Engineering functional thermostable proteins using ancestral sequence reconstruction.

Natural proteins are often only slightly more stable in the native state than the denatured state, and an increase in environmental temperature can easily shift the balance toward unfolding. Therefore, the engineering of proteins to improve protein stability is an area of intensive research. Thermostable proteins are required to withstand industrial process conditions, for increased shelf-life of protein therapeutics, for developing robust 'biobricks' for synthetic biology applications, and for research purposes (e.g., structure determination). In addition, thermostability buffers the often destabilizing effects of mutations introduced to improve other properties. Rational design approaches to engineering thermostability require structural information, but even with advanced computational methods, it is challenging to predict or parameterize all the relevant structural factors with sufficient precision to anticipate the results of a given mutation. Directed evolution is an alternative when structures are unavailable but requires extensive screening of mutant libraries. Recently, however, bioinspired approaches based on phylogenetic analyses have shown great promise. Leveraging the rapid expansion in sequence data and bioinformatic tools, ancestral sequence reconstruction can generate highly stable folds for novel applications in industrial chemistry, medicine, and synthetic biology. This review provides an overview of the factors important for successful inference of thermostable proteins by ancestral sequence reconstruction and what it can reveal about the determinants of stability in proteins.

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates.

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

Current Landscape of Methods to Evaluate Antimicrobial Activity of Natural Extracts.

Natural extracts have been and continue to be used to treat a wide range of medical conditions, from infectious diseases to cancer, based on their convenience and therapeutic potential. Natural products derived from microbes, plants, and animals offer a broad variety of molecules and chemical compounds. Natural products are not only one of the most important sources for innovative drug development for animal and human health, but they are also an inspiration for synthetic biology and chemistry scientists towards the discovery of new bioactive compounds and pharmaceuticals. This is particularly relevant in the current context, where antimicrobial resistance has risen as a global health problem. Thus, efforts are being directed toward studying natural compounds' chemical composition and bioactive potential to generate drugs with better efficacy and lower toxicity than existing molecules. Currently, a wide range of methodologies are used to analyze the in vitro activity of natural extracts to determine their suitability as antimicrobial agents. Despite traditional technologies being the most employed, technological advances have contributed to the implementation of methods able to circumvent issues related to analysis capacity, time, sensitivity, and reproducibility. This review produces an updated analysis of the conventional and current methods to evaluate the antimicrobial activity of natural compounds.

Chemical Properties and Biological Activity of Bee Pollen.

Pollen, a remarkably versatile natural compound collected by bees for its abundant source of proteins and nutrients, represents a rich reservoir of diverse bioactive compounds with noteworthy chemical and therapeutic potential. Its extensive biological effects have been known and exploited since ancient times. Today, there is an increased interest in finding natural compounds against oxidative stress, a factor that contributes to various diseases. Recent research has unraveled a multitude of biological activities associated with bee pollen, ranging from antioxidant, anti-inflammatory, antimicrobial, and antifungal properties to potential antiviral and anticancer applications. Comprehending the extensive repertoire of biological properties across various pollen sources remains challenging. By investigating a spectrum of pollen types and their chemical composition, this review produces an updated analysis of the bioactive constituents and the therapeutic prospects they offer. This review emphasizes the necessity for further exploration and standardization of diverse pollen sources and bioactive compounds that could contribute to the development of innovative therapies.

Evaluation of Biological Activity of Natural Compounds: Current Trends and Methods.

Natural compounds have diverse structures and are present in different forms of life. Metabolites such as tannins, anthocyanins, and alkaloids, among others, serve as a defense mechanism in live organisms and are undoubtedly compounds of interest for the food, cosmetic, and pharmaceutical industries. Plants, bacteria, and insects represent sources of biomolecules with diverse activities, which are in many cases poorly studied. To use these molecules for different applications, it is essential to know their structure, concentrations, and biological activity potential. In vitro techniques that evaluate the biological activity of the molecules of interest have been developed since the 1950s. Currently, different methodologies have emerged to overcome some of the limitations of these traditional techniques, mainly via reductions in time and costs. These emerging technologies continue to appear due to the urgent need to expand the analysis capacity of a growing number of reported biomolecules. This review presents an updated summary of the conventional and relevant methods to evaluate the natural compounds' biological activity in vitro.

Designing cytochrome P450 enzymes for use in cancer gene therapy.

Cancer is a significant global socioeconomic burden, as millions of new cases and deaths occur annually. In 2020, almost 10 million cancer deaths were recorded worldwide. Advancements in cancer gene therapy have revolutionized the landscape of cancer treatment. An approach with promising potential for cancer gene therapy is introducing genes to cancer cells that encode for chemotherapy prodrug metabolizing enzymes, such as Cytochrome P450 (CYP) enzymes, which can contribute to the effective elimination of cancer cells. This can be achieved through gene-directed enzyme prodrug therapy (GDEPT). CYP enzymes can be genetically engineered to improve anticancer prodrug conversion to its active metabolites and to minimize chemotherapy side effects by reducing the prodrug dosage. Rational design, directed evolution, and phylogenetic methods are some approaches to developing tailored CYP enzymes for cancer therapy. Here, we provide a compilation of genetic modifications performed on CYP enzymes aiming to build highly efficient therapeutic genes capable of bio-activating different chemotherapeutic prodrugs. Additionally, this review summarizes promising preclinical and clinical trials highlighting engineered CYP enzymes' potential in GDEPT. Finally, the challenges, limitations, and future directions of using CYP enzymes for GDEPT in cancer gene therapy are discussed.

