Biotechnological Applications Research Articles

Strategic Management of Eco-Innovation and Emerging Technologies for Sustainability in Agro-Based Industries

Eco-innovation and emerging technologies are increasingly central to driving sustainability in agro-based industries. This study examines the impact of technological advancements on sustainable agricultural practices, focusing on innovations that reduce environmental impacts while enhancing productivity. According to the USDA, precision agriculture systems implemented in the Midwest show a 15-20% reduction in fertilizer use and a 10-15% increase in crop yields. Furthermore, the adoption of renewable energy solutions, such as solar-powered irrigation in India, has led to a 30% decrease in energy costs and a 25% reduction in greenhouse gas emissions (GHGs), as reported by the International Renewable Energy Agency (IRENA). Biotechnology applications, such as genetically modified drought-resistant crops, have reduced pesticide use by up to 37% while increasing crop resilience, according to a study published in Nature. This paper also explores the socio-environmental implications of these technologies, emphasizing the need for balanced development that integrates technological progress with ecological sustainability. Case studies from Brazil, India, and Kenya provide further insights into the successful implementation of these innovations. The findings suggest that leveraging emerging technologies in agro-based industries can significantly enhance sustainability, contributing to a more resilient and equitable human-environment relationship.

Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting.

The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA-guided RNA cutting by Cas13d entered the scene as a highly promising alternative to RNA interference to engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, "collateral damage" by indiscriminate off-target cutting tampered enthusiasm for these systems. Yet, how collateral activity, or even RNA target reduction depends on Cas13d and guide RNA abundance has remained unclear due to the lack of expression-tuning studies to address this question. Here we use precise expression-tuning gene circuits to show that both nonspecific and specific, on-target RNA reduction depend on Cas13d and guide RNA levels, and that nonspecific RNA cutting from trans cleavage might contribute to on-target RNA reduction. Using RNA-level control techniques, we develop new Multi-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid (MONARCH) gene circuits that achieve a high dynamic range with low basal on-target RNA reduction while minimizing collateral activity in human kidney cells and green monkey cells most frequently used in human virology. MONARCH should bring RNA-guided RNA cutting systems to the forefront, as easily applicable, programmable tools for transcriptome engineering in biotechnological and medical applications.

Expanded genome and proteome reallocation in a novel, robust Bacillus coagulans strain capable of utilizing pentose and hexose sugars.

Bacillus coagulans, a Gram-positive thermophilic bacterium, is recognized for its probiotic properties and recent development as a microbial cell factory. Despite its importance for biotechnological applications, the current understanding of B. coagulans' robustness is limited, especially for undomesticated strains. To fill this knowledge gap, we characterized the metabolic capability and performed functional genomics and systems analysis of a novel, robust strain, B. coagulans B-768. Genome sequencing revealed that B-768 has the largest B. coagulans genome known to date (3.94 Mbp), about 0.63 Mbp larger than the average genome of sequenced B. coagulans strains, with expanded carbohydrate metabolism and mobilome. Functional genomics identified a well-equipped genetic portfolio for utilizing a wide range of C5 (xylose, arabinose), C6 (glucose, mannose, galactose), and C12 (cellobiose) sugars present in biomass hydrolysates, which was validated experimentally. For growth on individual xylose and glucose, the dominant sugars in biomass hydrolysates, B-768 exhibited distinct phenotypes and proteome profiles. Faster growth and glucose uptake rates resulted in lactate overflow metabolism, which makes B. coagulans a lactate overproducer; however, slower growth and xylose uptake diminished overflow metabolism due to the high energy demand for sugar assimilation. Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) made up 60%-65% of the measured proteomes but were allocated differently when growing on xylose and glucose. The trade-off in proteome reallocation, with high investment in COG-C over COG-G, explains the xylose growth phenotype with significant upregulation of xylose metabolism, pyruvate metabolism, and tricarboxylic acid (TCA) cycle. Strain B-768 tolerates and effectively utilizes inhibitory biomass hydrolysates containing mixed sugars and exhibits hierarchical sugar utilization with glucose as the preferential substrate.IMPORTANCEThe robustness of B. coagulans makes it a valuable microorganism for biotechnology applications; yet, this phenotype is not well understood at the cellular level. Through phenotypic characterization and systems analysis, this study elucidates the functional genomics and robustness of a novel, undomesticated strain, B. coagulans B-768, capable of utilizing inhibitory switchgrass biomass hydrolysates. The genome of B-768, enriched with carbohydrate metabolism genes, demonstrates high regulatory capacity. The coordination of proteome reallocation in Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) is critical for effective cell growth, sugar utilization, and lactate production via overflow metabolism. Overall, B-768 is a novel, robust, and promising B. coagulans strain that can be harnessed as a microbial biomanufacturing platform to produce chemicals and fuels from biomass hydrolysates.

