Biology subjects

Poborsky, M.

Publications and source records attributed to Poborsky, M..

4 recordsLinked to original sources

bioRxiv2026

Engineering orthogonal quorum sensing circuits using LuxR-type systems in yeast consortia

Engineered microbial communities hold significant biotechnological potential because their collective metabolism can produce functions beyond those achievable by individual strains. However, multicellular synthetic gene circuits require orthogonal communication systems that enable precise, programmable signaling between cells. Quorum sensing (QS), where cells both produce and detect small diffusible signal molecules, offers a natural framework for such intercellular communication. However, the construction of complex multicellular circuits for applications such as biobased production is currently hampered by the limited number of orthogonal QS channels available in yeast. Here, we expand the QS toolkit in Saccharomyces cerevisiae by characterizing four LuxR-type biosensors based on EsaR, LasR, TraR and RpaR, alongside the previously established LuxR biosensor. We functionally expressed acyl-CoA-dependent HSL synthases in yeast, producing a diverse range of aliphatic and aromatic HSL signals. LuxR and RpaR, were compatible with in vivo ligand production and established as orthogonal QS signaling pair with synthases MesI and RpaI, respectively. Co-culture experiments demonstrated QS-dependent intercellular signaling, with 3.9-fold and 6.4-fold induction relative to monocultures. Together, these results establish a modular and extensible platform for orthogonal intercellular communication in yeast, enabling the construction of multicellular synthetic gene circuits.

synthetic biology↗

bioRxiv2026

Utilizing raw rapeseed press cake in foods: A case study on sensory quality and profile of selected bitter compounds in snack bars

Todays canola quality rapeseed press cake (RPC) is a protein-rich co-product with potential as human food, but its application is limited due to antinutritional compounds and bitter taste. It remains, however, unknown how introduction of raw RPC to a food matrix affects sensory perception and which metabolites drive the sensation. Here, raw RPC from whole or dehulled seeds was introduced into snack bars at 0%, 7%, 14%, and 21%, and sensory responses were correlated to selected known RPC-derived bitter compounds. A trained panel evaluated 13 RPC-characteristic sensory attributes, and the bitter compounds sinapic acid, kaempferol 3-O-(2'''-O-sinapoyl-{beta}-sophoroside) (KSS), KSS-hexose, selected bitter glucosinolates, and goitrin were quantified using targeted LC-MS/MS. Most dose-dependent sensory responses increased up to 14% RPC and then plateaued, whereas astringent mouthfeel increased almost linearly across the full dose range. Dehulling intensified several odor- and flavor-related attributes but did not increase bitterness or protein content in the final product. Principal component analysis linked bitterness and astringency positively with KSS, KSS-hexose, and goitrin. Dose-over-threshold analysis further showed that goitrin, but not progoitrin, reached concentrations relevant for bitterness perception. Together, the results demonstrate that raw RPC contributes distinct dose-dependent sensory attributes and that metabolite transformations in the food matrix shape final sensory profiles. These findings provide a basis for developing RPC-containing foods and for breeding rapeseed lines with improved sensory characteristics. HIGHLIGHTSO_LIThis study presents the first sensory panel assessment of rapeseed press cake (RPC)-containing in food products (snack bars) made from whole and dehulled seeds. C_LIO_LI13 RPC-characteristic sensory attributes are identified. C_LIO_LISensory profiles of the tasted snack bars differed significantly, influenced by the dosage of RPC and by the dehulling treatment. Bitterness and astringency are positively correlated with the RPC dosage. C_LIO_LIGoitrin, kaempferol 3-O-(2'''-O-sinapoyl-{beta}-sophoroside) (KSS) and sinapic acid are RPC-derived bitter compounds that correlate with bitter taste of RPC-containing snack bars. C_LIO_LIApproximately 90% of glucosinolates introduced with the RPC are not detected in the snack bars, and goitrin levels in snack bars accounts for only [~]10% of introduced progoitrin. C_LIO_LIGoitrin is - for the first time - reported to contribute to the perceived bitterness of an RPC-containing food product. C_LI

plant biology↗

bioRxiv2026

Optimization of the glucosinolate core pathway for production of simple glucosinolates in Escherichia coli

Microbial biosynthesis of plant secondary metabolites aims to provide access to compounds of medicinal or industrial value independently of the native plant. Glucosinolates are plant secondary metabolites characteristic of brassicaceous plants and recognized as promoters of human health. However, plants often contain a complex mixture of glucosinolates with insufficient amounts to elicit a clinical effect through diet. Here, we demonstrate the biosynthesis of defined glucosinolate products in Escherichia coli through combinatorial screening of pathway enzyme homologs, tailoring the optimal biosynthetic route for each individual product. To achieve high product titers, we establish efficient P450 expression by membrane anchor truncation and engineer sulfate assimilation to increase the supply of a sulfate donor 3-phosphoadenosine-5-phosphosulfate. We use benzyl glucosinolate pathway as a model to test the engineering strategies and improve the titer 37-fold over our previous study. Extrapolating the best approaches to other simple glucosinolates, we establish the first microbial synthesis of tyrosine-derived p-glucosinolate. Showing the highest titer overall, we report production of 1250 {+/-} 91 {micro}M indolyl-3-methyl glucosinolate, a 500-fold increase over biosynthesis in yeast.

synthetic biology↗

bioRxiv2022

Systematic engineering of plant cytochrome P450 system identifies a comprehensive strategy for expression of highly functional P450 enzymes in Escherichia coli

Cytochrome P450s catalyse diverse and unique chemical reactions, which makes them invaluable enzymes in nature and industry. Metabolic engineers leverage these unique catalytic properties when refactoring plant biosynthetic pathways into microbial cell factories. However, due to their hydrophobic anchor, microbial expression of membrane-bound cytochrome P450s is challenging. An arsenal of protein engineering strategies was developed to improve their expression in Escherichia coli, but extensive screening is often necessary to tailor the engineering approach to an individual enzyme. Here, we propose a universal strategy that allows the expression of highly active cytochrome P450s in E. coli by systematically evaluating six common N-terminal modifications and their effect on in vivo activity of enzymes from the CYP79 and CYP83 families. We identified transmembrane domain truncation as the only strategy that had a significantly positive effect on all seven tested enzymes, increasing product titres between 2- to 170-fold. When comparing the changes in protein titre and product generation, we show that higher expression does not always translate to higher in vivo activity, thus making protein titre an unreliable screening target. Our results demonstrate that transmembrane domain truncation improves in vivo activity across a broad range of cytochrome P450s with diverse N-terminal sequences and could be applied as the modification-of-choice to avoid the time-consuming screening process and accelerate the future design of E. coli cell factories.

bioengineering↗