Environmental and nuclear influences on microalgal chloroplast gene expression.

Microalgal chloroplasts, such as those of the model organism Chlamydomonas reinhardtii, are emerging as a new platform to produce recombinant proteins, including industrial enzymes, diagnostics, as well as animal and human therapeutics. Improving transgene expression and final recombinant protein yields, at laboratory and industrial scales, require optimization of both environmental and cellular factors. Most studies on C. reinhardtii have focused on optimization of cellular factors. Here, we review the regulatory influences of environmental factors, including light (cycle time, intensity, and quality), carbon source (CO2 and organic), and temperature. In particular, we summarize their influence via the redox state, cis-elements, and trans-factors on biomass and recombinant protein production to support the advancement of emerging large-scale light-driven biotechnology applications.

CRISPR-Cas-Based Antimicrobials: Design, Challenges, and Bacterial Mechanisms of Resistance.

The emergence of antibiotic-resistant bacterial strains is a source of public health concern across the globe. As the discovery of new conventional antibiotics has stalled significantly over the past decade, there is an urgency to develop novel approaches to address drug resistance in infectious diseases. The use of a CRISPR-Cas-based system for the precise elimination of targeted bacterial populations holds promise as an innovative approach for new antimicrobial agent design. The CRISPR-Cas targeting system is celebrated for its high versatility and specificity, offering an excellent opportunity to fight antibiotic resistance in pathogens by selectively inactivating genes involved in antibiotic resistance, biofilm formation, pathogenicity, virulence, or bacterial viability. The CRISPR-Cas strategy can enact antimicrobial effects by two approaches: inactivation of chromosomal genes or curing of plasmids encoding antibiotic resistance. In this Review, we provide an overview of the main CRISPR-Cas systems utilized for the creation of these antimicrobials, as well as highlighting promising studies in the field. We also offer a detailed discussion about the most commonly used mechanisms for CRISPR-Cas delivery: bacteriophages, nanoparticles, and conjugative plasmids. Lastly, we address possible mechanisms of interference that should be considered during the intelligent design of these novel approaches.

Iron Oxide Nanoparticles: Green Synthesis and Their Antimicrobial Activity.

The rise of antimicrobial resistance caused by inappropriate use of these agents in various settings has become a global health threat. Nanotechnology offers the potential for the synthesis of nanoparticles (NPs) with antimicrobial activity, such as iron oxide nanoparticles (IONPs). The use of IONPs is a promising way to overcome antimicrobial resistance or pathogenicity because of their ability to interact with several biological molecules and to inhibit microbial growth. In this review, we outline the pivotal findings over the past decade concerning methods for the green synthesis of IONPs using bacteria, fungi, plants, and organic waste. Subsequently, we delve into the primary challenges encountered in green synthesis utilizing diverse organisms and organic materials. Furthermore, we compile the most common methods employed for the characterization of these IONPs. To conclude, we highlight the applications of these IONPs as promising antibacterial, antifungal, antiparasitic, and antiviral agents.

Crystal Structure, Hirshfeld Surface Analysis, and Biological Activities of Schiff-Base Derivatives of 4-Aminoantipyrine.

Eight Schiff bases, synthesized by the reaction of 4-aminoantipyrine with different cinnamaldehydes, were studied in the solid state by using vibrational spectroscopy (IR) and X-ray diffraction techniques. The analysis was extended to the solution phase through ultraviolet-vis, fluorescence spectroscopy, and cyclic voltammetry. Finally, the crystal structures of four compounds (3b, 3d, 3g, and 3h) were determined and studied. In addition to the experimental study, theoretical calculations using the semiempirical method PM6/ZDO were performed to understand better the compound's molecular properties, UV-vis, and infrared spectra. The primary difference is the angular conformation of the terminal phenyl rings around the corresponding linking C-N and C-C σ-bonds. Furthermore, as a result of extended bonding, the > C=N- azomethine group-containing Cpyr-N=(CH)-(CR)=(CH)-Cbz chain (with R=H for 3b, 3d, and 3h, and R=CH3 for 3g) is planar, nearly coplanar, with the mean plane of the pyrazole ring. Hirshfeld surface (HS) analysis was used to investigate the crystal packing and intermolecular interactions, which revealed that intermolecular C-H···O and C-H···N hydrogen bonds, π···π stacking, and C-H···π and C=O···π interactions stabilize the compounds. The energy contributions to the lattice energies of potential hydrogen bonds were primarily dispersive and repulsive. All derivatives were tested in vitro on LPS-stimulated mouse macrophages to assess their ability to suppress the LPS-induced inflammatory responses. Only a slight reduction in the level of NO production was found in activated macrophages treated with 3h. Additionally, the derivatives were tested for antimicrobial activity against several clinical bacteria and fungi strains, including three biofilm-forming microorganisms. Nevertheless, only Schiff base 3f showed interesting antibacterial activities with minimum inhibitory concentration (MIC) values as low as 15.6 µM against Enterobacter gergoviae. On the other hand, Schiff base 3f and, to a lesser extent, 3b and 3h showed antifungal activity against clinical isolates of Candida. The lowest MIC value was for 3f against Candida albicans (15.6 µM). It is interesting to note that the same Schiff bases exhibit the highest activity in both biological evaluations.