Genome mining reveals a minimum gene set for the biosynthesis of lactazoles, novel 32-membered macrocyclic thiopeptides

Thiopeptides are produced mainly by actinomycetes and typically contain highly modified sulfur-containing peptides, which have a characteristic macrocycle knotted with pyridine or piperidine, a six-membered nitrogen-containing ring. We used genome mining to identify a cryptic thiopeptide biosynthetic gene cluster responsible for biosynthesis of lactazoles from Streptomyces lactacystinaeus OM-6519. Lactazoles originate from the simplest cluster, containing only six unidirectional genes (lazA to lazF). It is the smallest cluster among the known thiopeptide biosynthetic gene clusters. The structure gene, lazAcontains the precursor peptide sequence, and it is classified into a phylogenetically distinct clade.

We demonstrate the heterologous expression of lactazole biosynthetic gene cluster in Streptomyces lividans, and determined the chemical structure of lactazoles. The ring size of macrocyclic thiopeptide had been limited to 26, 29, or 35 atoms, while lactazoles found to be structurally novel thiopeptides with a 32-membered macrocycle. The 2-oxazolyl-6-thiazolylpyridine core with the 3-position connected to tryptophan through an amide linkage also provides a unique structure in thiopeptides.

We also demonstrate overproduction of lactazoles by the promoter substitution, and the production of two analogs, S11C and W2S, by site-directed mutagenensis of the structure gene, godA. Thus, this compact biosynthetic machinery has high potency to lend large diversity to the thiopeptide core structures. Our approach facilitates the production of a more diverse set of thiopeptide structures, increasing the semisynthetic repertoire for use in drug development.

1. Onaka H et al. (2005) Microbiology 151(Pt 12):3923-3933.
2. Hayashi S et al. (2014) Chem Biol 21(5):679-688.