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.

The mevalonate pathway (MVA) is a central metabolic route responsible for the biosynthesis of isoprenoids, a vast and diverse class of biomolecules essential for cellular structure, signaling, and physiology. This review provides a comprehensive overview of the MVA pathway, addressing its distribution in different domains of life, evolutionary origins, and overall organization. We describe in detail its biochemical architecture, including enzymatic steps, catalytic mechanisms, structural characteristics, and multilayered regulatory strategies. In parallel, the methylerythritol phosphate pathway is presented as an alternative pathway to isoprenoid biosynthesis. The metabolic outputs of both pathways are explored, emphasizing the remarkable diversity of isoprenoid end products and their roles in membrane dynamics, protein modification, and cellular signaling. Furthermore, we analyze the biological functions and clinical relevance of the MVA pathway, including its involvement in human diseases and its variability in different kingdoms. The review also addresses recent advances in biotechnology, focusing on metabolic engineering and synthetic biology approaches for the microbial production of high-value isoprenoids. Finally, sustainable strategies for optimizing microbial cell factories and production processes are discussed, underscoring the growing importance of isoprenoid biosynthesis in industrial and pharmaceutical applications.

The main protease (Mpro) of SARS-CoV-2 plays a crucial role in the viral replication cycle and is a key target for COVID-19 antiviral drugs. In this review, we conducted an extensive bibliographic search on the main challenges and experimental limitations reported in the production and biochemical characterization of the Mpro. A large variability in the kinetic parameters was revealed, which compromises comparisons across various studies and the evaluation of inhibitor efficacy. The studies indicate that these discrepancies are mainly associated with experimental limitations, such as low substrate solubility, optical irregularity due to the inner-filter effect, and variations in the oligomeric state of Mpro. This review summarizes and discusses the different strategies for Mpro expression in Escherichia coli and purification, with emphasis on obtaining an N-terminal tag-free enzyme, essential for homodimerization into a highly active form for structural and functional characterization. The main FRET-based substrates used in enzymatic assays were compiled and analyzed, as well as alternative methodologies for assessing Mpro enzymatic activity. Furthermore, the effects of assay conditions, such as buffer composition, pH, and the presence of reducing agents were addressed. The intrinsic structural features of Mpro that impact its catalytic function are discussed, including functional asymmetry in substrate binding and the positive allosteric cooperation between monomers. Overall, this review provides a critical and integrated perspective on the main experimental limitations in the production and characterization of Mpro, applicable to other proteases, contributing to the standardization of approaches and advances in the development of more effective inhibitors.

The mevalonate (MVA) pathway is a central metabolic route responsible for the biosynthesis of isoprenoids with broad biological and biotechnological relevance. Due to its importance, the MVA pathway has attracted increasing interest in studies of enzymatic regulation, structural biology, metabolic engineering, and synthetic biology, particularly in fungi. This review provides a comprehensive overview of the MVA pathway, addressing its distribution across different domains of life, evolutionary aspects, and metabolic organization, with emphasis in fungi. Special attention is given to the biochemical and structural characterization of MVA-pathway enzymes, including catalytic mechanisms, structural features, and regulatory processes. The methylerythritol phosphate pathway is also presented as an alternative route for isoprenoid precursor biosynthesis and discussed in terms of its taxonomic distribution and metabolic significance. Recent advances in synthetic biology, enzyme regulation, and pathway engineering are highlighted, emphasizing their contributions to metabolic engineering and synthetic biology. Special emphasis is given to fungi, in which the MVA pathway plays a central role in ergosterol biosynthesis, protein prenylation, and secondary metabolite production. Advances in the engineering of fungal cells, including Saccharomyces cerevisiae and other emerging fungal species, are discussed in the context of sustainable isoprenoid production. Finally, strategies for optimizing microbial production are presented, highlighting the importance of fungal synthetic biology in advancing biotechnological applications.