Antifungal and Antibacterial Potential of Essential Oil of Boswellia sacra Resin in the Biological Control

<p><span lang="EN-US" style="font-size:12.0pt"><span style="background:white"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif"">This study focuses on extraction of Boswillia sacra resin essential oil that was performed using the hydrodistillation method, resulting in a yield of 2.45%. Phytochemical screening of the essential oil provided information about its major compounds, which include terpenes and anthocyanins. To assess the antioxidant and antimicrobial effects of the essential oil, multiple methods were employed. Positive results were obtained from tests such as DPPH and ABTS free radical scavenging, inhibition of bovine serum albumin protein denaturation, and antifungal and antibacterial assays. These tests confirmed the presence of antifungal, antibacterial and antioxidant activities and </span></span></span></span><span lang="EN-US" style="font-size:12.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif"">suggest numerous biotechnological applications of the essential oil.</span></span></span></p>

Characterizing and Engineering Rhamnose-Inducible Regulatory Systems for Dynamic Control of Metabolic Pathways in Streptomyces.

Fine-tuning gene expression is of great interest for synthetic biotechnological applications. This is particularly true for the genus Streptomyces, which is well-known as a prolific producer of diverse natural products. Currently, there is an increasing demand to develop effective gene induction systems. In this study, bioinformatic analysis revealed a putative rhamnose catabolic pathway in multiple Streptomyces species, and the removal of the pathway in the model organism Streptomyces coelicolor impaired its growth on minimal media with rhamnose as the sole carbon source. To unravel the regulatory mechanism of RhaR, a LacI family transcriptional regulator of the catabolic pathway, electrophoretic mobility shift assays (EMSAs) were performed to identify potential target promoters. Multiple sequence alignments retrieved a consensus sequence of the RhaR operator (rhaO). A synthetic biology-based strategy was then deployed to build rhamnose-inducible regulatory systems, referred to as rhaRS1 and rhaRS2, by assembling the repressor/operator pair RhaR/rhaO with the well-defined constitutive kasO* promoter. Both rhaRS1 and rhaRS2 exhibited a high level of induced reporter activity, with no leaky expression. rhaRS2 has been proven successful for the programmable production of actinorhodin and violacein in Streptomyces. Our study expanded the toolkit of inducible regulatory systems that will be broadly applicable to many other Streptomyces species.

Seminar Proceedings

This collection of articles includes recent research and innovations presented at a national seminar focused on the application of biotechnologies in agriculture. The topics covered include sustainable agricultural practices, genetic advancements, and biotechnological interventions aimed at improving crop productivity and environmental sustainability.

Tutorial Review on the Set-Up and Running of Quantum Mechanical Cluster Models for Enzymatic Reaction Mechanisms.

Enzymes turnover substrates into products with amazing efficiency and selectivity and as such have great potential for use in biotechnology and pharmaceutical applications. However, details of their catalytic cycles and the origins surrounding the regio- and chemoselectivity of enzymatic reaction processes remain unknown, which makes the engineering of enzymes and their use in biotechnology challenging. Computational modelling can assist experimental work in the field and establish the factors that influence the reaction rates and the product distributions. A popular approach in modelling is the use of quantum mechanical cluster models of enzymes that take the first- and second coordination sphere of the enzyme active site into consideration. These QM cluster models are widely applied but often the results obtained are dependent on model choice and model selection. Herein, we show that QM cluster models can give highly accurate results that reproduce experimental product distributions and free energies of activation within several kcal mol-1, regarded that large cluster models with >300 atoms are used that include key hydrogen bonding interactions and charged residues. In this tutorial review, we give general guidelines on the set-up and applications of the QM cluster method and discuss its accuracy and reproducibility. Finally, several representative QM cluster model examples on metal-containing enzymes are presented, which highlight the strength of the approach.

