Biotechnological Applications Research Articles

Synthetic translational coupling element for multiplexed signal processing and cellular control

Abstract Repurposing natural systems to develop customized functions in biological systems is one of the main thrusts of synthetic biology. Translational coupling is a common phenomenon in diverse polycistronic operons for efficient allocation of limited genetic space and cellular resources. These beneficial features of translation coupling can provide exciting opportunities for creating novel synthetic biological devices. Here, we introduce a modular synthetic translational coupling element (synTCE) and integrate this design with de novo designed riboregulators, toehold switches. A systematic exploration of sequence domain variants for synTCEs led to the identification of critical design considerations for improving the system performance. Next, this design approach was seamlessly integrated into logic computations and applied to construct multi-output transcripts with well-defined stoichiometric control. This module was further applied to signaling cascades for combined signal transduction and multi-input/multi-output synthetic devices. Further, the synTCEs can precisely manipulate the N-terminal ends of output proteins, facilitating effective protein localization and cellular population control. Therefore, the synTCEs could enhance computational capability and applicability of riboregulators for reprogramming biological systems, leading to future applications in synthetic biology, metabolic engineering and biotechnology.

The Kinetics of Carbon‐Carbon‐Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the b‐ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon‐carbon‐bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here, we report enzyme kinetics of the KS‐mediated carbon‐carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS is likely rate‐limiting to the fatty acid biosynthesis, its kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS‐mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. We identify positive cooperativity of the KS dimer, which we suggest to affect the conformational variability of the multienzyme. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

The Kinetics of Carbon‐Carbon‐Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the b‐ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon‐carbon‐bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here, we report enzyme kinetics of the KS‐mediated carbon‐carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS is likely rate‐limiting to the fatty acid biosynthesis, its kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS‐mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. We identify positive cooperativity of the KS dimer, which we suggest to affect the conformational variability of the multienzyme. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

Valuable Ca/P Sources Obtained from Tuna Species’ By-Products Derived from Industrial Processing: Physicochemical and Features of Skeleton Fractions

The global tuna canning industry generates substantial volumes of by-products, comprising 50% to 70% of the total processed material. Traditionally, these by-products have been utilized in low-value products such as fish oils and fishmeal. However, there is significant potential to extract high-value compounds from these by-products, such as calcium phosphates (CaP), which can have pharmaceutical, agricultural and biotechnological applications. This work explores the potential of tuna canning by-products, particularly mineral-rich fractions (central skeleton, head and fish bones) as sources of calcium phosphates (CaP), offering a sustainable alternative to conventional synthetic derivatives within a circular bioeconomy framework. By-products from two of the most exploited species (yellowfin and skipjack) were subjected to enzymatic hydrolysis and chemical extraction, followed by controlled calcination to obtain CaP. The content of organic matter, nitrogen, total proteins, lipids and amino acids in the cleaned bones, as well as the main chemical bonds, structure and elemental composition (FT-Raman, XRD, XRF) were evaluated. Results indicated that the highest recovery yield of wet bones was achieved using the chemical method, particularly from the dorsal and caudal fins of yellowfin tuna. The proximal composition, with ash content ranging from 52% to 66% and protein content varying between 30% and 53%, highlights the potential of tuna skeleton substrates for plant growth formulations. Furthermore, variations in crystalline structures of the substrates revealed significant differences depending on the by-product source and species. XRD and Raman results confirmed a monophase calcium phosphate composition in most samples from both species, primarily based on hydroxyapatite (central skeleton, caudal and dorsal fin) or whitlockite/β-tricalcium phosphate (viscera), whereas the heads exhibited a biphasic composition. Comparing the species, yellowfin tuna (YF) exhibited a hydroxyapatite structure in the branchial arch and scales, while skipjack (SKJ) had a biphasic composition in these same regions.

