Bacillus subtilis is widely underappreciated for its inherent biosynthetic potential. This report comprehensively summarizes the known bioactive secondary metabolites from B. subtilis and highlights potential applications as plant pathogen control agents, drugs, and biosurfactants. B. subtilis is well known for the production of cyclic lipopeptides exhibiting strong surfactant and antimicrobial activities, such as surfactins, iturins, and fengycins. Several polyketide-derived macrolides as well as nonribosomal peptides, dihydroisocoumarins, and linear lipopeptides with antimicrobial properties have been reported, demonstrating the biosynthetic arsenal of this bacterium. Promising efforts toward the application of B. subtilis strains and their natural products in areas of agriculture and medicine are underway. However, industrial-scale availability of these compounds is currently limited by low fermentation yields and challenging accessibility via synthesis, necessitating the development of genetically engineered strains and optimized cultivation processes. We hope that this review will attract renewed interest in this often-overlooked bacterium and its impressive biosynthetic skill set.

Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose‐1‐phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase‐catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate‐specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature‐dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis.

The biocatalytic synthesis of natural and modified nucleosides with nucleoside phosphorylases offers the protecting‐group‐free direct glycosylation of free nucleobases in transglycosylation reactions. This contribution presents guiding principles for nucleoside phosphorylase‐mediated transglycosylations alongside mathematical tools for straightforward yield optimization. We illustrate how product yields in these reactions can easily be estimated and optimized using the equilibrium constants of phosphorolysis of the nucleosides involved. Furthermore, the varying negative effects of phosphate on transglycosylation yields are demonstrated theoretically and experimentally with several examples. Practical considerations for these reactions from a synthetic perspective are presented, as well as freely available tools that serve to facilitate a reliable choice of reaction conditions to achieve maximum product yields in nucleoside transglycosylation reactions.

Efficient reaction monitoring is crucial for data acquisition in kinetic and mechanistic studies. However, for conversions of nucleosides to their corresponding nucleobases, as observed in enzymatically catalyzed nucleoside phosphorylation reactions, the current analytical arsenal does not meet modern requirements regarding cost, speed of analysis and high throughput. Herein, we present a UV/Vis spectroscopy-based assay employing an algorithm for spectral unmixing in a 96-well plate format. The algorithm relies on fitting of reference spectra of nucleosides and their bases to experimental spectra and allows determination of nucleoside/nucleobase ratios in solution with high precision. The experimental procedure includes appropriate dilution of a sample into aqueous alkaline solution, transfer to a multi-well plate, measurement of a UV/Vis spectrum and subsequent in silico spectral unmixing. This enables data collection in a high-throughput fashion and reduces costs compared to state-of-the-art HPLC analyses by approximately 5-fold while being 20-fold faster and offering comparable precision. Additionally, the method is robust regarding dilution and sample transfer errors as it only considers spectral form and not absolute intensity. It can be applied to all natural nucleosides and nucleobases and even unnatural ones as demonstrated by several examples.