Microbial lipases: propitious biocatalysts for various biotechnological applications

The use of microbial lipases is very important because they have a broad spectrum of catalytic reactions. Lipases catalyze reactions in aqueous and non-aqueous media and have advantages when compared to chemical catalysts, due to their specificity, enantioselectivity and stability at pH and temperature. The interest in research for microbial lipases occurs because of their applicability in various sectors and industries, such as food and beverage production, cosmetics, pharmaceuticals, surfactants, dairy products, treatment of effluents containing oils and fats, biofuels, among others. Biocatalysts can be obtained by submerged fermentation (SF) or solid-state fermentation (SSF), which has advantages over SF, because it is possible to use agroindustrial waste as substrate or growth support for microorganisms, becoming an alternative to reduce production costs. SSF is a promising technology for enzyme production, because besides using low-cost substrates, the biocatalyst can be produced in a more concentrated form, facilitating its recovery from the culture medium when necessary. Thus, this paper intends to discuss and review studies on microbial lipases, with direct application of the fermented solid (SSF), focusing on the main microorganisms, substrates and supports used in SSF, applicability of lipases in several industrial sectors, besides presenting conversion results using different microorganisms or/and substrates.

Substrate preference, RNA binding and active site versatility of Stenotrophomonas maltophilia nuclease SmNuc1, explained by a structural study.

Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5'-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

Structural basis of α-latrotoxin transition to a cation-selective pore

The potent neurotoxic venom of the black widow spider contains a cocktail of seven phylum-specific latrotoxins (LTXs), but only one, α-LTX, targets vertebrates. This 130 kDa toxin binds to receptors at presynaptic nerve terminals and triggers a massive release of neurotransmitters. It is widely accepted that LTXs tetramerize and insert into the presynaptic membrane, thereby forming Ca2+-conductive pores, but the underlying mechanism remains poorly understood. LTXs are homologous and consist of an N-terminal region with three distinct domains, along with a C-terminal domain containing up to 22 consecutive ankyrin repeats. Here we report cryoEM structures of the vertebrate-specific α-LTX tetramer in its prepore and pore state. Our structures, in combination with AlphaFold2-based structural modeling and molecular dynamics simulations, reveal dramatic conformational changes in the N-terminal region of the complex. Four distinct helical bundles rearrange and together form a highly stable, 15 nm long, cation-impermeable coiled-coil stalk. This stalk, in turn, positions an N-terminal pair of helices within the membrane, thereby enabling the assembly of a cation-permeable channel. Taken together, these data give insight into a unique mechanism for membrane insertion and channel formation, characteristic of the LTX family, and provide the necessary framework for advancing novel therapeutics and biotechnological applications.

Probing the function of C-terminal region of recombinant α-amylase BmaN1 from Bacillus megaterium NL3.

The α-amylase BmaN1 from Bacillus megaterium NL3 is a member of GH13_45 subfamily that has a conserved C-terminal region of approximately 30 residues. This region features a motif of five aromatic amino acids predicted to play a role in starch binding. This study aimed to unravel the role of the C-terminal region in starch hydrolysis. The full-length and C-terminally truncated forms of BmaN1 (BmaN1∆C) were expressed in Escherichia coli ArcticExpress (DE3), resulting in proteins with molecular weights of 56 kDa and 49 kDa, respectively. They exhibited comparable enzymatic activity in the hydrolysis of soluble starch, displaying versatility across a wide range of pH values, temperatures, and NaCl concentrations. BmaN1 and BmaN1∆C activities were inhibited by acarbose and were reduced by SDS and EDTA. In terms of binding and degrading the starch granules, BmaN1∆C showed lower affinity and activity in comparison to BmaN1. Our study indicates that the C-terminal region of BmaN1 significantly enhances its binding affinity and degrading the raw starches.IMPORTANCEα-Amylase (EC 3.2.1.1) stands as an endo-acting enzyme, essential for catalyzing the hydrolysis of α-1,4 glycosidic bonds within starch molecules. The relevance of α-amylases in biotechnological applications is substantial, constituting approximately 30% of the global enzyme market. Among these enzymes, BmaN1 was the first α-amylase identified to possess distinct catalytic residues within the GH13 family. BmaN1 from B. megaterium NL3 belongs to the GH13_45 subfamily. This subfamily is characterized by a conserved C-terminal region consisting of approximately 30 residues that contains a motif of five aromatic residues predicted to be involved in starch binding. Our study shows that the C-terminal effectively contributes to binding and degrading the raw starch granules. This pioneering research on BmaN1 expands our understanding of α-amylases and holds promise for innovative biotechnological advancements.

Exploring the Diversity and Function of Serine Proteases in Toxicofera Reptile Venoms: A Comprehensive Overview.

