Biology subjects

Naseri, G.

Publications and source records attributed to Naseri, G..

4 recordsLinked to original sources

bioRxiv2026

FASTOP - Fast editing toolkit for top expression sites in yeast

Budding yeast Saccharomyces cerevisiae is a workhorse chassis for producing added food and agricultural compounds. However, building multi-enzymatic pathways for these chemicals often requires iterative genomic integration, underscoring the need for efficient, rapid genome-editing tools that can reliably target transcriptionally active chromosomal regions. In this study, to accelerate strain construction, we established a genome-editing toolkit to rapidly engineer eight loci, highly expressed hot-spots, but nonessential genomic sites suitable for stable pathway assembly. Our approach integrates three key design features: (i) selectable markers to enable rapid screening of edited cells, (ii) extended homology arms that leverage the yeast homology-directed repair machinery for robust genomic integration, and (iii) co-delivery of Cas9 and guide RNAs to promote efficient double-stranded DNA breaks at specific integration sites. The sequence independence of FASTOP relies on the release of integration cassettes from integrative vectors, mediated by restriction digestion at two flanking multiple-cutting sites in the integration module to minimize the risk of introducing sequence errors during PCR amplification of the integration cassettes. Following the introduction of a fluorescent reporter cassette, we observed high integration efficiencies across the target sites. We then integrated the biosynthetic pathway of plant-derived flavonoid naringenin into the hot-spots of the yeast genome using the FASTOP toolkit. Our results demonstrated that upon expressing the five essential genes in simple shake flask culture, naringenin production reached 505.7 mg/L, representing a significant (69-fold) increase over previously reported titers for comparable minimal heterologous pathways in S. cerevisiae. Together, the FATSOP toolkit provides a user-friendly platform for reliably modifying hot-spot loci to rapidly construct multi-enzymatic metabolic pathways in S. cerevisiae, while achieving high production levels for high-value food-relevant metabolites.

synthetic biology↗

bioRxiv2026

Fine-tuned synthetic transcription factors for production of 3' phosphoadenosine-5'-phosphosulfate in yeast

Technologies developed over the past decade have made Saccharomyces cerevisiae a promising platform for producing various natural products. Balancing multi-enzyme expression, while maintaining robust microbial growth, remains a limiting factor for engineering long biosynthetic pathways in yeast. Here, we improved the transcriptional capacity of our previously developed isopropyl {beta}-D-1-thiogalactopyranoside (IPTG)-inducible synthetic transcription factors (synTFs) derived from the plant JUB1 DNA-binding domain. To this end, at cysteine positions within surface-exposed loop regions of a JUB1-derived DNA-binding scaffold, we introduced a short peptide to enhance loop flexibility while providing local stability and orientation. The generated synTFs, so-called JUB1-X synTFs, varying in strength, have been successfully used to improve the synthesis of 3-phosphoadenosine 5-phosphosulfate (PAPS), a universal sulfate donor necessary for the synthesis of bioactive molecules, including therapeutic glycosaminoglycans and sulfolipids. Using only this engineered yeast strain in simple batch culture, PAPS accumulation of 21.4 {+/-} 5.8 mg g-1 cdw was achieved after only 5 hours of inducing the expression of JUB1-X synTFs. Beyond PAPS production, the design principle demonstrated here provides a generalizable strategy to fine-tune other plant-derived synTFs, expanding the regulatory capabilities of existing synTF collections. Together, this work offers a modular, scalable approach to constructing high-performance gene circuits and supports the development of yeast cell factories for complex metabolic and synthetic biology applications.

synthetic biology↗

bioRxiv2022

A regulatory toolkit of arabinose-inducible artificial transcription factors for Gram-negative bacteria

The Gram-negative bacteria Salmonella Typhimurium and Escherichia coli are important model organisms, powerful prokaryotic expression platforms for biotechnological applications, and pathogenic strains constitute major public health threats. To facilitate new approaches for research, biomedicine, and biotechnological applications, we developed a set of arabinose-inducible artificial transcription factors (ATFs) using CRISPR/dCas9 and Arabidopsis-derived DNA-binding proteins, allowing to control gene expression in E. coli and Salmonella over a wide inducer concentration range. As a proof-of-concept, we employed the developed ATFs to engineer a Salmonella biosensor strain, SALSOR 0.2 (SALmonella biosenSOR 0.2), which responds to the presence of alkaloid drugs with quantifiable fluorescent output. We demonstrated that SALSOR 0.2 was able to detect the presence of the antitussive noscapine alkaloid with ~2.3-fold increased fluorescent signal over background noise compared to a previously described biosensor. Moreover, we used plant-derived ATFs to control {beta}-carotene biosynthesis in E. coli, which resulted in ~1.6-fold higher {beta}-carotene production compared to expression of the biosynthesis pathway using a strong constitutive promoter. The arabinose-inducible ATFs reported here thus enhance the synthetic biology repertoire of transcriptional regulatory modules that allow tuning protein expression in the Gram-negative model organisms Salmonella and E. coli.

synthetic biology↗

bioRxiv2021

BRED: bioluminescence energy transfer to dye for monitoring ceramide trafficking in cell

Bioluminescence resonance energy transfer (BRET) is a genetically encoded proximity-based tool to study biomolecular interactions. However, conventional BRET is usually restricted to only a few types of interactions like protein-protein or protein-ligand interactions. We here developed a spatially unbiased resonance energy transfer system, so-called BRED - bioluminescence resonance energy transfer to dye. BRED allows transferring energy from a genetically encoded bright human optimized luciferase to a fluorophore-labelled small molecule. The high efficiency of the system allows RET without specific interaction of donor and acceptor. Here, we applied BRED to monitor the trafficking of the signalling lipid ceramide, to the Golgi. This was enabled by an engineered Golgi-resident luciferase, which was used to sense the influx of BODIPY-labeled ceramide into the surrounding membrane. We demonstrated the implementation of the method via flow cytometry, thereby combining the sensitivity of bulk cell methods with the advantages of single-cell analysis. This toolbox enables simple and robust live-cell analysis of inhibitors of CERT-mediated ceramide transport. The design principle of our optogenetic tool can be applied to study intracellular trafficking of metabolites and screen for inhibitors of their key enzymes.

biochemistry↗