Biotechnological Applications Research Articles

LC–MS/MS quantification of bacterial and fungal signal peptides via direct injection: a case study of cross-kingdom communication

Abstract Bacteria and yeast use secreted signal peptides, also known as pheromones, for cell–cell communication within their respective species. Recently, genetic modification has allowed for the extension and exploitation of this type of communication, to communication between organisms from different species and even from different kingdoms. This innovative approach is intended to allow for the large-scale production of specific compounds for applications in medicine and biotechnology while producing reduced amounts of by-products. Until now, the detection of signal peptides, which are often short-lived and only present in small amounts, is usually qualitative, non-selective, and time-consuming and/or requires the presence of additional cell types. Here, an ESI-LC–MS/MS method for the specific quantification of signal peptides from yeast (α- and P-factor) and bacteria (CSF) using a TSKgel column operating under HILIC conditions has been demonstrated. The influence of different matrices, their adsorption behavior, and their stability were investigated. In matrix, LOQs of 0.05 µM, 0.03 µM, and 0.02 µM were obtained for CSF, α-factor, and P-factor, respectively. Subsequently, the developed method was applied to the detection of yeast- and bacteria-specific peptides secreted by genetically modified yeasts. It could be demonstrated that under overexpressing conditions, α-factor and P-factor concentrations of 1 µM were measured, while for CSF concentrations as high as 2.5 µM was reached. Finally, the established method permits the simultaneous, quantitative detection of signal peptides in different matrices and without pre-concentration in near-real time, thus advancing the possibility of tracking cross-kingdom communication. Graphical Abstract

Multiomics-based assessment of the impact of airflow on diverse plant callus cultures

Plant cell culture has multiple applications in biotechnology and horticulture, from plant propagation to the production of high-value biomolecules. However, the interplay between cellular diversity and ambient conditions influences the metabolism of cultured tissues; understanding these factors in detail will inform efforts to optimize culture conditions. This study presents multiomics datasets from callus cultures of tobacco (Nicotiana tabacum), rice (Oryza sativa), and two bamboo species (Phyllostachys nigra and P. bambusoides). Over four weeks, calli were cultured under continuous moisture without airflow or gradually reduced ambient moisture with airflow. For each sample, gene expression was profiled with high-throughput RNA sequencing, 442 metabolites were measured using liquid chromatography (LC) with triple-quadrupole mass spectrometry (LC–QqQMS), and 31 phytohormones were quantified using ultra-performance LC coupled with a tandem quadrupole mass spectrometer equipped with an electrospray interface (UPLC-ESI-qMS/MS) and ultra-high-performance LC–orbitrap MS (UHPLC-Orbitrap MS). These datasets highlight the impact of airflow on callus cultures, revealing differences between and within species, and provide a comprehensive resource to explore the physiology of callus growth.

A hammerhead ribozyme selects mechanically stable conformations for catalysis against viral RNA

Ribozymes, widely found in prokaryotes and eukaryotes, target nucleic acids and can be engineered as biotechnical tools or for gene regulation or immune therapy. Among them, hammerhead is the smallest and best characterized ribozyme. However, the structure and biochemical data of ribozymes have been disagreed on, making the understanding of its catalysis mechanism a longstanding issue. Particularly, the role of conformational dynamics in ribozyme catalysis remains elusive. Here, we use single-molecule magnetic tweezers to reveal a concerted catalysis mechanism of mechanical conformational selection for a mini hammerhead ribozyme against a viral RNA sequence from the SARS-CoV-2. We identify a conformational set containing five mechanical conformers of the mini ribozyme, where magnesium ions select the active one. Our results are supported by molecular dynamics simulations. Our understanding of the RNA catalytic mechanism will be beneficial for ribozyme’s biotechnological applications and as potential therapeutics against RNA viruses.

Folding Competition and Dynamic Transformation in DNA Origami: Parallel Versus Antiparallel Crossovers.

