Biology subjects

Kueffner, A. M.

Publications and source records attributed to Kueffner, A. M..

3 recordsLinked to original sources

bioRxiv2026

Form IF Rubiscos include highly active, specific, and small subunit-independent enzymes.

Plant-type (Form I) Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) suffers from inherent catalytic trade-offs and a strong dependency on other proteins--including an essential small subunit (SSU) and auxiliary chaperones--for assembly, constraining the enzymes evolutionary and engineering potential. Here, we investigated representatives from the newly discovered clade Form IF. These enzymes do not require specific chaperones to form functional complexes, exhibit high CO2-specificities (SC/O [~]50) while maintaining high turnover rates (up to kcat [~]11 s-1). Remarkably, two Form IF representatives (IF-1/IF-2) lost the dependency on the SSU and assemble into homo-octameric complexes without their cognate SSUs. While the SSU is not necessary for catalysis, its addition improves both activity and specificity in IF-1/IF-2. Our results show that complexity is actually not required to achieve highly active, specific and functional Rubisco variants--and that this complexity can even be reverted--which challenges our current thinking on the evolution and catalytic mechanism of Rubisco.

biochemistry↗

bioRxiv2024

Expanding the biotechnological scope of metabolic sensors through computation-aided designs

Metabolic sensors are microbial strains modified so that biomass formation correlates with the availability of specific metabolites. These sensors are essential for bioengineering (e.g. in growth-coupled designs) but creating them is often a time-consuming and low-throughput process that can potentially be streamlined by in silico analysis. Here, we present the systematic workflow of designing, implementing, and testing versatile Escherichia coli metabolic sensor strains. Glyoxylate, a key metabolite in (synthetic) CO2 fixation and carbon-conserving pathways, served as the test molecule. Through iterative screening of a compact metabolic model, we identified non-trivial growth-coupled designs that resulted in six metabolic sensors with a wide sensitivity range for glyoxylate, spanning three orders of magnitude in detected concentrations. We further adapted these E. coli strains for sensing glycolate and demonstrated their utility in both pathway engineering (testing a key metabolic module via glyoxylate) and applications in environmental monitoring (quantifying glycolate produced by photosynthetic microalgae). The versatility and ease of implementation of this workflow make it suitable for designing and building multiple metabolic sensors for diverse biotechnological applications. TeaserA streamlined workflow enables the rapid design of versatile E. coli metabolic sensors for detecting key metabolites in bioengineering and monitoring.

synthetic biology↗

bioRxiv2022

The interface of condensates of the hnRNPA1 low complexity domain promotes formation of amyloid fibrils

The maturation of liquid-like protein condensates into amyloid fibrils has been associated with neurodegenerative diseases. Here, we analyze the amyloid formation mediated by condensation of the low-complexity domain of hnRNPA1, a protein involved in Amyotrophic Lateral Sclerosis (ALS). We show that phase separation and fibrillation are connected but distinct processes which are independently mediated by different regions of the protein sequence. By monitoring the spatial and temporal evolution of amyloid formation we demonstrate that the formation of fibrils does not occur homogeneously inside the droplets but is promoted at the interface of the condensates. Consistently, we further show that coating the interface of the droplets with surfactant molecules inhibits fibril formation. Our results indicate that the interface of biomolecular condensates can act as an important catalyst for fibril formation, and therefore could represent a possible therapeutic target against the formation of aberrant amyloids mediated by condensation.

biophysics↗