Dr. rer. nat. Gita Naseri
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Eine umfassende Plattform für die nachhaltige Biosynthese von seltenen Naturstoffen in Pichia pastoris
Quelle ↗Förderer: DFG Nachwuchsgruppe Zeitraum: 04/2024 - 03/2030 Projektleitung: Dr. rer. nat. Gita Naseri
Eine umfassende Plattform für die nachhaltige Biosynthese von seltenen Naturstoffen in Pichia pastoris
Quelle ↗Förderer: DFG Nachwuchsgruppe Zeitraum: 04/2024 - 03/2030 Projektleitung: Dr. rer. nat. Gita Naseri
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Publikationen19
Top 25 nach Zitationen — Quelle: OpenAlex (BAAI/bge-m3 embedded für Matching).
Nature Communications · 173 Zitationen · DOI
In the first wave of synthetic biology, genetic elements, combined into simple circuits, are used to control individual cellular functions. In the second wave of synthetic biology, the simple circuits, combined into complex circuits, form systems-level functions. However, efforts to construct complex circuits are often impeded by our limited knowledge of the optimal combination of individual circuits. For example, a fundamental question in most metabolic engineering projects is the optimal level of enzymes for maximizing the output. To address this point, combinatorial optimization approaches have been established, allowing automatic optimization without prior knowledge of the best combination of expression levels of individual genes. This review focuses on current combinatorial optimization methods and emerging technologies facilitating their applications.
ACS Synthetic Biology · 52 Zitationen · DOI
Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.
Nature Communications · 47 Zitationen · DOI
Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing β-carotene and co-producing β-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.
Nature Communications · 33 Zitationen · DOI
Secondary natural products (NPs) are a rich source for drug discovery. However, the low abundance of NPs makes their extraction from nature inefficient, while chemical synthesis is challenging and unsustainable. Saccharomyces cerevisiae and Pichia pastoris are excellent manufacturing systems for the production of NPs. This Perspective discusses a comprehensive platform for sustainable production of NPs in the two yeasts through system-associated optimization at four levels: genetics, temporal controllers, productivity screening, and scalability. Additionally, it is pointed out critical metabolic building blocks in NP bioengineering can be identified through connecting multilevel data of the optimized system using deep learning.
AFRICAN JOURNAL OF BIOTECHNOLOGY · 23 Zitationen · DOI
Among the fungal diseases, sheath blight caused by Rhizoctonia solani Kuhn is one of the most important wide spread diseases found in all the rice plants ( Oryza sativa ) growing countries. The control of the disease with agricultural alternation and traditional breeding has not been successful so far. Application of fungicide is not recommended. Resistant genes which can cause immunity to the disease have not been found. D34 is a thaumatin-like protein (TLP) belonging to the group 5 of pathogen-related proteins (PRs). These proteins can probably change permeability of fungal membrane. In this research, an attempt was made to enhance the resistance to the sheath blight fungus on rice plant through the expression of TLP gene under the control of a CaMV35 promoter. To achieve this goal, rice plants ( O. sativa ) were transformed by the pAJ21-CaMV35S-tlpD34 construct via in planta method using Agrobacterium tumefaciens EHA101. The transformed plants were confirmed using polymerase chain reaction (PCR) amplifying a 710 bp fragment of the cloned gene. Keywords: In planta , pathogenesis-related protein, Rhizoctonia solani , rice, sheath blight, thaumatin-like protein, transformation
Frontiers in Bioengineering and Biotechnology · 13 Zitationen · DOI
The non-conventional yeast <i>Pichia pastoris</i> (syn. <i>Komagataella phaffii</i>) has become a powerful eukaryotic expression platform for biopharmaceutical and biotechnological applications on both laboratory and industrial scales. Despite the fundamental role that artificial transcription factors (ATFs) play in the orthogonal control of gene expression in synthetic biology, a limited number of ATFs are available for <i>P. pastoris</i>. To establish orthogonal regulators for use in <i>P. pastoris</i>, we characterized ATFs derived from Arabidopsis TFs. The plant-derived ATFs contain the binding domain of TFs from the plant <i>Arabidopsis thaliana</i>, in combination with the activation domains of yeast <i>GAL4</i> and plant <i>EDLL</i> and a synthetic promoter harboring the cognate <i>cis</i>-regulatory motifs. Chromosomally integrated ATFs and their binding sites (ATF/BSs) resulted in a wide spectrum of inducible transcriptional outputs in <i>P. pastoris</i>, ranging from as low as 1- to as high as ∼63-fold induction with only small growth defects. We demonstrated the application of ATF/BSs by generating <i>P. pastoris</i> cells that produce β-carotene. Notably, the productivity of β-carotene in <i>P. pastoris</i> was ∼4.8-fold higher than that in <i>S. cerevisiae</i>, reaching ∼59% of the β-carotene productivity obtained in a <i>S. cerevisiae</i> strain optimized for the production of the β-carotene precursor, farnesyl diphosphate, by rewiring the endogenous metabolic pathways using plant-derived ATF/BSs. Our data suggest that plant-derived regulators have a high degree of transferability from <i>S. cerevisiae</i> to <i>P. pastoris</i>. The plant-derived ATFs, together with their cognate binding sites, powerfully increase the repertoire of transcriptional regulatory modules for the tuning of protein expression levels required in metabolic engineering or synthetic biology in <i>P. pastoris</i>.
