| Summary: | <i>Actinosynnema</i> species produce diverse natural products with important biological activities, which represent an important resource of antibiotic discovery. Advances in genome sequencing and bioinformatics tools have accelerated the exploration of the biosynthetic gene clusters (BGCs) encoding natural products. Herein, the completed BGCs of dnacin B1 were first discovered in two <i>Actinosynnema pretiosum</i> subsp. <i>auranticum</i> strains DSM 44131<sup>T</sup> (hereafter abbreviated as strain DSM 44131<sup>T</sup>) and X47 by comparative genome mining strategy. The BGC for dnacin B1 contains 41 ORFs and spans a 66.9 kb DNA region in strain DSM 44131<sup>T</sup>. Its involvement in dnacin B1 biosynthesis was identified through the deletion of a 9.7 kb region. Based on the functional gene analysis, we proposed the biosynthetic pathway for dnacin B1. Moreover, <i>p</i>-amino-phenylalanine (PAPA) unit was found to be the dnacin B1 precursor for the quinone moiety formation, and this was confirmed by heterologous expression of <i>dinV</i>, <i>dinE</i> and <i>dinF</i> in <i>Escherichia coli</i>. Furthermore, nine potential PAPA aminotransferases (APAT) from the genome of strain DSM 44131<sup>T</sup> were explored and expressed. Biochemical evaluation of their amino group transformation ability was carried out with <i>p</i>-amino-phenylpyruvic acid (PAPP) or PAPA as the substrate for the final product formation. Two of those, APAT4 and APAT9, displayed intriguing aminotransferase ability for the formation of PAPA. The proposed dnacin B1 biosynthetic machinery and PAPA biosynthetic investigations not only enriched the knowledge of tetrahydroisoquinoline (THIQ) biosynthesis, but also provided PAPA building blocks to generate their structurally unique homologues.
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