Rotaxanes with a photoresponsive macrocycle modulate the lipid bilayers of large and giant unilamellar vesicles

Rotaxanes equipped with actuators hold great potential for developing highly functional molecular machines. Such systems could significantly enhance our ability to study and manipulate biological and artificial membranes. Here, we introduce a rotaxane with a ring featuring two azobenzene photoswitches, which retain their photoreversibility and can be stochastically shuttled along the axle in solution. Studies in model bilayers, supported by molecular dynamics simulations, show how azobenzene photoswitching alters the interaction of rotaxanes with surrounding lipids, leading to changes in lipid packing. Such changes in the lipid bilayer were leveraged to induce the light-triggered release of sulforhodamine B from large unilamellar vesicles. Additionally, light activation of the rotaxanes is shown to induce reversible contraction and expansion of giant unilamellar vesicles. The results provide novel insights into the interactions and operation of rotaxanes in lipid bilayers and their impact on membrane properties. This will aid in developing systems for precise membrane manipulation for applications in biomedicine and bioengineering.

Uncovering novel polyhydroxyalkanoate biosynthesis genes and unique pathway in yeast hanseniaspora valbyensis for sustainable bioplastic production

This study delves into the exploration of polyhydroxyalkanoate (PHA) biosynthesis genes within wild-type yeast strains, spotlighting the exceptional capabilities of isolate DMG-2. Through meticulous screening, DMG-2 emerged as a standout candidate, showcasing vivid red fluorescence indicative of prolific intracellular PHA granules. Characterization via FTIR spectroscopy unveiled a diverse biopolymer composition within DMG-2, featuring distinct functional groups associated with PHA and polyphosphate. Phylogenetic analysis placed DMG-2 within the Hanseniaspora valbyensis NRRL Y-1626 group, highlighting its distinct taxonomic classification. Subsequent investigation into DMG-2’s PHA biosynthesis genes yielded promising outcomes, with successful cloning and efficient PHA accumulation confirmed in transgenic E. coli cells. Protein analysis of ORF1 revealed its involvement in sugar metabolism, supported by its cellular localization and identification of functional motifs. Genomic analysis revealed regulatory elements within ORF1, shedding light on potential splice junctions and transcriptional networks influencing PHA synthesis pathways. Spectroscopic analysis of the biopolymer extracted from transgenic E. coli DMG2-1 provided insights into its co-polymer nature, comprising segments of PHB, PHV, and polyphosphate. GC-MS analysis further elucidated the intricate molecular architecture of the polymer. In conclusion, this study represents a pioneering endeavor in exploring PHA biosynthesis genes within yeast cells, with isolate DMG-2 demonstrating remarkable potential. The findings offer valuable insights for advancing sustainable bioplastic production and hold significant implications for biotechnological applications.

Extraction, characterization and antioxidative potentials of UV-screening compound, mycosporine-like amino acids from epilithic cyanobacterium Lyngbya sp. HKAR - 15.

Mycosporine-like amino acids (MAAs) are a unique class of UV-screening bioactive molecules with potent antioxidants and photoprotective properties, synthesized by various species of cyanobacteria in different habitats. The cyanobacterial biofilms play a crucial driver in the development of ecological communities. The current study examined the existence of the photoprotective MAAs in a novel epilithic cyanobacterium Lyngbya sp. strain HKAR-15 isolated from cyanobacterial biofilms on the rock surface. The isolated MAAs were identified, purified and characterized using UV-Vis spectroscopy, HPLC (High-Performance Liquid Chromatography), ESI-MS (Electrospray Ionization-Mass Spectrometry), FTIR (Fourier Transform Infrared Spectroscopy) and NMR (Nuclear Magnetic Resonance). The compounds were recognized as palythine (retention time (RT): 2.7min; UV λmax: 320nm; m/z: 245.02) and porphyra-334 (RT: 3.6min; UV λmax: 334nm; m/z: 347.1). FTIR spectroscopy analyses also revealed the presence of functional groups of both compounds. NMR spectroscopy analyses confirmed the presence of both palythine and porphyra-334. The UV-induced production of both MAAs was visualized under ultraviolet radiation (UVR) in contrast to the photosynthetically active radiation (PAR). The MAAs (palythine and porphyra-334) had a significant dose-dependent free radical scavenging capacity. The findings show that MAAs perform a dynamic role in the survival and photoprotection of cyanobacteria in hostile environments under high solar UV irradiances. These photoprotective compounds may have various biotechnological applications as well as role in the development of natural sunscreens.

