Reconstitution and Characterization of Biosynthetic Machinery for Parageocin I, a Novel Thiazole-Rich Peptide from the Thermophilic Bacterium <i>Parageobacillus caldoxylosilyticus</i>

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are the representative microbial peptidyl secondary metabolites including the class of linear azol(in)e-containing peptides (LAPs). A substantial proportion of LAPs have been identified in mesophilic microorganisms, including actinomycetes. In this study, we report the biosynthetic reconstitution and characterization of parageocin I, a novel thiazole-rich LAP derived from the thermophilic bacterium <i>Parageobacillus caldoxylosilyticus</i> KH1-5 which exhibits optimal growth around 60 °C. The biosynthetic gene cluster (<i>pgc</i>) consists of four genes: <i>pgcA</i>, <i>pgcB</i>, <i>pgcC</i>, and <i>pgcD</i>, encoding the precursor peptide, dehydrogenase, YcaO family cyclodehydratase, and biosynthetic scaffold protein, respectively. The precursor peptide PgcA possesses 13 Cys and 2 Ser residues, with regularly repeated sequences interspaced between Cys residues. We first reconstituted the biosynthesis heterologously in <i>Escherichia coli</i>. Mass spectrometry analysis of the synthesized peptide, coupled with mutational analyses of the modified PgcA, revealed that the final product, designated as parageocin I, harbors 13 thiazole rings derived from the cyclization of Cys residues, while Ser residues remain intact. Furthermore, mutational studies of PgcA revealed three key principles governing heterocyclization by PgcC: (i) Cys is acceptable, but Ser and Thr are not; (ii) the presence of an acidic amino acid preceding Cys is not permissible; and (iii) a minimum of two amino acids must separate Cys residues. In addition, we successfully reconstituted the biosynthesis in vitro using the purified recombinant enzymes. This is the first report of LAP biosynthesis in thermophilic Bacillaceae, thereby expanding our understanding of not only LAPs but also secondary metabolism in thermophiles.