Synthetic Biology Research Articles

Quinoline-hydrazone Conjugates: Recent Insights into Synthetic Strategies, Structure-activity Relationship, and Biological Activities

: The fusion of two distinct and free pharmacologically active chemical moieties into single conjugate molecules can result in synergized pharmacological action of both moieties into the new composite molecule. Ultimately, it increases the therapeutic potentialof the newly formed hybrid compound which is more than the combination of each specific moiety’s therapeutic potential. So nowadays, it is common practice to combine at least two pharmacophores to create a particular compound with a powerful therapeutic effects. Quinoline has been reported with multiple pharmacological activities and industrial applications. On the other hand, hydrazones are also found very useful as herbicides, acaricides, rodenticides, insecticides, and various therapeutic applications. The conjugate containing quinoline and hydrazone is also being used as an anticancer, antibacterial, antifungal, antimalarial, anticonvulsant, anti-inflammatory, and antioxidant. The combination of two moieties yields a better therapeutic effect because of excellent efficacy and fewer side effects. Several synthetic methods have been employed in recent times to synthesize quinoline-hydrazone conjugates which are listed in the manuscript with their merits and demerits. The structure-activity relationship relating to their pharmacological actions with molecular structure has also been highlighted. The article aims to provide a good toolkit and comprehension to the medicinal chemists, for their future work, comprising of quinoline-hydrazone hybrid compounds.

Transforming Lignocellulosic Biomass into Biofuels: Recent Innovations in Pretreatment and Bioconversion Techniques

Abstract: This scholarly review investigates contemporary advancements in the conversion of lignocellulosic biomass to biofuels, emphasizing innovative pretreatment and bioconversion technologies that aim to surmount the intrinsic challenges associated with the processing of this complex biomass. Lignocellulosic materials, which are predominantly comprised of cellulose, hemicellulose, and lignin, represent a renewable and plentiful source for biofuel production; however, their structural complexity necessitates sophisticated methodologies for the effective degradation of resistant components. The review scrutinizes a variety of pretreatment methodologies, encompassing physical, chemical, and burgeoning techniques such as plasma-assisted processing, which are engineered to augment cellulose accessibility for enzymatic hydrolysis. Additionally, it elucidates the progress made in bioconversion processes, concentrating on enzymatic hydrolysis, microbial fermentation, and consolidated bioprocessing (CBP), wherein recent endeavors in genetic engineering are refining microbial strains to enhance yield and efficiency. By addressing economic, technological, and environmental challenges, this article emphasizes the role of integrated biorefineries and innovative biotechnologies in facilitating scalable and cost-effective production of lignocellulosic biofuels. Prospective research trajectories include the formulation of sustainable pretreatment techniques and the advancement of synthetic biology to fully harness the potential of lignocellulosic biomass as a renewable energy resource. Ultimately, this review accentuates the significance of lignocellulosic biofuels as a feasible alternative to fossil fuels, thereby contributing to energy sustainability and climate change mitigation through diminished carbon emissions

Application of a Cell-Free Synthetic Biology Platform for the Reconstitution of Teleocidin B and UK-2A Precursor Biosynthetic Pathways.

We report the successful cell-free reconstitution of two natural product biosynthetic pathways of divergent complexity and structural classes. We first constructed the teleocidin biosynthetic pathway using our BY-2 (tobacco) cell-free protein synthesis (CFPS) system. We discovered a direct interaction between TleA and MbtH, and showed that the BY-2 system is capable of producing more than 80 mg/L teleocidin B-3 with cofactor supplementation and ∼20 mg/L with no cofactors supplemented, demonstrating the high metabolic activity of the system. We then extended our methodology and report the first successful cell-free biosynthesis of UK-2 diol (precursor to the commercially valuable secondary metabolite UK-2A) from simple building blocks by refactoring a complex pathway of 10 proteins in the wheat germ CFPS system. We show that plant CFPS systems are suitable for reconstructing pathways and identifying the functions of uncharacterized genes linked to biosynthetic gene clusters and rate-limiting biosynthetic steps.