Chemical Communications · 8 Zitationen · DOI
Acid sphingomyelinase (ASM) is a potential drug target and involved in rapid lipid signalling events. However, there are no tools available to adequately study such processes. Based on a non cell-permeable PtdIns(3,5)P2 inhibitor of ASM, we developed a compound with o-nitrobenzyl photocages and butyryl esters to transiently mask hydroxyl groups. This resulted in a potent light-inducible photocaged ASM inhibitor (PCAI). The first example of a time-resolved inhibition of ASM was shown in intact living cells.
Communications Biology · 6 Zitationen · DOI
The Gram-negative bacteria Salmonella enterica 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 and biotechnological applications, we here develop a set of arabinose-inducible artificial transcription factors (ATFs) using CRISPR/dCas9 and Arabidopsis-derived DNA-binding proteins to control gene expression in E. coli and Salmonella over a wide inducer concentration range. The transcriptional output of the different ATFs, in particular when expressed in Salmonella rewired for arabinose catabolism, varies over a wide spectrum (up to 35-fold gene activation). As a proof-of-concept, we use the developed ATFs to engineer a Salmonella two-input biosensor strain, SALSOR 0.2 (SALmonella biosenSOR 0.2), which detects and quantifies alkaloid drugs through a measurable fluorescent output. Moreover, we use plant-derived ATFs to regulate β-carotene biosynthesis in E. coli, resulting in ~2.1-fold higher β-carotene production compared to expression of the biosynthesis pathway using a strong constitutive promoter.
Methods in molecular biology · 3 Zitationen · DOI
Journal of Fungi · 2 Zitationen · DOI
Fungi play a crucial yet often unnoticed role in our lives and the health of our planet by breaking down organic matter through their diverse enzymes or eliminating environmental contamination, enhancing biomass pretreatment, and facilitating biofuel production. They offer transformative possibilities not only for improving the production of materials they naturally produce, but also for the production of non-native and even new-to-nature materials. However, despite these promising applications, the full potential of fungi remains untapped mainly due to limitations in our ability to control and optimize their complex biological systems. This review focuses on developments that address these challenges, with specific emphasis on fungal-derived rigid and flexible materials. To achieve this goal, the application of synthetic biology tools-such as programmable regulators, CRISPR-based genome editing, and combinatorial pathway optimization-in engineering fungal strains is highlighted, and how external environmental parameters can be tuned to influence material properties is discussed. This review positions filamentous fungi as promising platforms for sustainable bio-based technologies, contributing to a more sustainable future across various sectors.
