Biology subjects

De Mey, M.

Publications and source records attributed to De Mey, M..

6 recordsLinked to original sources

bioRxiv2026

Systematic mapping of orthogonality and domain-swap permissiveness across LysR-type transcriptional biosensors

Transcription factor-based biosensors monitor metabolites and control genetic programs, but their wider use is constrained by the limited repertoire of characterized, mutually compatible sensor parts. Here we combine a curated screen of natural LysR-type transcriptional regulators (LTTRs), the largest family of bacterial transcription factors, with systematic domain swapping. Using a standardized construction platform, we convert 17 LTTRs into whole-cell reporters in Escherichia coli. Of 16 viable circuits, nine show regulatory activity, including six ligand-inducible biosensors for acetate, benzoate, -ketoglutarate, chlorohydroquinone, L-homocysteine and salicylate. Mapping interactions across 11 LTTR systems identifies seven mutually orthogonal regulator pairs, providing, to our knowledge, the first orthogonality map for this family. We next construct 108 chimeras across three domain-swap architectures; 69 retain measurable activity, with functional outcomes enriched when the native hinge-ligand-binding-domain association is preserved. As proof of principle, we redesign a cross-reactive regulator: replacing its DNA-binding domain with one from an orthogonal regulator abolishes unwanted promoter crosstalk while preserving ligand-inducible activation of its own target, transferring orthogonality to a previously incompatible pair. Together, natural-diversity screening and domain swapping emerge as complementary routes to expand LTTR biosensor repertoires, revealing a strong link between connector architecture and chimera function.

synthetic biology↗

bioRxiv2025

Development and characterisation of a promoter library for Sulfolobus acidocaldarius

The hyperthermoacidophilic archaeon Sulfolobus acidocaldarius is a promising production host for industrial biotechnology applications due to its ability to thrive in extreme conditions. However, the lack of well-characterised genetic parts and tools, particularly promoters, limits its potential for metabolic engineering. In this study, we developed the first promoter library for S. acidocaldarius by randomising specific regions of the core promoter sequence of PSaci 2137, a promoter known to function in both S. acidocaldarius and Escherichia coli. The library was initially screened in E. coli using mKate2, a red fluorescent reporter protein, and seven promoters were selected for characterisation in S. acidocaldarius using the thermostable {beta}-galactosidase reporter LacS. The resulting promoter collection exhibited a 5-fold range of expression levels in S. acidocaldarius, spanning from low to high constitutive expression when compared to S. acidocaldarius promoters Psac7d and PmalE. This study demonstrates a successful workflow for generating and characterising S. acidocaldarius promoters, providing a valuable toolkit for fine-tuning gene expression and optimising metabolic pathways in this extremophilic archaeon. The design principles established here can be extended to other archaeal systems with similar promoter architectures.

synthetic biology↗

bioRxiv2025

An experimentally verified mechanistic model for predicting quorum sensing-based switches

Quorum sensing-based genetic circuits are gaining traction in synthetic biology as they link population-level behaviour to individual cell responses. However, tuning these circuits remains challenging due to complex dynamics, particularly during the Learn phase of the Design-Build-Test-Learn (DBTL) cycle. To accelerate this process, we developed a mathematical model to predict how varying expression levels of the transcription factor and synthase affect the response of the EsaI/EsaR quorum sensing system. A strain library was constructed, and experimental data were used to optimize the model. The final model could successfully differentiate between the effects of these expression levels on the response of the bidirectional promoter. It allowed visualization of all potential system outcomes and emphasized the transcription factors critical role in tuning the circuit. This model offers a valuable tool for fine-tuning EsaI/EsaR-based systems for synthetic biology applications. Moreover, given the homology within the LuxR-family quorum sensing systems, this modelling approach may serve as a foundation for model-based tuning of other quorum sensing systems.

synthetic biology↗

bioRxiv2025

Characterization and orthogonality assessment of two quorum sensing systems for synthetic biology applications

