Transaminases are a class of enzymes that catalyze the transfer of amino between amino acids and keto acids, playing an important role in the biosynthesis of organic amines and the corresponding derivatives. However, natural enzymes often have low catalytic efficiency against non-natural substrates, which limits their widespread applications. Enzyme engineering serves as an effective approach to improve the catalytic properties and thereby expand the application scope of transaminases. In this study, a high-throughput screening method for transaminases was established based on the fluorescent color reaction between methoxy-2-aminobenzoxime (PMA) and ketones. According to the changes in fluorescence intensity, the concentration changes of ketones could be easily monitored. The efficiency, sensitivity, and accuracy of the screening method were improved by optimization of the system. With 4-hydroxy-2-butanone as the substrate, the mutant library of the transaminase from Actinobacteria sp. was established and a mutant with increased activity was successfully obtained, which improved the production efficiency of (R)-3-aminobutanol by enzyme-catalyzed synthesis. This study laid an important foundation for efficient screening, modification, and application of transaminase.
Transaminases/metabolism* ; Fluorescent Dyes/chemistry* ; High-Throughput Screening Assays/methods* ; Ketones/metabolism* ; Actinobacteria/enzymology*

Tunicamycin, a potent reversible translocase I inhibitor, is produced by several Actinomycetes species. The tunicamycin structure is highly unusual, and contains an 11-carbon dialdose sugar and an α, β-1″,11'-glycosidic linkage. Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression (HHE) strategy combined with a bioassay. Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains, demonstrating the role of the genes for the biosynthesis of tunicamycins. Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes (tunA-tunL). Amongst these is a putative radical SAM enzyme (Tun B) with a potentially unique role in biosynthetic carbon-carbon bond formation. Hence, a seven-step novel pathway is proposed for tunicamycin biosynthesis. Moreover, two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827. These data provide clarification of the novel mechanisms for tunicamycin biosynthesis, and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.
Actinobacteria ; enzymology ; genetics ; Base Sequence ; Biological Assay ; Carbohydrate Sequence ; Carbohydrates ; biosynthesis ; genetics ; Cloning, Molecular ; Gene Deletion ; Gene Library ; High-Throughput Screening Assays ; Molecular Sequence Data ; Multigene Family ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; Streptomyces ; enzymology ; genetics ; Tunicamycin ; biosynthesis ; chemistry ; genetics