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In vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure. We demonstrated the utility of our selection system by performing directed evolution experiments using TEM β-lactamase as the model enzyme. Five evolutionary rounds afforded a highly active variant exhibiting 440-fold improvement in catalytic efficiency. We also showed that, without the cis repressor, the selection system cannot provide sufficient selection pressure required for evolving highly efficient TEM β-lactamase. The selection system should be applicable for the exploration of catalytic perfection of a wide range of enzymes.

The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targets with antibody-like affinity. However, in many applications the use of these aptamers is limited by nuclease-mediated degradation. In contrast, oligonucleotides that are modified at their sugar C2ʹ positions with methoxy or fluorine substituents are stable to nucleases, but they cannot be synthesized by natural polymerases. Here we report the development of a polymerase-evolution system and its use to evolve thermostable polymerases that efficiently interconvert C2ʹ-OMe-modified oligonucleotides and their DNA counterparts via ‘transcription’ and ‘reverse transcription’ or, more importantly, that PCR-amplify partially C2ʹ-OMe- or C2ʹ-F-modified oligonucleotides. A mechanistic analysis demonstrates that the ability to amplify the modified oligonucleotides evolved by optimizing interdomain interactions that stabilize the catalytically competent closed conformation of the polymerase. The evolved polymerases should find practical applications and the developed evolution system should be a powerful tool for tailoring polymerases to have other types of novel function. Naturally occurring DNA polymerases can amplify DNA efficiently via PCR, but they cannot utilize C2′-modified substrates to make non-natural nucleic acids. Such C2′-modified nucleic acids are of interest as they are resistant to nucleases. Now, a Stoffel fragment DNA polymerase has been evolved to transcribe C2′-modified DNA from a DNA template, reverse transcribe C2′-modified DNA back into DNA, and PCR-amplify C2′-modified DNA.

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function.

Key success for regenerative endodontics is an effective removal of bacteria inside the root canal during the treatment. Ciprofloxacin (CIP) is one of the triple antibiotics commonly used as the root canal medicament. However, the rapid clearance of antibiotics causes bacterial reinfection. Nanoparticles (NPs) provide an alternative approach for antibiotics delivery system to improve the drug stability and release control. In the previous study, modified dextran was successfully formed the ester linkage with vinyl decanoate via lipase-catalyzed transesterification. The amphiphilic dextran, dextran-decanoate (Dex-D), has been synthesized with the degree of substitution of 83-88% and were applied for CIP-loaded nanoparticles. The suitable technique for producing antibiotic carriers considering by size, shape, and drug encapsulation efficiency was investigated in this work. The preparation methods were compared between nanoprecipitation and solid-in-oil-in-water (s/o/w) ion pairing technique. We found that the obtained Dex-D nanoparticles had stable monodispersed with spherical shape in both techniques. However, Dex-D nanoparticles formed by s/o/w ion pairing technique provided smaller size with better encapsulation efficiency. The antimicrobial activity of CIP-loaded Dex-D nanoparticles against oral pathogens showed satisfactory outcome with the MIC equal to 0.7 μg/mL. Also, the compatibility of nanoparticles and dental stem cells was observed in this work.

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile arrow right Thr, Leu arrow right Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function.