Quantitative Characterization of Gene Regulatory Circuits Associated With Fungal Secondary Metabolism to Discover Novel Natural Products.

Microbial genetic circuits are vital for regulating gene expression and synthesizing bioactive compounds. However, assessing their strength and timing, especially in multicellular fungi, remains challenging. Here, an advanced microfluidic platform is combined with a mathematical model enabling precise characterization of fungal gene regulatory circuits (GRCs) at the single-cell level. Utilizing this platform, the expression intensity and timing of 30 transcription factor-promoter combinations derived from two representative fungal GRCs, using the model fungus Aspergillus nidulans are determined. As a proof of concept, the selected GRC combination is utilized to successfully refactor the biosynthetic pathways of bioactive molecules, precisely control their production, and activate the expression of the silenced biosynthetic gene clusters (BGCs). This study provides insights into microbial gene regulation and highlights the potential of platform in fungal synthetic biology applications and the discovery of novel natural products.

Deep learning revealed the distribution and evolution patterns for invertible promoters across bacterial lineages.

Invertible promoters (invertons) are crucial regulatory elements in bacteria, facilitating gene expression changes under stress. Despite their importance, their prevalence and the range of regulated gene functions are largely unknown. We introduced DeepInverton, a deep learning model that identifies invertons across a broad phylogenetic spectrum without using sequencing reads. By analyzing 68 733 bacterial genomes and 9382 metagenomes, we have uncovered over 200 000 nonredundant invertons and have also highlighted their abundance in pathogens. Additionally, we identified a post-Cambrian Explosion increase of invertons, paralleling species diversification. Furthermore, we revealed that invertons regulate diverse functions, including antimicrobial resistance and biofilm formation, underscoring their role in environmental adaptation. Notably, the majority of inverton identifications by DeepInverton have been confirmed by the in vitro experiments. The comprehensive inverton profiles have deepened our understanding of invertons at pan-genome and pan-metagenome scales, enabling a broad spectrum of applications in microbial ecology and synthetic biology.

Differential amino acid usage leads to ubiquitous edge effect in proteomes across domains of life that can be explained by amino acid secondary structure propensities

Amino acids are the building blocks of proteins and enzymes which are essential for life. Understanding amino acid usage offers insights into protein function and molecular mechanisms underlying life histories. However, genome-wide patterns of amino acid usage across domains of life remain poorly understood. Here, we analysed the proteomes of 5590 species across four domains and found that only a few amino acids are consistently the most and least used. This differential usage results in lower amino acid usage diversity at the most and least frequent ranks, creating a ubiquitous inverted U-shape pattern of amino acid diversity and rank which we call an ‘edge effect’ across proteomes and domains of life. This effect likely stems from protein secondary structural constraints, not the evolutionary chronology of amino acid incorporation into the genetic code, highlighting the functional rather than evolutionary influences on amino acid usage. We also tested other contemporary hypotheses regarding amino acid usage in proteomes and found that amino acid usage varies across life’s domains and is only weakly influenced by growth temperature. Our findings reveal a novel and pervasive amino acid usage pattern across genomes with the potential to help us probe deep evolutionary relationships and advance synthetic biology.

Current Technologies in Snake Venom Analysis and Applications

This comprehensive review explores the cutting-edge advancements in snake venom research, focusing on the integration of proteomics, genomics, transcriptomics, and bioinformatics. Highlighting the transformative impact of these technologies, the review delves into the genetic and ecological factors driving venom evolution, the complex molecular composition of venoms, and the regulatory mechanisms underlying toxin production. The application of synthetic biology and multi-omics approaches, collectively known as venomics, has revolutionized the field, providing deeper insights into venom function and its therapeutic potential. Despite significant progress, challenges such as the functional characterization of toxins and the development of cost-effective antivenoms remain. This review also discusses the future directions of venom research, emphasizing the need for interdisciplinary collaborations and new technologies (mRNAs, cryo-electron microscopy for structural determinations of toxin complexes, synthetic biology, and other technologies) to fully harness the biomedical potential of venoms and toxins from snakes and other animals.