Quorum sensing systems have a broad range of applications within the field of synthetic biology. However, a bottleneck is the optimization and tuning of these systems due to the lack of standardization and complete characterization. In this research, two quorum sensing systems, namely the LasI/LasR and the EsaI/EsaR system, were fully characterized in the model host organism Escherichia coli. Furthermore, insight was gained in the interplay between the various parts of these systems. To further expand the range of possibilities with these quorum sensing systems, the orthogonality of the two systems was assessed to allow simultaneous use within the same cell without interfering crosstalk. This assessment was performed on three levels: promoter, signal and synthase crosstalk. It was demonstrated that LasR is able to interact with the promoter of the EsaI/EsaR system, albeit to a low extent. Additionally, LasR was able to respond to the autoinducers produced by EsaI. To solve the promoter crosstalk, a nucleotide change was introduced into the binding site of EsaR within the promoter region. Additionally, LasR mutants were created rationally and screened for decreased response to EsaI while retaining functionality. The best performing mutant, LasR(P117S), was further characterized. In conclusion, we have further unlocked the potential of quorum sensing systems for synthetic biology applications by obtaining two functional, characterized and orthogonal quorum sensing systems. Highlights- Characterization of two LuxR-type quorum sensing systems - Assessing the orthogonality of the EsaI/EsaR and LasI/LasR quorum sensing system - Eliminating the crosstalk between the EsaI/EsaR and LasI/LasR quorum sensing system

synthetic biology↗

bioRxiv2025

Effects of genomic location on ectopic integration and gene expression of a reporter gene cassette in Sulfolobus acidocaldarius

In eukaryotes and bacteria, it is well-established that the genomic location of ectopic gene integration influences the expression level due to replication-associated gene dosage effects as well as effects mediated by chromatin organization. In contrast, in archaea, the impact of genomic location on gene expression remained unexplored. Here, we investigated this impact in the model archaeon Sulfolobus acidocaldarius, a crenarchaeal species that has a chromatin architecture with mixed eukaryotic-like and bacterial-like features. We aimed to integrate a standardized {beta}-galactosidase (lacS) reporter cassette into diverse loci in the genome of S. acidocaldarius SK-1 for a comparative analysis. Nine integration mutant strains were successfully obtained, for which qRT-PCR analysis and lacS reporter gene assays revealed significant variation in transcriptional and translational expression of the reporter, respectively, demonstrating that genomic location strongly influences gene expression in S. acidocaldarius. However, variability in transcription levels and its regulation was shown to be primarily driven by transcriptional activity of neighboring genes, due to the high coding density in the S. acidocaldarius genome as well as a lack of insulator elements. Interestingly, translational activity exhibited a more apparent correlation with the distance to the closest origin of replication (R2 = 0.432) as compared to transcriptional activity (R2 = 0.026). In conclusion, this study not only provides insights into genome context effects, but also provides inspiration for the future design of genomic knock-in constructions in S. acidocaldarius.

microbiology↗

bioRxiv2025

Understanding bottlenecks in the microbial production of partially acetylated chitooligosaccharides

Chitooligosaccharides (COS) are versatile biomolecules with applications across food, pharmaceutical, and cosmetic industries. Expanding the COS portfolio, particularly with partially acetylated COS (paCOS) of defined degree and pattern of acetylation, is essential to unlocking their full potential. This study investigates the co-expression of chitin deacetylases (CDAs) with a chitooligosaccharide synthase (CHS) RhNodC in E. coli for in vivo paCOS production. While this approach shows promise, it is hampered by reduced overall (pa)COS yields and incomplete conversion of fully acetylated COS to paCOS. Our findings reveal that RhNodC and CDAs co-localize, suggesting potential interactions that influence production efficiency. Additionally, CDA expression induces significant stress responses, including upregulation of ibpA and cpxP promoters linked to inclusion body formation and membrane stress, respectively. This is accompanied by pronounced cellular elongation, further indicating cellular distress. These bottlenecks highlight the need for deeper exploration of RhNodC-CDA interactions and stress mitigation strategies to optimize scalable in vivo paCOS production. Highlights- Co-expression of rhizobial NodC and chitin deacetylases reduces chitooligosaccharide yield. - Conversion of COS into paCOS remains incomplete upon co-expression. - Upregulation of stress responses suggests protein misfolding and membrane stress. - NodC and chitin deacetylases possibly co-localize and affect cellular localization. - Chitin deacetylase expression causes cell elongation up to 30 micrometers. - Further study needed on rhizobial NodC-chitin deacetylase and substrate interactions.

synthetic biology↗