DNA is a versatile abiomaterial for constructing nanostructures with biomedical and biotechnological applications. Among the methods available, DNA origami is a robust and widely recognized technique. Traditionally, most origami designs adopt antiparallel crossovers in both scaffold and staple strands, with less emphasis on parallel crossovers, which offer advantages like enhanced nuclease resistance and single-strand routing potential. Here, a DNA origami nanostructure is designed, featuring two rotational panels that can be locked into configurations based on either antiparallel or parallel crossovers. By systematically varying the length and arrangement of these key staples, 36 pairs of antiparallel and parallel designs are studied in competitive folding tests, providing insights into the relative preference for each design. The 12 antiparallel and parallel designs are ranked, their folding pathways are examined, and nuclease resistance is assessed. The results reveal that the arrangement of staples near the central scaffold crossover is crucial for shifting betweenparallel and antiparallel conformations. Additionally, a two-way isothermal transformation between antiparallel and parallel origami driven by toehold-mediated displacement reactions is demonstrated, highlighting the potential of parallel designs as dynamic nanodevices for temperature-sensitive environments. This study offers valuable insights into - dynamics in antiparallel and parallel DNA origami, opening opportunities for designing nanodevices based on parallel crossovers.

Phenanthrene degradation by a flavoprotein monooxygenase from Phanerodontia chrysosporium.

Phenanthrene (PHEN), a polycyclic aromatic hydrocarbon (PAH), is degraded by white-rot fungi like Phanerochaete chrysosporium (the fungus has been renamed as Phanerodontia chrysosporium). PHEN is metabolized by P. chrysosporium and transformed into various monohydroxylated and dihydroxylated products. These intermediates are further degraded by cleavage of the aromatic ring. However, the enzymes involved in PHEN conversion in P. chrysosporium remain largely unidentified. We aimed to identify and characterize the P. chrysosporium enzymes involved in the degradation of PHEN and its intermediates. Recombinant P. chrysosporium flavoprotein monooxygenase 11 (FPMO11), a homolog of the salicylate 1-monooxygenase from the naphthalene-degrading bacterium Pseudomonas putida G7, was overexpressed in Escherichia coli. FPMO11 catalyzes the oxidative decarboxylation of 1-hydroxy-2-naphthoate (1H2N) and 2-hydroxy-1-naphthoate (2H1N) to 1,2-dihydroxynaphthalene (1,2DHN). To the best of our knowledge, this is the first study to identify and characterize enzymes with 1H2N and 2H1N monooxygenase activities in members of the FPMO superfamily. Additionally, our search for a dioxygenase with the ability to catalyze the aromatic ring cleavage of 1,2DHN led to the identification of intradiol dioxygenase (IDD) 1 and IDD2 from P. chrysosporium, which catalyzes the ring cleavage of 1,2DHN. Thus, this study also identified, for the first time, intradiol 1,2DHN dioxygenase activity in members of the IDD superfamily. The findings highlight the unique substrate spectra of FPMO11 and IDDs, rendering them attractive candidates for biotechnological applications, especially mitigation of environmental and health risks associated with PAH pollution.IMPORTANCEPhenanthrene (PHEN), a polycyclic aromatic hydrocarbon (PAH), is a widely studied pollutant in environmental science and toxicology due to its presence in fossil fuels, tobacco smoke, and as a byproduct of incomplete combustion processes. White-rot fungi like P. chrysosporium can degrade PHEN through the production of extracellular oxidative enzymes. We investigated the properties of PHEN-degrading enzymes in P. chrysosporium, specifically one flavoprotein monooxygenase (FPMO11) and two intradiol dioxygenases (IDD1 and IDD2). Our findings indicate that the enzymes catalyze the aromatic ring cleavage of PHEN, using the intermediates as substrates, transforming them into less harmful and more biodegradable compounds. This could help reduce environmental pollution and mitigate health risks associated with PAH exposure. The potential of these enzymes for biotechnological applications is also highlighted, emphasizing their critical role in understanding PAH degradation by white-rot fungi.