Gene Delivery via Octadecylamine-Based Nanoparticles for iPSC Generation from CCD1072-SK Fibroblast Cells

This study presents a novel biotechnological approach using octadecylamine-based solid lipid nanoparticles (OCTNPs) for the first-time reprogramming of human CCD1072-SK fibroblast cells into induced pluripotent stem cells (iPSCs). OCTNPs, with an average size of 178.9 nm and a positive zeta potential of 22.8 mV, were synthesized, thoroughly characterized, and utilized as a non-viral vector to efficiently deliver reprogramming factors, achieving a remarkable transfection efficiency of 82.0%. iPSCs were characterized through immunofluorescence, flow cytometry, and RT-qPCR, confirming the expression of key pluripotency markers such as OCT4, SOX2, and KLF4, with alkaline phosphatase activity further validating their pluripotent state. Following this comprehensive characterization, the iPSCs were successfully differentiated into cardiomyocyte-like cells using 5-azacytidine. Our research highlights the innovative application of OCTNPs as a safe and effective alternative to viral vectors, addressing key limitations of iPSC reprogramming. The novel application of OCTNPs for efficient gene delivery demonstrates a powerful tool for advancing stem cell technologies, minimizing risks associated with viral vectors. These findings pave the way for further innovations in biotechnological applications, particularly in tissue engineering and personalized medicine.

Production and Antimicrobial Activities of Pyocyanin Extracts from Pseudomonas aeruginosa

Pyocyanin is a blue-green phenazine pigment synthesized by Pseudomonas aeruginosa that has significant biotechnological applications. The present study aims to investigate pyocyanin extracts of multi-drug resistant P. aeruginosa isolates sourced from hospital wastewater and evaluate their antimicrobial activity against a panel of clinically relevant pathogens such as Staphylococcus aureus, Salmonella typhi, Achromobacter xylosoxidans and Candida albicans. Nutrient broth and King’s A broth supplemented with 1% nutrient supplements such as rice water and groundnut cake powder were used as a production medium. Extracted pyocyanin was confirmed by FTIR analysis. The isolates P8 and P9 demonstrated of varying concentrations of pyocyanin in different media. Isolate P8 showed maximum pyocyanin production in King’s A broth compared to nutrient broth with pyocyanin yields 14.34 (µg/mL) and 5.63 (µg/mL), respectively, without the nutrient supplements. Preliminary antimicrobial activity of the pyocyanin extracts exhibited substantial inhibition of tested bacterial culture at a concentration of 25 mg/µl; however, did not show any antifungal activity against tested fungi.

Genomic insights into the cold adaptation and secondary metabolite potential of Pseudoalteromonas sp. WY3 from Antarctic krill

In the Antarctic marine ecosystem, krill play a pivotal role, yet the intricate microbial community intertwined with these diminutive crustaceans remains largely unmapped. In this study, we successfully isolated and characterized a unique bacterial strain, Pseudoalteromonas sp. WY3, from Antarctic krill. Genomic analysis revealed that WY3 harbors a multitude of genes associated with cold shock proteins, oxidoreductases, and enzymes involved in the osmotic stress response, equipping it with a robust molecular arsenal to withstand frigid Antarctic conditions. Furthermore, the presence of two distinct biosynthesis-related gene clusters suggests that WY3 has the potential to synthesize diverse secondary metabolites, including aryl polyenes and ribosomally synthesized and post-translationally modified peptides. Notably, the identification of genes encoding enzymes crucial for biological immunity pathways, such as apeH and ubiC, hints at a complex symbiotic relationship between WY3 and its krill host. This comprehensive study highlights the robust potential of WY3 for secondary metabolite production and its remarkable ability to thrive at extremely low temperatures in the Antarctic ecosystem, shedding light on the interplay between culturable microorganisms and their hosts in harsh environments, and providing insights into the underexplored microbial communities associated with Antarctic marine organisms and their role in environmental adaptation and biotechnological applications.

