Biology subjects

Beliaev, A.

Publications and source records attributed to Beliaev, A..

4 recordsLinked to original sources

bioRxiv2026

Lanthanide protein biosensors with a single ion-binding site

Rising demand for rare earth elements, including lanthanides (Lns), has intensified environmental pressures and supply-chain vulnerabilities, motivating the development of bio-based methods for their extraction and separation. However, the lack of high-throughput assays for analysing the selectivity of lanthanide-binding proteins remains a key bottleneck in engineering bio-based Ln-extraction systems. Here, we report the development of high-throughput assays based on Ln-responsive protein biosensors. These {beta}-lactamase-based biosensors contain receptors with a single Ln-binding site derived from either lanmodulin or the AI-designed protein RF2. We established multiplexed colourimetric assays that quantify biosensor activity and selectivity in vitro and in the periplasm of E. coli. We further demonstrate that E. coli cells expressing these biosensors exhibit Ln-dependent survival in the presence of {beta}-lactam antibiotics. These platforms enable large-scale testing of Ln biosensors and Ln-binding proteins.

synthetic biology↗

bioRxiv2026

ChatGEM: An Agentic Architecture Enabling Interactive Simulation of Genome-Scale Metabolic Models

Genome-scale metabolic models (GEMs) are powerful tools for predicting cellular phenotypes and guiding microbial strain engineering, yet broad adoption remains challenging due to the computational expertise required. To overcome that, we present ChatGEM, an agentic platform that enables interactive GEM simulation through natural language. Built on the multi-agent ADEPT framework, ChatGEM integrates COBRApy within a retrieval-augmented generation (RAG) architecture that coordinates code generation and execution through specialized agents. Benchmarking across three tasks of increasing complexity showed that RAG-enabled code generation improved the mean overall performance score from 2.63 to 4.20 while reducing the execution time significantly starting from routine to complex tasks. Application of ChatGEM using an enzyme-constrained GEM (ecGEM) for four engineered Pseudomonas putida KT2440 strains identified the constitutive strain as the optimal chassis for succinate overproduction using a succinate leakage index - a prediction observed experimentally. Therefore, ChatGEM democratizes metabolic modeling by enabling researchers without computational expertise to perform sophisticated GEM-based analyses through natural language, and, hence, accelerating scientific discovery.

systems biology↗

bioRxiv2025

Cellulase secretion by engineered Pseudomonas putida enables growth on cellulose oligomers.

Pseudomonas putida is an attractive synthetic biology platform organism for chemical synthesis from low-grade feedstocks due to its high tolerance to chemical solvents and lignin-derived small molecules that are often inhibitory to other biotechnologically relevant microorganisms. However, there are few molecular tools available for engineering P. putida and other gram-negative bacteria to secrete non-native enzymes for extracellular feedstock depolymerisation. In this study P. putida was transformed to secrete cellulase enzymes and evaluated for growth on polymeric or oligomeric cellulose substrates. Active exo- and endocellulase enzymes were secreted into the culture supernatant, and a preferred set of twin-arginine translocase secretion signal peptides were identified. Extracellular cellulase activity was sufficient to support growth of P. putida using cellotriose or cellotetraose as the sole source of carbon and energy. This work supports progress towards consolidated bioprocessing of cellulosic materials using P. putida, and advances the state of engineered protein secretion in gram negative bacteria. Key PointsO_LIEngineered Pseudomonas putida secreted cellulase enzymes into the culture medium C_LIO_LICellulase activity was sufficient to support growth on cellulose oligomers C_LI

synthetic biology↗

bioRxiv2024

Carbon-conserving Bioproduction of Malate in an E. coli-based Cell-Free System

Formate, a biologically accessible form of CO2, has attracted interest as a renewable feedstock for bioproduction. However, approaches are needed to investigate efficient routes for biological formate assimilation due to its toxicity and limited utilization by microorganisms. Cell-free systems hold promise due to their potential for efficient use of carbon and energy sources and compatibility with diverse feedstocks. However, bioproduction using purified cell-free systems is limited by costly enzyme purification, whereas lysate-based systems must overcome loss of flux to background reactions in the cell extract. Here, we engineer an E. coli-based system for an eight-enzyme pathway from DNA and incorporate strategies to regenerate cofactors and minimize loss of flux through background reactions. We produce the industrial di-acid malate from glycine, bicarbonate, and formate by engineering the carbon-conserving reductive TCA and formate assimilation pathways. We show that in situ regeneration of NADH drives metabolic flux towards malate, improving titer by 15-fold. Background reactions can also be reduced 6-fold by diluting the lysate following expression and introducing chemical inhibitors of competing reactions. Together, these results establish a carbon-conserving, lysate-based cell-free platform for malate production, producing 64 M malate after 8 hours. This system conserves 43% of carbon otherwise lost as CO2 and incorporates 0.13 mol CO2 equivalents/mol glycine fed. Finally, techno-economic analysis of cell-free malate production from formate revealed that the high cost of lysate is a key challenge to the economic feasibility of the process, even assuming efficient cofactor recycling. This work demonstrates the capabilities of cell-free expression systems for both the prototyping of carbon-conserving pathways and the sustainable bioproduction of platform chemicals. HighlightsO_LISuccessfully engineered the carbon-conserving reductive TCA and formate assimilation pathways in a lysate-based cell-free system for production of the C4 industrial di-acid malate from C1 and C2 feedstocks. C_LIO_LIAchieved a 6-fold reduction in competition from the endogenous cell-free metabolism by blocking TCA activity using small-molecule inhibitors and lysate dilution. C_LIO_LIIncreased accumulation of malate by 15-fold in a single-step reaction using cell-free expression of an enzymatic cofactor regeneration system. C_LIO_LITechno-economic analysis identified routes for economically feasible production of malate from renewable feedstocks in a cell-free system by improving conversion efficiency and reducing lysate cost. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/623433v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@3c89adorg.highwire.dtl.DTLVardef@72d9b3org.highwire.dtl.DTLVardef@327e2eorg.highwire.dtl.DTLVardef@fa0964_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