Exploring the Fermentation Potential of Kluyveromyces marxianus NS127 for Single-Cell Protein Production

Kluyveromyces marxianus is a food-grade yeast known for its diverse beneficial traits, making it an attractive candidate for both food and biotechnology applications. This study explores the potential of Kluyveromyces marxianus as a promising alternative protein source for single-cell protein (SCP) production. Various Kluyveromyces strains were isolated and screened from traditional fermented dairy products, with Kluyveromyces marxianus NS127 identified as the most promising strain due to its superior growth characteristics, high SCP yield, and environmental tolerance. Notably, Kluyveromyces marxianus NS127 demonstrated significant substrate conversion capacity with a biomass yield of 0.63 g biomass/g molasses, achieving a dry biomass concentration of 66.64 g/L and a protein yield of 28.37 g/L. The protein extracted from the dry biomass exhibited excellent solubility (62.55%) and emulsification properties (13.15 m2/g) under neutral conditions, alongside high foaming stability (93.70–99.20%) across a broad pH range (3–11). These results underscore the potential of Kluyveromyces marxianus NS127 as a viable alternative protein source and provide a solid theoretical foundation for its industrial application.

Stress-Induced Response and Adaptation Mechanisms in Bacillus licheniformis PSKA1 Exposed With Abiotic and Antibiotic Stresses.

Soil ecosystems consist of diverse microbial communities with great potential for ecological and biotechnological applications. These communities encounter various abiotic stresses, which expedite the activation of transient overexpression of heat shock proteins (HSPs). In the present study, a soil bacterium was isolated and identified as Bacillus licheniformis strain PSK.A1, and its growth parameters were optimized before exposing it to heat, salt, pH, and antibiotic stress conditions. Comparative protein expression was analyzed using SDS-PAGE, protein stabilization via protein aggregation assays, and survival through single spot dilution and colony-counting methods under various stress conditions. The pre-treatment of short stress dosage showed endured overall tolerance of bacterium to lethal conditions, as evidenced by moderately enhanced total soluble intracellular protein content, better protein stabilization, comparatively over-expressed HSPs, and relatively enhanced cell survival. The findings highlighted that cells grown under optimal conditions were more susceptible to lethal environments than stressed cells, with their enhanced tolerance linked to the overexpression of 20 distinct HSPs of 17-91 kD. These insights offer the potential for developing strategies to enhance microbial resilience for various applications including bacterial bioprocessing, bio-remediation, and infectious disease management.

Diversity of Potential (Bio)technological Applications of Amino Acid-Based Ionic Liquids

This review explores the emerging potential of amino acid-based ionic liquids (AA ILs) in various (bio)applications, emphasizing their unique properties and versatility. It provides a comprehensive analysis of recent advancements, covering applications in drug delivery, catalysis, environmental remediation, and biotechnology. The review also offers an overview of the synthetic methods for preparing AA ILs, highlighting both traditional and innovative approaches, and examines key physicochemical properties—such as biocompatibility, stability, and tunability—that make AA ILs highly attractive for diverse applications. Additionally, challenges hindering their widespread adoption, including high production costs, toxicity concerns, scalability issues, and environmental impact, are discussed. This review concludes with perspectives on future research directions and strategies to overcome these challenges, unlocking the full potential of AA ILs in both scientific and industrial contexts.

Engineering the Marine Pseudoalteromonas haloplanktis TAC125 via the pMEGA Plasmid Targeted Curing Using PTasRNA Technology

Marine bacteria that have adapted to thrive in extreme environments, such as Pseudoalteromonas haloplanktis TAC125 (PhTAC125), offer a unique biotechnological potential. The discovery of an endogenous megaplasmid (pMEGA) raises questions about its metabolic impact and functional role in that strain. This study aimed at streamlining the host genetic background by curing PhTAC125 of the pMEGA plasmid using a sequential genetic approach. We combined homologous recombination by exploiting a suicide vector, with the PTasRNA gene-silencing technology interfering with pMEGA replication machinery. This approach led to the construction of the novel PhTAC125 KrPL2 strain, cured of the pMEGA plasmid, which exhibited no significant differences in growth behavior, though showcasing enhanced resistance to oxidative stress and a reduced capacity for biofilm formation. These findings represent a significant achievement in developing our understanding of the role of the pMEGA plasmid and the biotechnological applications of PhTAC125 in recombinant protein production. This opens up the possibility of exploiting valuable pMEGA genetic elements and further advancing the genetic tools for PhTAC125.

