Presentation Information
[P01-093]Biosynthesis of a thiazole-rich peptide in thermophilic microorganisms
○Hiroya Tomita1, Ayane Yano1, Ayano Hayami1, Kentaro Miyazaki1, Kohsuke Honda1 (1. The University of Osaka (Japan))
Keywords:
biosynthesis,ribosomally synthesized and post-translationally modified peptides,thermophile
Thermophiles are generally defined as organisms which have an optimal growth temperature exceeding 45°C. In comparison to mesophilic bacteria (such as actinomycetes), fungi, and plants, which are well-known sources of secondary metabolites, thermophiles have a limited number of secondary metabolite biosynthetic gene clusters. However, recent findings have suggested that many strains belonging to the thermophilic bacterial genus Geobacillus possess the potential to produce ribosomally synthesized and post-translationally modified peptides (RiPPs).
RiPPs are the representative microbial peptidyl secondary metabolites including the class of linear azol(in)e-containing peptides (LAPs). In the biosynthetic mechanisms of LAPs, Cys and Ser/Thr residues are cyclized and oxidized to thiazole and oxazole rings, respectively. A substantial proportion of LAPs have been identified in mesophilic microorganisms, including actinomycetes. Here, we carried out the biosynthetic reconstitution and characterization of parageocin I, a novel thiazole-rich LAP derived from the thermophilic bacterium Parageobacillus caldoxylosilyticus KH1-5 which exhibits optimal growth around 60°C. We found the biosynthetic gene cluster (pgc) consists of four genes necessary for azole ring formation: pgcA, pgcB, pgcC, and pgcD, encoding the precursor peptide, dehydrogenase, 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 reconstituted the biosynthesis heterologously in Escherichia coli. MALDI-TOF-MS analysis of the synthesized peptide, coupled with mutational analyses of the modified PgcA, revealed that the final product 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 at 60°C, which exceeds reaction temperatures of all previously reported LAP biosynthetic systems. This study expands our understanding of not only LAPs but also secondary metabolism in thermophiles and contributes to design novel artificial peptides.
RiPPs are the representative microbial peptidyl secondary metabolites including the class of linear azol(in)e-containing peptides (LAPs). In the biosynthetic mechanisms of LAPs, Cys and Ser/Thr residues are cyclized and oxidized to thiazole and oxazole rings, respectively. A substantial proportion of LAPs have been identified in mesophilic microorganisms, including actinomycetes. Here, we carried out the biosynthetic reconstitution and characterization of parageocin I, a novel thiazole-rich LAP derived from the thermophilic bacterium Parageobacillus caldoxylosilyticus KH1-5 which exhibits optimal growth around 60°C. We found the biosynthetic gene cluster (pgc) consists of four genes necessary for azole ring formation: pgcA, pgcB, pgcC, and pgcD, encoding the precursor peptide, dehydrogenase, 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 reconstituted the biosynthesis heterologously in Escherichia coli. MALDI-TOF-MS analysis of the synthesized peptide, coupled with mutational analyses of the modified PgcA, revealed that the final product 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 at 60°C, which exceeds reaction temperatures of all previously reported LAP biosynthetic systems. This study expands our understanding of not only LAPs but also secondary metabolism in thermophiles and contributes to design novel artificial peptides.
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