Calcium‐Dependent Lipopeptide Antibiotics against Drug‐Resistant Pathogens Discovered via Host‐Dependent Heterologous Expression of a Cloned Biosynthetic Gene Cluster

AbstractHistorically, small molecules biosynthesised by bacteria have been an excellent source for antibacterial drugs. Today, however, the rediscovery of known compounds is a significant hurdle to developing new antimicrobials. Here we use a genome mining and synthetic biology approach to discover the ambocidins: calcium‐dependent lipodepsipeptides that are active against drug‐resistant Gram‐positive pathogens. By cloning a silent biosynthetic gene cluster (the amb cluster) from Streptomyces ambofaciens ATCC 2387 and integrating this into the chromosome of Streptomyces avermitilis we induce expression of ambocidin A and B: two new Nϵ‐hydroxyarginine‐containing cyclic lipodepsipeptides active against drug‐resistant Gram‐positive pathogens. Using a panel of Streptomyces host strains, we show that the choice of heterologous host is critical for producing the biologically active compounds, and that inappropriate host choice leads to aberrant production inactive derivatives. We show that Nϵ‐hydroxyarginine is the product of a heme‐dependent oxygenase and that it enhances biological activity. Ambocidin A inhibits cell wall biosynthesis by binding to Lipid II at a different site than vancomycin. Furthermore, unlike daptomycin, ambocidin A retains potent antimicrobial activity in the presence of lung surfactant, giving it the potential to treat bacterial pneumonia. Our work expands the family of calcium‐dependent lipopeptide antibiotics with a new member exhibiting a distinct mechanism of action.

Development of a xylose-inducible and glucose-insensitive expression system for Parageobacillus thermoglucosidasius

Inducible expression systems are pivotal for governing gene expression in strain engineering and synthetic biotechnological applications. Therefore, a critical need persists for the development of versatile and efficient inducible expression mechanisms. In this study, the xylose-responsive promoter xylA5p and its transcriptional regulator XylR were identified in Parageobacillus thermoglucosidasius DSM 2542. By combining promoter xylA5p with its regulator XylR, fine-tuning the expression strength of XylR, and reducing the glucose catabolite repression on xylose uptake, we successfully devised a xylose-inducible and glucose-insensitive expression system, denoted as IExyl*. This system exhibited diverse promoter strengths upon induction with xylose at varying concentrations and remained unhindered in the presence of glucose. Moreover, we showed the applicability of IExyl* in P. thermoglucosidasius by redirecting metabolic flux towards riboflavin biosynthesis, culminating in a 2.8-fold increase in riboflavin production compared to that of the starting strain. This glucose-insensitive and xylose-responsive expression system provides valuable tools for designing optimized biosynthetic pathways for high-value products and facilitates future synthetic biology investigations in Parageobacillus.Key points• A xylose-inducible and glucose-insensitive expression system IExyl* was developed.• IExyl* was applied to enhance the riboflavin production in P. thermoglucosidasius• A tool for metabolic engineering and synthetic biology research in Parageobacillus strains.

The heterologous expression of novel recombinant protein composed of HN and F moieties of Newcastle disease virus and immunogenicity evaluation in mouse model

Background and Objectives: The rapid spread of Newcastle disease (ND), driven by extensive commercial exchange in the poultry industry, necessitates urgent preventive measures. Although effective vaccines against the Newcastle disease virus (NDV) have been used since 1940, recent outbreaks and the limitations of current vaccines highlight the need for improved solutions. Advances in synthetic biology, reverse vaccinology, molecular biology, and recombinant DNA technology over the past 20 years have led to the development of recombinant vaccines, which offer enhanced protection and broader immuno- genic coverage against NDV. This study aimed to express the immunogenic domains of Hemagglutinin Neuraminidase (HN) and Fusion (F) glycoproteins, linked to the heat-labile enterotoxin B subunit (LTB) bio-adjuvant, to develop an effective and reliable recombinant vaccine for NDV. Materials and Methods: In this study, the L(HN)2F protein, composed of the LTB bio-adjuvant and the immunogenic regions of the doubled Hemagglutinin Neuraminidase (HN-HN) and Fusion (F) epitope, was expressed in Escherichia coli. Subcutaneous injection was used to evaluate the humoral immune response in mice and the result was compared with B1 vaccine. Results: The induction of strong humoral immune responses proved the strong immunoreactivity of the recombinant protein. Conclusion: The IgG elicited by the recombinant proteins was comparable to that of the commercial B1 vaccine against NDV, indicating its potential as a viable candidate for further development and evaluation as a recombinant vaccine against NDV.

