Valinomycin is a nonribosomal peptide that was discovered from Streptomyces in 1955. Over the past more than six decades, it has received continuous attention due to its special chemical structure and broad biological activities. Although many research papers have been published on valinomycin, there has not yet been a comprehensive review that summarizes the diverse studies ranging from structural characterization, biogenesis, and bioactivity to the identification of biosynthetic gene clusters and heterologous biosynthesis. In this review, we aim to provide an overview of valinomycin to address this gap, covering from 1955 to 2020. First, we introduce the chemical structure of valinomycin together with its chemical properties. Then, we summarize the broad spectrum of bioactivities of valinomycin. Finally, we describe the valinomycin biosynthetic gene cluster and reconstituted biosynthesis of valinomycin. With that, we discuss possible opportunities for the future research and development of valinomycin.

Serratiopeptidase is a clinical therapeutic protein for the treatment of human diseases such as arthritis, bronchitis, and thrombosis. Yet production of this protein in a heterologous host (e.g., Escherichia coli) is difficult due to the issue of protein insolubility and the requirement of laborious refolding procedures. Cell-free protein synthesis (CFPS) systems, derived from crude cell extracts, are effective platforms for the expression of recombinant proteins in vitro. Here, we report a new method to produce serratiopeptidase by using an E. coli-based CFPS system. After rational selection of cell extracts and construction of expression vectors, soluble expression of serratiopeptidase was achieved and the enzyme activity could be readily tested in the cell-free reaction mixture. By further optimizing the key parameters, optimum conditions for the enzyme activity assay were obtained, including the pH value at 5, reaction temperature at 45 °C, substrate concentration at 10 mg/mL, and supplementing Ca²⁺ ions at 5 mM. Moreover, the CFPS mixture was freeze-dried and the activity of serratiopeptidase could be regenerated by hydration without losing activity. Overall, the CFPS system enabled soluble expression of serratiopeptidase with catalytic activity, providing a new and promising approach for this enzyme production. Our work extends the utility of the cell-free platform to produce therapeutic proteins with clinical applications.

Serine integrases are emerging as one of the most powerful biological tools for biotechnology. Over the past decade, many research papers have been published on the use of serine integrases in synthetic biology. In this review, we aim to systematically summarize the various studies ranging from structure and the catalytic mechanism to genetic design and interdisciplinary applications. First, we introduce the classification, structure, and catalytic model of serine integrases. Second, we present a timeline with milestones that describes the representative achievements. Then, we summarize the applications of serine integrases in genome engineering, genetic design, and DNA assembly. Finally, we discuss the potential of serine integrases for advancing interdisciplinary research. We anticipate that serine integrases will be further expanded as a versatile genetic toolbox for synthetic biology applications.

Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this work, we used cell-free protein synthesis (CFPS) to express enzymes in an aqueous-organic biphasic system for the construction of an artificial enzymatic pathway. CFPS-expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to ( S )-1-phenyl-1,2-ethanediol (two steps in one pot) with 100% conversion. In addition, our CFPS system showed great tolerance to different organic solvents and, importantly, the entire biocatalytic system can be consistently scaled up without reduction of the substrate conversion rate. We therefore anticipate that our cell-free approach will make possible cost-effective, high-yielding synthesis of valuable chemicals.