Research Square · DOI
bioRxiv (Cold Spring Harbor Laboratory) · DOI
ABSTRACT 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 β-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.
edoc Publication server (Humboldt University of Berlin) · DOI
Fungi play a crucial yet often unnoticed role in our lives and the health of our planet by breaking down organic matter through their diverse enzymes or eliminating environmental contamination, enhancing biomass pretreatment, and facilitating biofuel production. They offer transformative possibilities not only for improving the production of materials they naturally produce, but also for the production of non-native and even new-to-nature materials. However, despite these promising applications, the full potential of fungi remains untapped mainly due to limitations in our ability to control and optimize their complex biological systems. This review focuses on developments that address these challenges, with specific emphasis on fungal-derived rigid and flexible materials. To achieve this goal, the application of synthetic biology tools—such as programmable regulators, CRISPR-based genome editing, and combinatorial pathway optimization—in engineering fungal strains is highlighted, and how external environmental parameters can be tuned to influence material properties is discussed. This review positions filamentous fungi as promising platforms for sustainable bio-based technologies, contributing to a more sustainable future across various sectors.
bioRxiv (Cold Spring Harbor Laboratory) · DOI
Abstract 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 biosen sor 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 β-carotene biosynthesis in E. coli , which resulted in ~1.6-fold higher β-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 .
bioRxiv (Cold Spring Harbor Laboratory) · DOI
ABSTRACT 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.
Zenodo (CERN European Organization for Nuclear Research) · DOI
Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in <em>Saccharomyces cerevisiae</em>. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing β-carotene and co-producing β-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.
publish.UP (University of Potsdam) · DOI
Bereits seit 9000 Jahren verwendet die Menschheit die Bäckerhefe Saccharomyces cerevisiae für das Brauen von Bier, aber erst seit 150 Jahren wissen wir, dass es sich bei diesem unermüdlichen Helfer im Brauprozess um einzellige, lebende Organismen handelt. Und die Bäckerhefe kann noch viel mehr. Im Rahmen des Forschungsgebietes der Synthetischen Biologie soll unter anderem die Bäckerhefe als innovatives Werkzeug für die biobasierte Herstellung verschiedenster Substanzen etabliert werden. Zu diesen Substanzen zählen unter anderem Feinchemikalien, Biokraftstoffe und Biopolymere sowie pharmakologisch und medizinisch interessante Pflanzenstoffe. Damit diese verschiedensten Substanzen in der Bäckerhefe hergestellt werden können, müssen große Mengen an Produktionsinformationen zum Beispiel aus Pflanzen in die Hefezellen übertragen werden. Darüber hinaus müssen die neu eingebrachten Biosynthesewege reguliert und kontrolliert in den Zellen ablaufen. Auch Optimierungsprozesse zur Erhöhung der Produktivität sind notwendig. Für alle diese Arbeitsschritte mangelt es bis heute an anwendungsbereiten Technologien und umfassenden Plattformen. Daher wurden im Rahmen dieser Doktorarbeit verschiedene Technologien und Plattformen zur Informationsübertragung, Regulation und Prozessoptimierung geplant und erzeugt. Für die Konstruktion von Biosynthesewegen in der Bäckerhefe wurde als erstes eine Plattform aus neuartigen Regulatoren und Kontrollelementen auf der Basis pflanzlicher Kontrollelemente generiert und charakterisiert. Im zweiten Schritt erfolgte die Entwicklung einer Technologie zur kombinatorischen Verwendung der Regulatoren in der Planung und Optimierung von Biosynthesewegen (COMPASS). Abschließend wurde eine Technologie für die Prozessoptimierung der veränderten Hefezellen entwickelt (CapRedit). Die Leistungsfähigkeit der entwickelten Plattformen und Technologien wurde durch eine Optimierung der Produktion von Carotenoiden (Beta-Carotin und Beta-Ionon) und Flavonoiden (Naringenin) in Hefezellen nachgewiesen. Die im Rahmen der Arbeit etablierten neuartigen Plattformen und innovativen Technologien sind ein wertvoller Grundbaustein für die Erweiterung der Nutzbarkeit der Bäckerhefe. Sie ermöglichen den Einsatz der Hefezellen in kosteneffizienten Produktionswegen und alternativen chemischen Wertschöpfungsketten. Dadurch können zum Beispiel Biokraftstoffe und pharmakologisch interessante Pflanzenstoffe unter Verwendung von nachwachsenden Rohstoffen, Reststoffen und Nebenprodukten hergestellt werden. Darüber hinaus ergeben sich Anwendungsmöglichkeiten zur Bodensanierung und Wasseraufbereitung.
New Biotechnology · DOI
Journal of Biotechnology & Biomaterials
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- Dr. rer. nat. Gita Naseri
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- Dr. rer. nat.
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- Lebenswissenschaftliche Fakultät
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- Institut für Biologie
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- NWG COMPLATn
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