Toxicofera reptile venoms are composed of several toxins, including serine proteases. These proteases are glycosylated enzymes that affect the prey's hemostatic system. Their actions extend across the coagulation cascade, the kallikrein-kinin system, and platelet activation. Despite their specificity for different substrates, these enzymes are homologous across all toxicoferans and display high sequence similarity. The aim of this review is to compile decades of knowledge about venom serine proteases, showing the diversity of biochemically and biophysically characterized enzymes, their structural characteristics, advances in understanding their origin and evolution, as well as methods of obtaining enzymes and their biotechnological applications.

Optimization of Sugar Extraction Process from Date Waste Using Full Factorial Design Toward Its Use for New Biotechnological Applications.

In Tunisia, the date industry generates a large quantity of waste, raising environmental concerns. However, dates are rich in sugars, which offer a renewable source of nutrients for various applications. In this study, sugar extraction from two low-grade pitted date fruits (Alig and Kentichi) under ultrasound, was optimized using full factorial design. At 40 °C, for20 min, and with a liquid-to-solid ratio of 10 mL/g, the optimum sugar contents were 60.87% and 50.79% for the varieties Alig and Kentichi, respectively. The date extracts were chemically analyzed, revealing low fat and protein contents, but significant polyphenol and mineral contents in both varieties. HPLC-IR analysis revealed more inverted sugars (glucose and fructose) in the Alig variety and more sucrose in the Kentichi variety. FTIR and SEM analysis showed the efficiency of the ultrasonic treatment of the biomass in terms of improving mass transfer diffusion through ultrasonic cavitation. Thus, ultrasound-assisted extraction constitutes an effective method for the recovery of sugar from date waste.

Development of a New Isoxsuprine Hydrochloride-Based Hydroxylated Compound with Potent Antioxidant and Anti-Inflammatory Activities.

The scientific community actively pursuits novel compounds with biological activities. In this context, our study utilized the predicted data mining approach (PDMA), which can efficiently screen out biotransformable precursor candidates to produce new bioactive compounds. The PDMA was applied to Bacillus megaterium tyrosinase (BmTYR) to form new bioactive hydroxyl compounds from isoxsuprine hydrochloride (isoxsuprine). The results show that isoxsuprine could be biotransformed by BmTYR to form a new compound, 3''-hydroxyisoxsuprine. 3''-Hydroxyisoxsuprine exhibited 40- fold and 10-fold higher potent antioxidant and anti-inflammation activities than the precursor, isoxsuprine. The 3''-hydroxyisoxsuprine effectively mitigates the hyperimmune response in RAW 264.7 macrophages by inhibiting the upregulation of pro-inflammatory cytokine (IL-1β and IL-6) and inflammatory enzyme COX-2 gene expression triggered by LPS stimulation. This study illustrates that PDMA is an effective strategy for screening known natural and chemical compounds and for generating new bioactive compounds through biotransformation. Our newly produced compound has potential future applications in pharmacology and biotechnology.

Equimolar Cross-Coupling Using Reactive Coiled Coils for Covalent Protein Assemblies.

Biocompatible cross-coupling reactions enable the efficient covalent attachment of large biomolecules at near-stoichiometric ratios, ensuring the stability and integrity of the resulting products. We present an affinity-based peptide platform utilizing coiled coils containing reactive side chains for proximity-driven protein cross-coupling in the presence of a cross-linking agent. This platform supports both chemical synthesis and recombinant expression, using canonical amino acids to generate reactive affinity tags. Employing the E3/R3 coiled coil pair as a scaffold, we design four complementary coils with cysteine residues as cross-linking sites, achieving >90% conversion to covalent heterodimeric coupling products using 3,4-dibromomaleimide. Equimolar mixtures of proteins with reactive coils at their termini yield near-quantitative heterodimeric cross-coupling products. The strategic selection of complementary coiled coil pairs and cross-linking agents enables orthogonal assembly of macromolecules with diverse architectures. This method offers a versatile approach for creating covalent fusion proteins, enhancing their stability and functionality for applications in chemical biology, biotechnology, and medicine.

