Biology subjects

Liese, A.

Publications and source records attributed to Liese, A..

3 recordsLinked to original sources

bioRxiv2026

Electrostatic Engineering of Phosphoketolase Enhances Activity on Small Non-phosphorylated Sugars and Improves Cell-Free ATP Regeneration from Inexpensive C2-Substrates

Phosphoketolases can be used to convert non-phosphorylated sugars to the high energy compound acetyl phosphate and the versatile metabolic precursor acetyl-CoA. The performance of these pathways is limited by low catalytic activity of natural phosphoketolases towards these sugars. Here, we report the rational engineering of the phosphoketolase from Bifidobacterium adolescentis (Bad.F6Pkt) to enhance its activity and affinity towards glycoaldehyde (GA) and D-erythrulose (ERU) through re-organisation of the protein electric field to reproduce the role of terminal phosphate groups in cognate substrates. Guided by predicted induced side-chain pKa shifts, visualisation of electrostatic potential difference maps alongside molecular modelling and sequence variation analyses, we identified mutations that could promote in situ ring opening of the pre-dominant cyclic GA dimer form in solution. This approach to the electrostatic inverse design problem yielded the GA-specific double mutant H142N:E153D, exhibiting a ten-fold improved affinity and slightly enhanced catalytic efficiency (KM = 4.4 mM, kcat/ KM = 26.3 s-1 M-1) compared to the previously reported H142N variant (KM = 42.3 mM, kcat/ KM = 20.6 s-1 M-1). We additionally constructed a H256Y:H260Y:H548Y variant comprising long-range electrostatic mutations with a 3.8-fold increased catalytic efficiency (kcat/ KM = 49.6 s-1 M-1) on the acylic four-carbon ERU ketose compared to the wild-type enzyme. The engineered enzymes were evaluated in cell-free enzyme cascades for ATP regeneration via acetyl phosphate formation. The H142N variant enabled efficient ATP regeneration from GA and ethylene glycol, whereas the H142N:E153D mutant exhibited reduced stability under synthesis conditions. Furthermore, coupling of a highly GA-specific D-threose aldolase and a D-threose isomerase with the PKT triple mutant enabled rapid conversion of GA into C4 sugar intermediates and significantly improved ATP regeneration from GA.

synthetic biology↗

bioRxiv2025

A Combined Chemo-Enzymatic Treatment for the Oxidation of Epoxy-Based Carbon Fiber-Reinforced Polymers (CFRPs)

Carbon fiber-reinforced polymers (CFRPs), particularly epoxy-based composites, have become essential in the aerospace, automotive, and wind energy industries due to their robust mechanical properties, and lightweight nature. However, there is a lack of recycling technologies that are environmentally sustainable while also ensuring the recovery of carbon fibers in their original state. Although certain bacterial and fungal strains can colonize epoxy polymers, enzymes capable of efficiently degrading these materials have not yet been reported. Consequently, there is an urgent need for an effective, sustainable, and biologically inspired solution for CFRP recycling. Here, a chemo-enzymatic two-step oxidation process was developed. A chemical pre-treatment with propionic acid and hydrogen peroxide was used to recover imbedded carbon fibers. Additionally, three novel bacterial laccases isolated from the European spruce bark beetle (Ips typographus) demonstrated the ability to degrade three epoxy resin scaffolds from Hexflow(R) RTM6, used in aircraft applications. The sequential combination of both oxidative steps enabled the retrieval of clean carbon fibers and partial modification of epoxy functional groups, with the release of defined products over time. This bio-inspired approach renders the process more environmentally friendly and marks an initial step toward developing a bio-based recycling method for epoxy CFRPs.

biochemistry↗

bioRxiv2023

Imaging of plant calcium-sensor kinase conformation monitors real time calcium decoding in planta

Changes in cytosolic calcium concentration are among the earliest reactions to a multitude of stress cues. Whereas a plethora of calcium-permeable channels may generate distinct calcium signatures and contribute to response specificities, the mechanisms by which calcium signatures are decoded is poorly understood. Here we develop a genetically encoded, FRET-based reporter that visualizes the conformational change of calcium-dependent protein kinases (CDPKs/CPKs), preceding kinase activation, for calcium-dependent AtCPK21 and calcium-independent AtCPK23. In pollen tubes, naturally displaying a physiological calcium range, CPK21-FRET, but not CPK23-FRET, report activity oscillations with similar features to cytosolic calcium, suggesting an isoform-specific calcium dependency and reversibility of the conformational change. In guard cells CPK21-FRET identifies CPK21 as a decoder of signal-specific calcium signatures in response to ABA and flg22. Based on this data, CDPK-FRET stands as a novel approach for tackling real-time live-cell calcium decoding in a multitude of plant developmental and stress responses.

biochemistry↗