Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

Complementary Cell-Free Translational Assay for Quantification of Amino Acids.

In this study, we present a simple and economical method that enables rapid quantification of amino acids based on their polymerization into a signal-generating protein. This method harnesses amino acid-deficient cell-free protein synthesis systems that generate fluorescence signals in response to exogenous amino acids. When premixed with assay samples containing the amino acids in question, incubation of the cell-free synthesis reaction mixture rapidly resulted in the production of sfGFP, the fluorescence intensity of which was linearly proportional to the concentration of the amino acids. The assay method achieved a limit of detection as low as ∼100 nM and was successfully applied to the quantification of disease-related amino acids in biological samples. Compared with standard methods in current use that require chemical derivatization of amino acids and chromatographic equipment, the complementation assay method developed in this work enables the direct translation of amino acid titer into measurable biofluorescence intensity in a much shorter period, providing a more affordable and flexible option for the quantification of amino acids.

Cell-free synthesis of functional phospholipase A1 from Serratia sp.

BACKGROUND: Phospholipase A1 is an enzyme that hydrolyzes phospholipids at the sn-1 position. It has potential applications across diverse fields including food, pharmaceutical, and biofuel industries. Although there has been increasing interest in the use of phospholipase A1 for degumming of plant oils during biodiesel production, production of recombinant phospholipase A1 has been hampered by low efficiency of gene expression and its toxicity to the host cell. RESULTS: While expression of phospholipase A1 in Escherichia coli resulted in extremely low productivity associated with inhibition of transformed cell growth, drastically higher production of functional phospholipase A1 was achieved in a cell-free protein synthesis system where enzyme expression is decoupled from cell physiology. Compared with expression in E. coli, cell-free synthesis resulted in an over 1000-fold higher titer of functional phospholipase A1. Cell-free produced phospholipase A1 was also used for successfully degumming crude plant oil. CONCLUSIONS: We demonstrate successful production of Serratia sp. phospholipase A1 in a cell-free protein synthesis system. Including the phospholipase A1 investigated in this study, many industrial enzymes can interfere with the regular physiology of cells, making cellular production of them problematic. With the experimental results presented herewith, we believe that cell-free protein synthesis will provide a viable option for rapid production of important industrial biocatalysts.