitochondria into a large ball-like structure adjacent to the nucleus in premeioticDrosophila melanogasterspermatocytes, which we term “mitoball”. Mitoballs are transient structures that colocalize with the endoplasmic reticulum, Golgi bodies, and the fusome. We observed similar premeiotic mitochondrial clusters in a wide range of insect species, including mosquitos and cockroaches. Through a genetic screen, we identified that Milton, an adaptor protein that links mitochondria to microtubule-based motors, mediates mitoball formation. Flies lacking a 54 amino acid region in the C terminus of Milton completely lacked mitoballs, had swollen mitochondria in their spermatocytes, and showed reduced male fertility. We suggest that the premeiotic mitochondrial clustering is a conserved feature of insect spermatogenesis that supports sperm development.

Efficient separation and sensitive identification of pathogenic bacterial strains is essential for a prosperous modern society, with direct applications in medical diagnostics, drug discovery, biodefense, and food safety. We developed a fast and reliable method for antibody-based selective immobilization of bacteria from suspension onto a gold-plated glass surface, followed by detection using strain-specific antibodies linked to gold nanoparticles decorated with a reporter molecule. The reporter molecules are subsequently detected by surface-enhanced Raman spectroscopy (SERS). Such a multi-functionalized nanoparticle is called a SERS-tag. The presented procedure uses widely accessible and cheap materials for manufacturing and functionalization of the nanoparticles and the immobilization surfaces. Here, we exemplify the use of the produced SERS-tags for sensitive single-cell detection of opportunistic pathogen Escherichia coli, and we demonstrate the selectivity of our method using two other bacterial strains, Staphylococcus aureus and Serratia marcescens, as negative controls. We believe that the described approach has a potential to inspire the development of novel medical diagnostic tools for rapid identification of bacterial pathogens.