Engineering Yarrowia lipolytica to Produce l-Malic Acid from Glycerol.

The declining availability of cheap fossil-based resources has sparked growing interest in the sustainable biosynthesis of organic acids. l-Malic acid, a crucial four-carbon dicarboxylic acid, finds extensive applications in the food, chemical, and pharmaceutical industries. Synthetic biology and metabolic engineering have enabled the efficient microbial production of l-malic acid, albeit not in Yarrowia lipolytica, an important industrial microorganism. The present study aimed to explore the potential of this fungal species for the production of l-malic acid. First, endogenous biosynthetic genes and heterologous transporter genes were overexpressed in Y. lipolytica to identify bottlenecks in the l-malic acid biosynthesis pathway grown on glycerol. Second, overexpression of isocitrate lyase, malate synthase, and malate dehydrogenase in the glyoxylate cycle pathway and introduction of a malate transporter from Schizosaccharomyces pombe significantly boosted l-malic acid production, which reached 27.0 g/L. A subsequent increase to 37.0 g/L was attained through shake flask medium optimization. Third, adaptive laboratory evolution allowed the engineered strain Po1g-CEE2+Sp to tolerate a lower pH and to accumulate a higher amount of l-malic acid (56.0 g/L). Finally, when scaling up to a 5 L bioreactor, a titer of 112.5 g/L was attained. In conclusion, this study demonstrates for the first time the successful production of l-malic acid in Y. lipolytica by combining metabolic engineering and laboratory evolution, paving the way for large-scale sustainable biosynthesis of this and other organic acids.

Planetary scale climatic change through bioengineering the microbial world. A technofix imaginary

This article critically analyses the imaginary that is emerging from attempts to mitigate planetary scale climatic change through bioengineering the microbial world, that is, through synthetic biology. We explore how engineering microbial life is put forth by its advocates as a viable means of mitigating climate change and thus conferring benefit for future human and more-than-human life. We discuss how these promises, while far-reaching, translate into a technofix imaginary, whereby a future existence of human and untold more-than-human life is contingent on the realisation of such promises. Moreover, we illustrate how the engineering imaginary of synthetic biology is not limited to life alone, but is rather the byproduct of an aspiration to control global ecosystems and climates, and even the course of evolution, via the manufacturing and control of life at the microbial level. We conclude with a critique of how such an imaginary remains incommensurate with fundamental aspects of life that elude human control, and will arguably continue to do so. Thus, the technofix imaginary of synthetic biology is likely to be scaled down in order to reflect the actual achievements of the field, and not its speculative applications.

Chemical Communication between Giant Vesicles and Gated Nanoparticles for Strip-Based Sensing.

Inspired by nature, the development of artificial micro/nanosystems capable of communicating has become an emergent topic in nanotechnology, synthetic biology, and related areas. However, the demonstration of actual applications still has to come. Here, we demonstrate how chemical communication between micro- and nanoparticles can be used for the design of sensing systems. To realize this, we synergistically combine two different types of particles: i.e., giant unilamellar vesicles (GUVs) as senders and gated mesoporous nanoparticles as receivers. The use of engineered GUVs allows the detection of analytes based on responsive membranes, while the use of gated nanoparticles allows a straightforward application on test strips with smartphone-based detection. In addition, we demonstrate that the combined communication system exhibits signal amplification and its application in real samples employing the bacterial toxin α-hemolysin as target analyte. Altogether, our report presents a new route for engineering sensing systems based on the combination of communicative micro/nanoparticles.