Recent advances in modifications, biotechnology, and biomedical applications of chitosan-based materials: A review.

Chitosan, a natural polysaccharide with recognized biocompatibility, non-toxicity, and cost-effectiveness, is primarily sourced from crustacean exoskeletons. Its inherent limitations such as poor water solubility, low thermal stability, and inadequate mechanical strength have hindered its widespread application. However, through modifications, chitosan can exhibit enhanced properties such as water solubility, antibacterial and antioxidant activities, adsorption capacity, and film-forming ability, opening up avenues for diverse applications. Despite these advancements, realizing the full potential of modified chitosan remains a challenge across various fields. The purpose of this review article is to conduct a comprehensive evaluation of the chemical modification techniques of chitosan and their applications in biotechnology and biomedical fields. It aims to overcome the inherent limitations of chitosan, such as low water solubility, poor thermal stability, and inadequate mechanical strength, thereby expanding its application potential across various domains. This review is structured into two main sections. The first part delves into the latest chemical modification techniques for chitosan derivatives, encompassing quaternization, Schiff base formation, acylation, carboxylation, and alkylation reactions. The second part provides an overview of the applications of chitosan and its derivatives in biotechnology and biomedicine, spanning areas such as wastewater treatment, the textile and food industries, agriculture, antibacterial and antiviral activities, drug delivery systems, wound dressings, dental materials, and tissue engineering. Additionally, the review discusses the challenges associated with these modifications and offers insights into potential future developments in chitosan-based materials. This review is anticipated to offer theoretical insights and practical guidance to scientists engaged in biotechnology and biomedical research.

Engineering of the genetic code.

The genetic code is a universally conserved mechanism that translates genetic information into proteins, consisting of 64 codons formed by four nucleotide bases. With a few exceptions, the genetic code universally encodes 20 canonical amino acids (AAs) and three stop signals, with many AAs represented by multiple codons. Genetic engineering has expanded this system through approaches like codon reassignment and synthetic base pair introduction, allowing for the incorporation of noncanonical AAs (ncAAs) into proteins, known as genetic code expansion (GCE). These ncAAs add novel functionalities, such as bio-orthogonal handles, fluorophores, and redox-active ncAAs, enhancing the diversity of proteins. Recent advancements include genome-wide recoding, evolution of orthogonal translation components, and synthetic genetic alphabet, with a focus on improving translational efficiency and reducing off-target effects. This review emphasizes strategies for modifying nucleotides, reassessing codons, and engineering translational enzymes, highlighting innovations that tackle challenges in GCE and promote new protein chemistries and biotechnological applications.

Application and Recent Trends of Biotechnology in Sustainable Agriculture: A Brief Review

Biotechnology has emerged as one of the key tools in crop improvement and molecular breeding programmes. CRISPR, plant tissue culture, molecular breeding, transgenic development, marker assisted selection (MAS), and animal cloning are regarded as key methods in biotechnology. Over the last two decades this technology has abetted in improved quality, yield, tolerance to both biotic and abiotic stress; and plays a pivotal role in introduction of several new features in crop development programmes. In agricultural sector, it has multifarious applications in crop improvement, fisheries and in livestock enhancement. The introduction of GM and biofortified Crops has proved to be noteworthy in overall crop development programme. Biotechnology in agriculture not only has boosted farmer income in both developed and developing countries but also played a remarkable role in agricultural sustainability. To improve farmers' lifestyles and the status of agriculture while preserving international standards of food quality and nutritional value, it is essential to promote the use of biotechnology in agriculture and related fields. The article opens by defining sustainability and emphasizing the critical role of biotechnology in different fields in attainment of sustainable agriculture.

