Ecteinascidin 743 (ET-743) is a tetrahydroisoquinolone natural product that displays potent anti-cancer activity. Isolated in very low yields from the marine colonial tunicate Ecteinascidia turbinata, ET-743 has been developed by PharmaMar as a cancer chemotherapeutic agent currently marketed in Europe as trabectedin (Yondelis(r)) for the treatment of soft tissue sarcomas and refractory ovarian cancer. Like many natural products, sourcing of ET-743 has presented a significant challenge. Due to its chemically complex structure, total chemical synthesis of ET-743 has proven not viable to support industrial scale production. Currently, manufacturing of ET-743 relies on a semi-synthetic process whereby the structurally related natural product cyanosafracin B is isolated from Pseudomonas fluorescens fermentation and further transformed in a low yielding 17-step chemical sequence to the desired ET-743 compound. To improve upon ET-743 production, Alluvium Biosciences proposes in this Phase I SBIR to initiate the development of a heterologous bio-production system capable of generating this clinically important natural product. This work is made possible due to recent metagenomic DNA sequencing and bioinformatics efforts that have identified, for the first time, the entire ET-743 biosynthetic pathway that drives ET-743 biosynthesis in E. frumentensis, an endosymbiotic bacterium of E. turbinata. Unfortunately, this endosymbiont is not amenable to isolation or laboratory culture; thus, direct fermentation of the native bacterial host cannot be employed for the sustainable production of ET-743. As an alternative to chemical or semi-chemical synthesis, Alluvium proposes to evaluate the feasibility of heterologously producing ET-743 in Streptomyces lavendulae, a bacterium that produces the ET-743 related natural product saframycin A. To establish proof-of-concept of this novel ET-743 production strategy, a codon optimized NRPS encoding gene that has been identified within the ET-743 pathway, EtuA1, will functionally replace a homologous NRPS gene within the known saframycin A pathway in S. lavendulae. In addition, a synthetic operon containing four codon optimized glycolic acid biosynthetic genes, whose encoded proteins generate the EtuA1 requisite glycolic acid substrate, will also be genetically incorporated into the S. lavendulae genome. In total, the sum of these genetic integrations will result in a hybrid saframycin A/ET-743 biosynthetic that is expected to produce a glycolic acid containing analog of saframycin A in fermentation of S. lavendulae. Success in this initial proof-of-concept approach will establish the ability of the heterologous host to expres functional ET-743 pathway enzymes, thereby providing the foundation for Phase II genetic engineering efforts aimed at pursuing the transfer of the entire set of ET-743 biosynthetic genes for heterologous production of this clinically valuable anti-cancer natural product. When completed, this R&D program is expected to result in a powerful biotechnology tool that can be applied toward the sustainable, bio- production of ET-743, as well as novel structural analogs not readily accessible through chemical synthesis.
Cancer represents a significant global human health concern that justifies substantial research investments for the discovery and development of novel treatments. ET-743 is a known, potent anti-cancer natural product compound that is currently approved to treat rare forms of cancer. This proposed research seeks to improve upon the production of this important, and costly, therapeutic so that it can be generated in a more efficient and economical manner to enable broader patient access.