Bacterial live therapeutics for human diseases.

The genomic revolution has fueled rapid progress in synthetic and systems biology, opening up new possibilities for using live biotherapeutic products (LBP) to treat, attenuate or prevent human diseases. Among LBP, bacteria-based therapies are particularly promising due to their ability to colonize diverse human tissues, modulate the immune system and secrete or deliver complex biological products. These bacterial LBP include engineered pathogenic species designed to target specific diseases, and microbiota species that promote microbial balance and immune system homeostasis, either through local administration or the gut-body axes. This review focuses on recent advancements in preclinical and clinical trials of bacteria-based LBP, highlighting both on-site and long-reaching strategies.

Difficult-to-treat Takayasu arteritis: a case-based review.

Takayasu arteritis is a rare chronic inflammatory large vessel vasculitis which affects the aorta and its large branches. The diagnosis is based on the 2022 ACR/EULAR classification criteria for Takayasu arteritis. The management of this vasculitis is challenging. Although it is corticosteroid-responsive, relapses and disease progression are common. Thus, it is possible to resort to alternative conventional synthetic disease-modifying anti-rheumatic drugs and biologics, as second-line such as tumor necrosis factor-alpha inhibitors, tocilizumab, or JAK inhibitors as second-line agents is possible. Nevertheless, in some complex cases, the vasculitis remains active despite different proposed therapeutic lines, and a multitarget approach could induce sustained remission. We report herewith a case of 33-female patient with a refractory Takayasu arteritis which remained active after three different therapeutic lines with tocilizumab, then infliximab, then Upadacitinib. Finally, we consider a successful multitarget approach with a combination of infliximab, Upadacitinib, and methotrexate.

A Review of Antimicrobial Peptides: Structure, Mechanism of Action, and Molecular Optimization Strategies

Antimicrobial peptides (AMPs) are bioactive macromolecules that exhibit antibacterial, antiviral, and immunomodulatory functions. They come from a wide range of sources and are found in all forms of life, from bacteria to plants, vertebrates, and invertebrates, and play an important role in controlling the spread of pathogens, promoting wound healing and treating tumors. Consequently, AMPs have emerged as promising alternatives to next-generation antibiotics. With advancements in systems biology and synthetic biology technologies, it has become possible to synthesize AMPs artificially. We can better understand their functional activities for further modification and development by investigating the mechanism of action underlying their antimicrobial properties. This review focuses on the structural aspects of AMPs while highlighting their significance for biological activity. Furthermore, it elucidates the membrane targeting mechanism and intracellular targets of these peptides while summarizing molecular modification approaches aimed at enhancing their antibacterial efficacy. Finally, this article outlines future challenges in the functional development of AMPs along with proposed strategies to overcome them.

De Novo Synthesis of Anticholinergic Hyoscyamine and Scopolamine in Nicotiana benthamiana Based on Elucidating Tropane Alkaloid Biosynthetic Pathway of Anisodus luridus

Anisodus luridus, a perennial herb belonging to the genus Anisodus of the Solanaceae family, is an important Tibetan medicinal plant that produces pharmaceutical tropane alkaloids (TAs) including hyoscyamine and scopolamine. Its high yield of hyoscyamine makes A. luridus a valuable plant source for commercially producing TAs. In this study, we conduct homologous gene research across transcriptome data of different tissues together with functionally tested sequences in Atropa belladonna as a reference and identify 13 candidate genes for TAs biosynthesis in A. luridus. The results show that these 13 TAs biosynthesis genes identified in A. luridus were highly conserved in terms of sequence similarity and gene expression patterns compared to A. belladonna, suggesting that the two species may share the same biosynthetic pathway for TAs biosynthesis. Furthermore, scopolamine was detected in Nicotiana benthamiana leaves when these 13 enzymes were co-expressed in N. benthamiana, which confirmed that these 13 TAs biosynthesis genes are involved in the biosynthesis of TAs. The results of our study not only systematically elucidate the tropane alkaloid biosynthetic pathway of A. luridus, but also realize the de novo synthesis of TAs in N. benthamiana for the first time. It is now possible to make N. benthamiana a potential source for TAs production through synthetic biology techniques.