Impact of water stress to plant epigenetic mechanisms in stress and adaptation.

Water is the basic molecule in living beings, and it has a major impact on vital processes. Plants are sessile organisms with a sophisticated regulatory network that regulates how resources are distributed between developmental and adaptation processes. Drought-stressed plants can change their survival strategies to adapt to this unfavorable situation. Indeed, plants modify, change, and modulate gene expression when grown in a low-water environment. This adaptation occurs through several mechanisms that affect the expression of genes, allowing these plants to resist in dry regions. Epigenetic modulation has emerged as a major factor in the transcription regulation of drought stress-related genes. Moreover, specific molecular and epigenetic modifications in the expression of certain genetic networks lead to adapted responses that aid a plant's acclimatization and survival during repeated stress. Indeed, understanding plant responses to severe environmental stresses, including drought, is critical for biotechnological applications. Here, we first focused on drought stress in plants and their general adaptation mechanisms to this stress. We also discussed plant epigenetic regulation when exposed to water stress and how this adaptation can be passed down through generations.

Separation of life stages within anaerobic fungi (Neocallimastigomycota) highlights differences in global transcription and metabolism.

Anaerobic gut fungi of the phylum Neocallimastigomycota are microbes proficient in valorizing low-cost but difficult-to-breakdown lignocellulosic plant biomass. Characterization of different fungal life stages and how they contribute to biomass breakdown are critical for biotechnological applications, yet we lack foundational knowledge about the transcriptional, metabolic, and enzyme secretion behavior of different life stages of anaerobic gut fungi: zoospores, germlings, immature thalli, and mature zoosporangia. A Miracloth-based technique was developed to enrich cell pellets with zoospores - the free-swimming, flagellated, young life stage of anaerobic gut fungi. By contrast, fungal mats contained relatively more vegetative, encysted, mature sporangia that form films. Global gene expression profiles were compared from two sample types (zoospore-enriched cell pellets vs. mature mats) harvested from the anaerobic gut fungal strain Neocallimastix californiae G1. Despite cultures being grown on glucose, the fungal zoospore-enriched samples were transcriptionally primed to encounter plant matter substrate, as evidenced by upregulation of catabolic carbohydrate-active enzymes and putative carbohydrate transporters. Furthermore, we report significant differential gene expression for gene annotation groups, including putative secondary metabolites and transcription factors. Understanding global gene expression differences between the fungal zoospore-enriched cells and mature fungi aid in characterizing fungal development, unmasking gene function, and guiding cultivation conditions and engineering targets to promote enzyme secretion.

Systematic characterization of horizontally transferred biosynthetic gene clusters in the human gut microbiota using HTBGC‐Finder

AbstractThe human gut microbiota contains biosynthetic gene clusters (BGCs) that encode bioactive secondary metabolites, which play pivotal roles in microbe‐microbe and host‐microbe interactions and serve as a rich source of pharmaceutical lead compounds. Understanding the horizontal transfer of BGCs can reveal insights into microbial adaptation, resource utilization, and evolutionary mechanisms, thereby advancing biotechnological applications. Despite its importance, horizontal transfer of BGCs within the gut microbiota remains poorly understood. In this study, we introduce a novel tool, the Horizontally Transferred Biosynthetic Gene Clusters Finder (HTBGC‐Finder), designed to systematically identify potential horizontally transferred BGCs (HTBGCs) within the extensive human gut microbiota. Using HTBGC‐Finder, we identified 81 potential HTBGCs, underscoring the prevalence and significance of horizontal gene transfer in shaping the genetic landscape of the gut microbiome. Remarkably, ribosomally synthesized and post‐translationally modified peptides (RiPPs) constituted the majority of these HTBGCs (76 out of 81, 93.83%), exhibiting a significantly higher transfer rate compared to non‐RiPPs (Chi‐squared test, p < 0.001). Upon detailed examination of BGCs, cyclic‐lactone‐autoinducer (CLA) and RiPP recognition element (RRE)‐containing BGCs were predominant, representing nearly three‐quarters of the total (45, or 55.56%, and 14, or 17.28%, respectively). Notably, CLA BGCs also demonstrated a higher transfer rate than non‐CLA BGCs (Chi‐squared test, p < 0.001). Taxonomy profiling revealed that horizontal BGC transfer occurred exclusively in the phyla Bacteroidota (synonym Bacteroidetes) and Bacillota (synonym Firmicutes), with 50 and 31 instances, respectively. Furthermore, cross‐phylum transfer events were observed, highlighting the complex interactions between the gut microbiota and host health. These findings offer valuable insights into the horizontal transfer dynamics of BGCs within the gut microbiome and their potential implications for host‐microbiota interactions.

