Elloramycin

Background/goal/aim: The tetracenomycins are aromatic anticancer polyketides that inhibit peptide translation via binding to the large ribosomal subunit. Here, we expressed the elloramycin biosynthetic gene cluster in the heterologous host Streptomyces coelicolor M1146 to facilitate the downstream production of tetracenomycin analogs. Main methods and major results: We developed a BioBricks genetic toolbox of genetic parts for substrate precursor engineering in S. coelicolor M1146::cos16F4iE. We cloned a series of integrating vectors based on the VWB, TG1, and SV1 integrase systems to interrogate gene expression in the chromosome. We genetically engineered three separate genetic constructs to modulate tetracenomycin biosynthesis: (1) the vhb hemoglobin from obligate aerobe Vitreoscilla stercoraria to improve oxygen utilization; (2) the accA2BE acetyl-CoA carboxylase to enhance condensation of malonyl-CoA; (3) lastly, the sco6196 acyltransferase, which is a "metabolic regulatory switch" responsible for mobilizing triacylglycerols to β-oxidation machinery for acetyl-CoA. In addition, we engineered the tcmO 8-O-methyltransferase and newly identified tcmD 12-O-methyltransferase from Amycolatopsis sp. A23 to generate tetracenomycins C and X. We also co-expressed the tcmO methyltransferase with oxygenase urdE to generate the analog 6-hydroxy-tetracenomycin C. Conclusions and implications: Altogether, this system is compatible with the BioBricks [RFC 10] cloning standard for the co-expression of multiple gene sets for metabolic engineering of Streptomyces coelicolor M1146::cos16F4iE. This production platform improves access to potent analogs, such as tetracenomycin X, and sets the stage for the production of new tetracenomycins via combinatorial biosynthesis.

The glycosyltransferase ElmGT from Streptomyces olivaceus is involved in the biosynthesis of the antitumor drug elloramycin, and it has been shown to possess a broad deoxysugar recognition pattern, being able to transfer different l- and d-deoxysugars to 8-demethyl-tetracenomycin C, the elloramycin aglycone. Site-directed mutagenesis in residues L309 and N312, located in the alpha/beta/alpha motif within the nucleoside diphosphate-sugar binding region, can be used to modulate the substrate flexibility of ElmGT, making it more precise for transfer of specific deoxysugars.

Elloramycin is an anthracycline-like antitumour drug produced by Streptomyces olivaceus Tü2353. Cosmid cos16F4 has been previously shown to direct the biosynthesis of the elloramycin aglycon 8-demethyltetracenomycin C (8-DMTC), but not elloramycin. Sequencing of the 24.2 kb insert in cos16F4 shows the presence of 17 genes involved in elloramycin biosynthesis (elm genes) together with another additional eight ORFs probably not involved in elloramycin biosynthesis. The 17 genes would code for the biosynthesis of the polyketide moiety, sugar transfer, methylation of the tetracyclic ring and the sugar moiety, and export. Four genes (rhaA, rhaB, rhaC and rhaD) encoding the enzymic activities required for the biosynthesis of the sugar l-rhamnose were also identified in the S. olivaceus chromosome. The involvement of this rhamnose gene cluster in elloramycin biosynthesis was demonstrated by insertional inactivation of the rhaB gene, generating a non-producer mutant that accumulates the 8-DMTC C aglycon. Coexpression of cos16F4 with pEM4RO (expressing the four rhamnose biosynthesis genes) in Streptomyces lividans led to the formation of elloramycin, demonstrating that both subclusters are required for elloramycin biosynthesis. These results demonstrate that, in contrast to most of the biosynthesis gene clusters from actinomycetes, genes involved in the biosynthesis of elloramycin are located in two chromosomal loci.