Risk Propensity and Acceptance of Gene-edited and Genetically Modified Food among US Consumers: A Comparison between Plants and Animal Products

Abstract Utilizing online survey data of US consumers, this study examines the extent to which consumers' acceptance of genetically modified (GM) and gene-edited (GE) food is driven by their risk attitudes. Our results indicate that individuals with high-risk propensity are more likely to accept both GM and GE food than individuals with low- and medium-risk propensity. Our results also find differences in consumers' attitudes toward plants and animal products in the context of both GM and GE. Intriguingly, these attitudinal differences can be explained by consumers' risk propensities. Specifically, both low- and medium-risk propensity consumers differentiate between plants and animal products; the latter is less acceptable than the former, indicating a tendency to have more concerns about the application of biotechnology to animals than plants. However, individuals with high-risk propensity do not differentiate between GM and GE plants and animal products. Our results suggest that policymakers, the food industry, and researchers need to consider these attitudinal differences while studying consumer attitudes toward GM and GE food. Failing to capture these attitudinal differences in studies focusing on consumer behavior toward GM and GE food may result in either overestimating or underestimating consumer response.

Investigation of γ-polyglutamic acid production via asynchronous saccharification and fermentation of raw corn starch.

Starch, a crucial raw material, has been extensively investigated for biotechnological applications. However, its application in γ-polyglutamic acid (γ-PGA) production remains unexplored. Based on γ-PGA output of Bacillus subtilis SCP010-1, a novel asynchronous saccharification and fermentation process for γ-PGA synthesis was implemented. The results revealed that a starch concentration of 20%, α-amylase dosage of 75 U/g, liquefaction temperature of 72℃, and γ-PGA yield of 36.31g/L was achieved. At a glucoamylase dosage of 100 U/g, saccharification 38h at 60℃, the yield of γ-PGA increased to 48.88g/L. The contents of total sugar, glucose, maltose and oligosaccharide in saccharified liquid were determined. Through batch fermentation of saccharified liquid in fermentor, the γ-PGA output was elevated to 116.08g/L. This study can offer a potential cost reduction of 40%, which can be a promising advancement in industrial γ-PGA production. Moreover, our approach can be applied in other starch-based fermentation industries.

Systematic Literature Review on Application of Biotechnology on Salak (Salacca spp.) Breeding Program Acceleration: The Unsolved Mysteries

Systematic Literature Review on Application of Biotechnology on Salak (Salacca spp.) Breeding Program Acceleration: The Unsolved Mysteries

Assessing the transcriptional landscape of Pseudomonas phage 201ϕ2-1: Uncovering the small regulatory details of a giant phage.

The transcriptional architecture of phages can deepen our understanding of the phage-host infection process and can be of key importance for phage engineering and biotechnological applications. Here, we applied ONT-cappable-sequencing, a long-read RNA-sequencing technique, to study the regulatory mechanisms of Pseudomonas infecting giant phage 201ϕ2-1. We identified 67 promoters and 132 terminators that together represent 92 transcriptional units. A full comparison of these data to the transcriptome of model Pseudomonas phage ϕKZ confirmed that the transcriptional programs of these prototypes of the Serwervirus and Phikzvirus genera are largely conserved, despite some subtle regulatory differences. Evidence supporting these shared mechanisms include the identification of highly similar sequence motifs for regulatory elements in both phages and the conservation of regulatory elements loci relative to homologous genes in each phage. Moreover, we discovered a sRNA in 201ϕ2-1 that is highly conserved among prototype members of different giant phage genera. Sequencing of the 201ϕ2-1 host genome resulted in its reclassification as Pseudomonas atacamensis, a close relative of the important agricultural biocontrol agent Pseudomonas chlororaphis. Finally, we conducted invivo assays of eight 201ϕ2-1 terminators and found them to strongly terminate transcription in P. chlororaphis. Control elements from phage transcriptional programs have a rich history for applications in biotechnology. In these studies, we demonstrate new insight into the transcriptional program of 201ϕ2-1 and demonstrate the potential of its regulatory elements for novel and useful tools for synthetic biology circuitry.

Dynamical stiffening of dsDNA confined and stretched in a nanochannel

The internal dynamics of double-stranded DNA (dsDNA) confined within channels of diameters ranging from the persistence length to twice that length were investigated using fluorescence microscopy. By analysing spatiotemporal intensity fluctuations, we derived the intermediate dynamic structure factor. The structure factor consistently exhibited behaviour characteristic of Rouse dynamics, regardless of the channel diameter. Notably, as the channel diameter decreased, the DNA molecules became increasingly elongated along the channel, leading to shorter relaxation times. These findings indicate a dynamic stiffening effect, where the effective spring constant of a stretched polymer chain increases. We propose that this effect has significant implications for controlling DNA motion in biological and biotechnological applications.