Biochemical and structural insights into pinoresinol hydroxylase from Pseudomonas sp.

The vanillyl alcohol oxidase/p-cresol methylhydroxylase (VAO/PCMH) flavoprotein family comprises a broad spectrum of enzymes capable of catalyzing the oxidative bioconversions of various substrates. Among them, pinoresinol hydroxylase (PinH) from the 4-alkylphenol oxidizing subgroup initiates the oxidative degradation of (+)-pinoresinol, a lignan important for both lignin structure and plant defense. In this study, we present a detailed biochemical and structural characterization of PinH from Pseudomonas sp., with focus on its substrate specificity and product formation. PinH was expressed in E. coli and purified as FAD-containing, soluble protein. The flavoenzyme catalyzes the hydroxylation of both (+)-pinoresinol and eugenol. Structural analysis reveals its dimeric form, non-covalent flavin binding, and a large active site. AlphaFold models of the PinH-cytochrome complex demonstrate cytochrome's dual role in electron transfer and modulating PinH’s conformation. A distinctive feature of PinH is a large cavity that hosts its multi-ring (+)-pinoresinol substrate. The capability of converting bulky lignans is particularly attractive for biotechnological applications aimed at producing high-value compounds from phenolic precursors. These insights expand our knowledge on the structure and mechanism of the VAO/PCMH flavoenzyme family members.

Biotechnological uses of purified and characterized alkaline cellulase from extremophilic Bacillus pumilus VLC7 from Lake Van

The present study focuses on the isolation and characterization of a bacterium from Lake Van adapted to an alkaline pH environment with significant cellulase production potential. The identified isolate, Bacillus pumilus VLC7 (GenBank Acc No: OR415888.1), exhibited the highest cellulase activity among other alkaliphilic isolates. The purification employing protein precipitation by ammonium sulfate, ultrafiltration, and ion exchange chromatography resulted in 20-fold purification with a specific activity of 16 U/mg protein. Cellulase had a molecular weight of 76 kDa, 3.13 mM Km and 0.160 U/mg Vmax values. The enzyme displayed maximum activity at pH 9.0 and 40 °C and retained at least 80% of its activity at temperatures of 25–60 °C for 90 min and pH 4–12 for one hour. Stability tests also revealed the enzyme’s resilience to various reagents, metal ions, organic solvents, and detergents. Furthermore, the biotechnological applications of cellulase were explored, demonstrating its effectiveness in fabric biopolishing (removing pilling), as well as in the removal of fabric dyes. The research findings underscore the potential of B. pumilus VLC7 as a valuable source for eco-friendly industrial biotechnology applications.Graphical