Omic-driven strategies to unveil microbiome potential for biodegradation of plastics: a review.

Plastic waste accumulation has lately been identified as the leading and pervasive environmental concern, harming all living beings, natural habitats, and the global market. Given this issue, developing ecologically friendly solutions, such as biodegradation instead of standard disposal, is critical. To effectively address and develop better strategies, it is critical to understand the inter-relationship between microorganisms and plastic, the role of genes and enzymes involved in this process. However, the complex nature of microbial communities and the diverse mechanisms involved in plastic biodegradation have hindered the development of efficient plastic waste degradation strategies. Omics-driven approaches, encompassing genomics, transcriptomics and proteomics have revolutionized our understanding of microbial ecology and biotechnology. Therefore, this review explores the application of omics technologies in plastic degradation studies and discusses the key findings, challenges, and future prospects of omics-based approaches in identifying novel plastic-degrading microorganisms, enzymes, and metabolic pathways. The integration of omics technologies with advanced molecular technologies such as the recombinant DNA technology and synthetic biology would guide in the optimization of microbial consortia and engineering the microbial systems for enhanced plastic biodegradation under various environmental conditions.

Role of Biocontrol Agents in Suppressing Plant Pathogen: A Compressive Analysis

Biocontrol agents have emerged as important tools in the quest for sustainable agriculture, offering environmentally friendly alternatives to chemical pesticides for managing plant pathogens and pests. This comprehensive review explores the advancements in biocontrol agent research, focusing on the exploration of new agents, genetic engineering, and integration into Integrated Pest Management (IPM) systems. The discovery of novel biocontrol agents from diverse environments, including plant microbiomes and marine ecosystems, has expanded the arsenal available for agricultural use, while genetic engineering and synthetic biology have enhanced the efficacy of existing agents by improving their production of antimicrobial metabolites and stress tolerance. Additionally, the development of synthetic microbial consortia and innovative delivery systems, such as encapsulation and nanotechnology, has improved the stability and application efficiency of biocontrol agents in various agricultural settings. The review also highlights the importance of sustainable and eco-friendly approaches, such as the use of organic amendments and crop rotation, to enhance the effectiveness of biocontrol strategies. These methods not only improve soil health and biodiversity but also reduce the reliance on chemical inputs. However, the widespread adoption of biocontrol agents faces challenges, including environmental variability, host specificity, and the need for supportive regulatory frameworks. Harmonizing regulatory processes, promoting public awareness, and providing incentives for sustainable practices are essential for overcoming these barriers. International cooperation and knowledge exchange are crucial for advancing research and ensuring that biocontrol agents become integral components of modern agriculture. This review underscores the potential of biocontrol agents to contribute significantly to global food security and environmental sustainability, provided that ongoing research and innovation continue to address existing challenges and expand their practical applications in diverse agricultural contexts.

D’Arcy Thompson and Synthetic Biology—Then and Now

AbstractThough often presented as a recent scientific endeavor, synthetic biology began in the 19th century and was a particularly active field in the years preceding the publication of D’Arcy Thompson’s On Growth and Form. Much synthetic biology of the era was devoted to the construction of nonliving chemical systems that would undergo morphogenesis or dynamic behaviors which had been observed in living organisms. The point was to show that “life-like” structure and behavior could be generated by physicochemical laws and required no vitalist element. D’Arcy Thompson’s careful analysis of physicochemical morphogenetic mechanisms as possible explanations of organic form links closely to this way of thinking. In the modern era, when we can genetically engineer cells to undergo specific behaviors, and program cells to undergo simple morphogenetic behaviors of the kind that Thompson and others felt might underly natural morphogenesis, it is possible to test whether they will in fact produce a predictable multicellular shape. This addresses essentially the same questions about the morphogenetic role of physicochemical forces, such as surface tension, but does so “the other way round”: physicochemical mechanisms are not being used as models for morphogenesis by natural cells but rather as a means to engineer cells to make designed forms.