TPGPred: A Mixed-Feature-Driven Approach for Identifying Thermophilic Proteins Based on GradientBoosting

Thermophilic proteins maintain their stability and functionality under extreme high-temperature conditions, making them of significant importance in both fundamental biological research and biotechnological applications. In this study, we developed a machine learning-based thermophilic protein GradientBoosting prediction model, TPGPred, designed to predict thermophilic proteins by leveraging a large-scale dataset of both thermophilic and non-thermophilic protein sequences. By combining various machine learning algorithms with feature-engineering methods, we systematically evaluated the classification performance of the model, identifying the optimal feature combinations and classification models. Trained on a large public dataset of 5652 samples, TPGPred achieved an Accuracy score greater than 0.95 and an Area Under the Receiver Operating Characteristic Curve (AUROC) score greater than 0.98 on an independent test set of 627 samples. Our findings offer new insights into the identification and classification of thermophilic proteins and provide a solid foundation for their industrial application development.

Extremophiles and Extremophilic Behaviour—New Insights and Perspectives

Extremophiles, throughout evolutionary time, have evolved a plethora of unique strategies to overcome hardships associated with the environments they are found in. Modifying their genome, showing a bias towards certain amino acids, redesigning their proteins, and enhancing their membranes and other organelles with specialised chemical compounds are only some of those strategies. Scientists can utilise such attributes of theirs for a plethora of biotechnological and astrobiological applications. Moreover, the rigorous study of such microorganisms regarding their evolution and ecological niche can offer deep insight into science’s most paramount inquiries such as how life originated on Earth and whether we are alone in the universe. The intensification of studies involving extremophiles in the future can prove to be highly beneficial for humanity, even potentially ameliorating modern problems such as those related to climate change while also expanding our knowledge about the complex biochemical reactions that ultimately resulted in life as we know it today.

Antifungal Activity of Mexican Oregano (Lippia graveolens Kunth) Extracts from Industrial Waste Residues on Fusarium spp. in Bean Seeds (Phaseolus vulgaris L.)

Food security is essential to ensure everyone can access sufficient nutritious food. Cereals and legumes are fundamental foods worldwide. Phaseolus vulgaris L., the common bean, is an essential staple food in many nations worldwide. However, it is vulnerable to fusariosis, a disease caused by the fungus Fusarium spp. that can significantly decrease crop quality and yield. To combat plant diseases, industrial residues and plant residues are valuable due to their bioactive compounds with biotechnological applications. This study proposes using ethanolic extracts with phytochemical compounds, such as flavonoids, different from those reported in essential oils, to reduce the growth of Fusarium species both in vitro and in vivo. Industrial residues that are produced after extracting essential oils offer a promising alternative to develop organic biopesticides, promoting more sustainable and environmentally friendly agriculture.

Harnessing the optimization of enzyme catalytic rates in engineering of metabolic phenotypes.