Assessing the role of deep eutectic solvents in Yarrowia lipolytica inhibition

Yarrowia lipolytica has gained recognition as a microorganism with biological relevance and extensive biotechnological applications. Some of its features include a high enzyme secretion capacity and a high cell-density fermentation mode. Hexokinase (YlHxk) is a vital enzyme in Y. lipolytica growth since it catalyzes glucose metabolism through phosphorylation in the glycolytic pathway. Given the potential application of deep eutectic solvents (DES) as novel solvents in biotechnological processes, this study evaluated the influence of eighteen DES on the growth of Y. lipolytica. Furthermore, this work examined the effects of individual ions on the YlHxk enzyme by analyzing its enzymatic tunnel structure, molecule transport, and molecular docking. The results revealed a significant reduction in yeast growth in the presence of most DES compared to the control (medium without DES), with the exception of the [N8881]Cl: hexanoic acid (1:1) DES. The growth varied between 11.95 ± 0.60 and 0.68 ± 0.17g dry cell weight L-1. According to the enzymatic tunnel analysis, DES components associated with the lowest microbial growth values were transported through tunnel 1. On the other hand, DES components had their pathway facilitated through tunnel 2 ([N8881]+ and hexanoic acid) and showed growth values close to the control. Molecular docking analysis identified a similarity between all the ligands in this tunnel (including substrate and product), presenting binding interactions with the ASN273 amino acid of the YlHxk active site. Combining experimental results with computational tools provided promising insights at the molecular level, while also potentially reducing analysis costs and time, paving the way for similar approaches in broad biocatalytic reactions.

A scalable and autoclavable oxygen nanosensor platform for metabolic monitoring of Saccharomyces cerevisiae in a bioreactor and other in situ systems.

Polymer-encapsulated dye nanoparticle sensors are a valuable approach to achieving in situ analyte measurements with luminescence; however, typical emulsion-based nanosensors are poorly suited for large-scale biological samples due to limitations of synthesis scalability and stability. Branched polyethylenimine (PEI) is a versatile polymer scaffold ideal for constructing nanoparticles with various covalently conjugated moieties due to their high density of reactive primary amines, high water solubility, and biological stability. In this work, we used branched polyethylenimine as a scaffold-based approach for making a stable and scalable ratiometric oxygen sensor. Pt (II) tetracarboxyporphine was usedas an oxygen-sensing dye and coumarin 343 as a reference dye, all covalently linked to the PEI scaffold producing a product that could withstand sterilization procedures and easily be scaled. To minimize toxicity from the PEI scaffold, we conjugated it with 2000MW PEG.Theapplicability of the sensors was demonstratedin a 200mL Saccharomyces cerevisiae yeast culture, using orthogonal luminescent and electrochemical oxygen measurements to validate sensor response and measure the metabolic activity of the yeast in our culture. This approach was able to match the sensitivity of our electrochemical measurements while improving upon drawbacks of other luminescent methods of oxygen detection, demonstrating effective monitoring for at least 20h. Our scaffold-based approach is a modular and easily translatable technology that could be useful in various biotechnological applications.

Engineered PepFect14 analog for efficient cellular delivery of oligonucleotides.

The broad use of oligonucleotides (ON) in therapeutic and biotechnological applications is limited due to inefficient delivery methods. In parallel with lipids and polymeric carriers, cell-penetrating peptides (CPPs) are efficient vehicles for delivering nucleic acids of various types and activity into cells. In the current work, we examined the structural motifs required for the high efficacy of PepFect14, an often-used CPP for ON delivery, by introducing point mutations into the peptide sequence. We predicted the characteristics of modified CPPs, and analyzed their structure and ability to condense ONs into nanoparticles (NPs) using biophysical methods. We evaluated the ability of new PF14 analogs to deliver splicing switching oligonucleotides (SCO) and small interfering RNA (siRNA) in reporter cell lines, as well as microRNA miR-146a in human primary keratinocytes and in a mouse skin inflammation in vivo. Our findings indicate that the α-helical structure of PF14 is essential for efficient ON delivery, and mutations that disrupt the hydrophobic or cationic face in the peptide abolish NP formation and cellular internalization. PF14-Lys, an analog containing lysine residues instead of original ornithines, yielded a higher biological response to SCO and siRNA in the respective reporter cells than PF14. Furthermore, PF14-Lys efficiently delivered miRNA into keratinocytes and led to the subsequent downregulation of the target genes. Importantly, subcutaneously administered PF14-Lys-miR-146a NPs suppressed the inflammatory responses in mouse model of irritant contact dermatitis. In conclusion, our results suggest that PF14-Lys is a highly promising delivery vector for various oligonucleotides, applicable both in vitro and in vivo.