Biology subjects

Tietze, L.

Publications and source records attributed to Tietze, L..

7 recordsLinked to original sources

bioRxiv2025

Engineering Artificial 5' Regulatory Sequences for Thermostable Protein Expression in the Extremophile Thermus thermophilus

The utilisation of biocatalysts in biotechnological applications often necessitates their heterologous expression in suitable host organisms. However, the range of standardised microbial hosts for recombinant protein production remains limited, with most being mesophilic and suboptimal for certain protein types. Although the thermophilic bacterium Thermus thermophilus has long been established as a valuable extremophile host, thanks to its high-temperature tolerance, robust growth, and extensively characterised proteome, its genetic toolkit has predominantly depended on a limited set of native promoters. To overcome this bottleneck, we have expanded the available regulatory repertoire in T. thermophilus by developing novel artificial 5' regulatory sequences. In this study, we applied our Gene Expression Engineering platform to engineer 53 artificial 5' regulatory sequences (ARES) in T. thermophilus. These ARES, which comprise both promoter and 5' untranslated regions (UTRs), were functionally characterised in both T. thermophilus and Escherichia coli, revealing distinct host-specific expression patterns. Furthermore, we demonstrated the utility of these ARES by demonstrating high-level expression of thermostable proteins, including {beta}-galactosidase, a superfolder citrine fluorescent protein, and phytoene synthase. A bioinformatic analysis of the novel sequences has also being carried out indicating that the ARES possess markedly lower GC content compared to native promoters. This study contributes to expanding the genetic toolkit for recombinant protein production by providing a set of functionally validated ARES, enhancing the versatility of T. thermophilus as a synthetic biology chassis for thermostable protein expression.

synthetic biology↗

bioRxiv2024

High-resolution mapping of Sigma Factor DNA Binding Sequences using Artificial Promoters, RNA Aptamers and Deep Sequencing

The variable sigma ({sigma}) subunit of the bacterial RNA polymerase holoenzyme determines promoter specificity and facilitate open complex formation during transcription initiation. Understanding {sigma}-factor binding sequences is therefore crucial for deciphering bacterial gene regulation. Here, we present a data-driven high-throughput approach that utilizes an extensive library of 1.54 million DNA templates providing artificial promoters and 5' UTR sequences for {sigma}-factor DNA binding motif discovery. This method combines the generation of extensive DNA libraries, in vitro transcription, RNA aptamer selection, and deep DNA and RNA sequencing. It allows direct assessment of promoter activity, identification of transcription start sites, and quantification of promoter strength based on mRNA production levels. We applied this approach to map {sigma}54 DNA binding sequences in Pseudomonas putida. Deep sequencing of the enriched RNA pool revealed 64,966 distinct {sigma}54 binding motifs, significantly expanding the known repertoire. This data-driven approach surpasses traditional methods by directly evaluating promoter function and avoiding selection bias based solely on binding affinity. This comprehensive dataset enhances our understanding of {sigma}-factor binding sequences and their regulatory roles, opening avenues for new research in biology and biotechnology.

biochemistry↗

bioRxiv2021

mCherry contains a fluorescent protein isoform that interferes with its reporter function

Fluorescent proteins are essential reporters in cell biology and molecular biology. Here, we reveal that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform. The short isoform creates significant background fluorescence that biases the outcome of expression studies. Our investigation identifies the short protein isoform, traces its origin, and determines the extent of the issue within the family of red fluorescent protein. Our analysis shows that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. Finally, we provide a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

molecular biology↗

bioRxiv2020

Hypothalamic glucocorticoid receptor in CRF neurons is essential for HPA axis habituation to repeated stressor

Short abstractHabituation of the hypothalamic-pituitary-adrenal (HPA) axis to repeated homotypic stressors is crucial for the organisms well-being. Many physiological and psychological disorders are associated with HPA axis dysfunction. Here, we show that glucocorticoid receptors in CRF neurons of the hypothalamic paraventricular nucleus are essential for HPA habituation. By increasing inhibitory tone onto CRF neurons, glucocorticoid receptors led to essential cellular modulation and hypothalamic-pituitary-adrenal axis activation dampening, when re-exposed to the same stressor.