The increasing availability of enzyme turnover number measurements from experiments and of turnover number predictions from deep learning models prompts the use of these enzyme parameters in precise metabolic engineering. Yet, there is no computational approach that allows the prediction of metabolic engineering strategies that rely on the modification of turnover numbers. It is also unclear if modifications of turnover numbers without alterations in the host's transcriptional regulatory machinery suffice to increase the production of chemicals of interest. Here, we present a constraint-based modeling approach, termed Overcoming Kinetic rate Obstacles (OKO), that uses enzyme-constrained metabolic models to predict in silico strategies to increase the production of a given chemical, while ensuring specified cell growth. We demonstrate that the application of OKO to enzyme-constrained metabolic models of Escherichia coli and Saccharomyces cerevisiae results in strategies that can at least double the production of over 40 compounds with little penalty to growth. Interestingly, we show that the overproduction of compounds of interest does not entail only an increase in the values of turnover numbers. Lastly, we demonstrate that a refinement of OKO, allowing also for manipulation of enzyme abundance, facilitates the usage of the available compendia and deep learning models of turnover numbers in the design of precise metabolic engineering strategies. Our results expand the usage of genome-scale metabolic models toward the identification of targets for protein engineering, allowing their direct usage in the generation of innovative metabolic engineering designs for various biotechnological applications.

Plant Growth Regulators: An Overview of WOX Gene Family

The adaptation of plants to land requires sophisticated biological processes and signaling. Transcription factors (TFs) regulate several cellular and metabolic activities, as well as signaling pathways in plants during stress and growth and development. The WUSCHEL-RELATED HOMEOBOX (WOX) genes are TFs that are part of the homeodomain (HD) family, which is important for the maintenance of apical meristem, stem cell niche, and other cellular processes. The WOX gene family is divided into three clades: ancient, intermediate, and modern (WUS) based on historical evolution linkage. The number of WOX genes in the plant body increases as plants grow more complex and varies in different species. Numerous research studies have discovered that the WOX gene family play a role in the whole plant’s growth and development, such as in the stem, embryo, root, flower, and leaf. This review comprehensively analyzes roles of the WOX gene family across various plant species, highlighting the evolutionary significance and potential biotechnological applications in stress resistance and crop improvement.

Gibberellin biosynthesis, its roles in plant physiology and abiotic stress responses

Gibberellins (GAs) are a group of phytohormones that significantly impact plant growth and development processes, including seed germination, stem elongation, and fruit development. These diterpenoid acids are influenced by environmental factors such as light and temperature, which can alter their concentration and activity. Initially discovered in the 1920s in the fungus Gibberella fujikuroi, over 136 GAs have been identified in various plants, fungi, and bacteria. A particularly active form of gibberellin, GA3, is widely used in agriculture to promote cell elongation, induce the grow of seedless fruits and improve crop yields. The synthesis and regulation of gibberellins in plants involve complex biosynthetic pathways that integrate both endogenous and environmental signals. Additionally, GAs play a role in stress response, helping plants adapt to challenging conditions like salinity and water scarcity. Research continues to explore the biotechnological applications of GAs, including their use in floriculture, fruit production, and improving plant resilience under stress conditions, making them essential for sustainable agriculture.

Crucial Roles of Brassinosteroids in Cell Wall Composition and Structure Across Species: New Insights and Biotechnological Applications.

Brassinosteroids (BR) are steroidal phytohormones essential for plant growth, development, and stress resistance. They fulfil this role partially by modulating cell wall structure and composition through the control of gene expression involved in primary and secondary cell wall biosynthesis and metabolism. This affects the deposition of cellulose, lignin, and other components, and modifies the inner architecture of the wall, allowing it to adapt to the developmental status and environmental conditions. This review focuses on the effects that BR exerts on the main components of the cell wall, cellulose, hemicellulose, pectin and lignin, in multiple and relevant plant species. We summarize the outcomes that result from modifying cell wall components by altering BR gene expression, applying exogenous BR and utilizing natural variability in BR content and describing new roles of BR in cell wall structure. Additionally, we discuss the potential use of BR to address pressing needs, such as increasing crop yield and quality, enhancing stress resistance and improving wood production through cell wall modulation.

