A Heterotrimeric Dehydrogenase Complex Functions with 2 Distinct YcaO Proteins to Install 5 Azole Heterocycles into 35-Membered Sulfomycin Thiopeptides.

Sulfomycins are sulfur-rich, ribosomally synthesized, and post-translationally modified peptides (RiPPs) that are characterized by a 35-membered macrocyclic ring system with a pyridine domain central to five azoles and additional dehydroamino acids. The pathway through which these large thiopeptide antibiotics are formed in Streptomyces viridochromogene remains elusive. Starting with the cloning of the biosynthetic gene cluster of sulfomycins, we here dissect a two-stage process in which an unusual dehydrogenase heterotrimer functions with two distinct YcaO proteins to install five azole heterocycles into the core peptide sequence of the precursor peptide. The first stage involves the activity of a typical cyclodehydratase complex composed of a didomain E1-YcaO protein and an F-protein partner to heterocyclize distant residues l-Cys2 and l-Thr9 and then the activity of the heterotrimeric dehydrogenase complex that converts the resulting azolines to azoles. In the second stage, this dehydrogenase complex associates with a discrete YcaO protein to form an atypical, four-component azole synthase complex, which is capable of sequentially converting residues l-Cys7, l-Thr5, and l-Ser12 to azoles in a distinct manner. During this process, an E1-like partner protein plays a critical role and functions through the two stages to mediate a variety of specific protein-protein interactions. This partner protein participates in the formation of the active dehydrogenase heterotrimer and the engagement of discrete YcaO activity to form the azole synthase heterotetramer. The findings in this study advance the understanding in the biosynthesis of different azole-containing RiPPs and set the stage for the discovery, engineering, and creation of new thiopeptides using genome mining and synthetic biology approaches.

Identification and characterization of a new erythromycin biosynthetic gene cluster in Actinopolyspora erythraea YIM90600, a novel erythronolide-producing halophilic actinomycete isolated from salt field.

Erythromycins (Ers) are clinically potent macrolide antibiotics in treating pathogenic bacterial infections. Microorganisms capable of producing Ers, represented by Saccharopolyspora erythraea, are mainly soil-dwelling actinomycetes. So far, Actinopolyspora erythraea YIM90600, a halophilic actinomycete isolated from Baicheng salt field, is the only known Er-producing extremophile. In this study, we have reported the draft genome sequence of Ac. erythraea YIM90600, genome mining of which has revealed a new Er biosynthetic gene cluster encoding several novel Er metabolites. This Er gene cluster shares high identity and similarity with the one of Sa. erythraea NRRL2338, except for two absent genes, eryBI and eryG. By correlating genotype and chemotype, the biosynthetic pathways of 3'-demethyl-erythromycin C, erythronolide H (EH) and erythronolide I have been proposed. The formation of EH is supposed to be sequentially biosynthesized via C-6/C-18 epoxidation and C-14 hydroxylation from 6-deoxyerythronolide B. Although an in vitro enzymatic activity assay has provided limited evidence for the involvement of the cytochrome P450 oxidase EryFAc (derived from Ac. erythraea YIM90600) in the catalysis of a two-step oxidation, resulting in an epoxy moiety, the attempt to construct an EH-producing Sa. erythraea mutant via gene complementation was not successful. Characterization of EryKAc (derived from Ac. erythraea YIM90600) in vitro has confirmed its unique role as a C-12 hydroxylase, rather than a C-14 hydroxylase of the erythronolide. Genomic characterization of the halophile Ac. erythraea YIM90600 will assist us to explore the great potential of extremophiles, and promote the understanding of EH formation, which will shed new insights into the biosynthesis of Er metabolites.