neuroscience↗

bioRxiv2019

Galactose-modified duocarmycin prodrugs as senolytics

Senescence is a stable growth arrest that impairs the replication of damaged, old or preneoplastic cells, therefore contributing to tissue homeostasis. Senescent cells accumulate during ageing and are associated with diseases, such as cancer, fibrosis and many age-related pathologies. Recent evidence suggests that the selective elimination of senescent cells can be effective on the treatment of many of these senescence-associated diseases. A universal characteristic of senescent cells is that they display elevated activity of the lysosomal {beta}-galactosidase this has been exploited as a marker for senescence (senescence-associated {beta}-galactosidase activity). Consequently, we hypothesised that galactose-modified cytotoxic prodrugs will be preferentially processed by senescent cells, resulting in their selective killing. Here, we show that different galactose-modified duocarmycin (GMD) derivatives preferentially kill senescent cells. GMD prodrugs induce selective apoptosis of senescent cells in a lysosomal {beta}-galactosidase (GLB1)-dependent manner. GMD prodrugs can eliminate a broad range of senescent cells in culture, and treatment with a GMD prodrug enhances the elimination of bystander senescent cells that accumulate upon whole body irradiation or doxorubicin treatment of mice. Moreover, taking advantage of a mouse model of human adamantinomatous craniopharyngioma (ACP), we show that treatment with a GMD pro-drug result selectively reduced the number of {beta}-catenin-positive preneoplastic senescent cells, what could have therapeutic implications. In summary, the above results show that galactose-modified duocarmycin prodrugs behave as senolytics, suggesting that they could be used to treat a wide range of senescence-related pathologies.

cell biology↗

bioRxiv2019

A universal method for gene expression engineering

The precise expression of genes is one of the foundations of biotechnology. Here we present GeneEE, a straightforward method for generating artificial gene expression systems. We demonstrate that GeneEE segments, containing a 200 nucleotide DNA with random nucleotide composition, can facilitate constitutive and inducible gene expression. To highlight the universal character of our method, we demonstrate GeneEE-mediated gene and protein expression in six bacterial species and Bakers yeast.

bioengineering↗

bioRxiv2017

The role of m6A-RNA methylation in stress response regulation

N6-Methyladenosine (m6A) is an abundant internal RNA modification that regulates transcript processing and translation. The regulation of brain m6A by stressful stimuli in vivo and its role in the stress response are currently unknown.\n\nHere, we provide a detailed analysis of the stress-epitranscriptome using m6A-Seq, global and gene-specific m6A measurements. We show that stress exposure and glucocorticoids alter m6A and its regulatory network in a region- and time-specific manner. We demonstrate that depletion of the methyltransferase Mettl3 and the demethylase Fto in adult neurons increases fear memory, and alters the transcriptome response to fear as well as synaptic plasticity. Finally, we report that regulation of m6A is impaired in major depressive disorder patients following glucocorticoid receptor activation.\n\nOur findings indicate that brain m6A represents a novel layer of complexity in gene expression regulation after stress and that dysregulation of the m6A-response may contribute to the pathophysiology of stress-related psychiatric disorders.\n\nHighlightsO_LIm6A RNA methylation in adult mouse brain is regulated by stress\nC_LIO_LIBrain m6A levels are temporally and spatially regulated by stress\nC_LIO_LIMettl3 and Fto-KO alter fear memory, transcriptome response and synaptic plasticity\nC_LIO_LIThe m6A-glucocorticoid-response is impaired in major depressive disorder patients\nC_LI\n\neTOC blurbEngel et al. demonstrate a brain-area-specific and time-dependent role for the mRNA modification, m6A, in stress-response regulation. Manipulating m6A-enzymes alters fear-memory, transcriptome-response and synaptic-plasticity. Altered m6A dynamics in depressed patients suggest an involvement of m6A-modifications in stress-related psychiatric disorders.

neuroscience↗