Self‐Driving Lab for Solid‐Phase Extraction Process Optimization and Application to Nucleic Acid Purification

Sorptive bioprocesses are the basis for numerous biotechnological applications such as enzyme immobilization, biosensors, controlled drug delivery, water treatment, and molecular purification. Yet due to the complexity of these processes, their optimization is still time, labor, and cost‐intensive. This research presents a flexible self‐driving laboratory (SDL) designed for the accelerated development and optimization of solid‐phase extraction processes. As a use case, the SDL was used to optimize a DNA purification process using silica magnetic beads. Through the integration of robotics, machine learning, and data‐driven experimentation, the SDL demonstrates a highly accelerated process optimization with minimal human intervention. In the multistep purification approach, the system is able to optimize buffer compositions for DNA extraction from complex samples, demonstrating effectiveness in both conventional chaotropic salt‐based methods and innovative chaotropic salt‐free buffers. The study highlights the SDL's capability to autonomously refine process parameters, achieving significant enhancements in yield and purity of the product. This blueprint for future self‐driving optimization of bioprocess parameters showcases the potential of autonomous systems to revolutionize biochemical process development, offering insights into scalable, environmentally sustainable, and cost‐effective solutions.

Synthesis and evaluation of self‐healing polyvinyl alcohol‐tannic acid membranes for skin bio‐applications

AbstractAutonomic self‐healing films have shown limited mechanical strength, hindering their application as biomaterials, particularly in skin bioengineering. To address this challenge, we developed polyvinyl alcohol (PVA)‐tannic acid (TA)‐based membranes with improved self‐healing properties and strong mechanical performance. Using a blade coating method and freeze–thaw cycles (−10°C for 1 h, followed by room temperature thawing), the synthesized membranes demonstrated a tensile strength of 8.8 MPa before healing and 7.6 MPa after healing, achieving an 88% healing efficiency. Membrane thickness showed a slight reduction from 465 to 432 μm posthealing. After 3000 bending cycles at a 1 cm radius, the membranes maintained flexibility with no significant changes in water vapor transmission rate (WVTR) (7.66 g/m2·day) or oxygen transmission rate (OTR) (0.13 cm3/m2·day·bar). These results underscore the membranes' suitability for dynamic skin environments, combining robust mechanical properties with excellent self‐healing capabilities. The PVA‐TA membranes present a promising solution for advanced skin bioengineering applications.

Characterization of a novel salt- and solvent-tolerant esterase Dhs82 from soil metagenome capable of hydrolyzing estrogenic phthalate esters

Esterases that can function under extreme conditions are important for industrial processing and environmental remediation. Here, we report the identification of a salt- and solvent-tolerant esterase, Dhs82, from a soil metagenomic library. Dhs82 prefers short-chain p-nitrophenyl (p-NP) esters and exhibits enzymatic activity up to 1460 ± 61 U/mg towards p-NP butyrate. Meanwhile, Dhs82 can catalyze the hydrolysis of dialkyl phthalate esters, especially the widely-used diethyl phthalate (DEP), dipropyl phthalate (DPP) and di-n-butyl phthalate (DBP). Importantly, as an acidic protein with negative charges dominating its surface, Dhs82 is highly active and extraordinarily stable at high salinity. This property is quite rare among previously reported esterases/hydrolases capable of degrading phthalate esters (PAEs). In addition, Dhs82 activity can be significantly enhanced in the presence of solvents over a concentration range of 10–30 % (v/v). Notably, Dhs82 also showed high stability towards these solvents and solvent concentrations as high as 50–60 % (v/v) are required to inactivate Dhs82. Furthermore, molecular docking revealed the key residues, including the catalytic triad (Ser156, His281, and Asp251) and the surrounding Gly84 and Gly85, involved in the interaction of Dhs82 with DBP, depicting how Dhs82 degrades PAEs as a family IV esterase. Together, these diverse properties make Dhs82 a valuable candidate for both basic research and biotechnological applications.