Development of a heterologous bio-production system for the clinically important anti-cancer natural product compound ET-743

DESCRIPTION provided by applicant Ecteinascidin ET is a tetrahydroisoquinolone natural product that displays potent anti cancer activity Isolated in very low yields from the marine colonial tunicate Ecteinascidia turbinata ET 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 has presented a significant challenge Due to its chemically complex structure total chemical synthesis of ET has proven not viable to support industrial scale production Currently manufacturing of ET 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 step chemical sequence to the desired ET compound To improve upon ET 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 biosynthetic pathway that drives ET 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 As an alternative to chemical or semi chemical synthesis Alluvium proposes to evaluate the feasibility of heterologously producing ET in Streptomyces lavendulae a bacterium that produces the ET related natural product saframycin A To establish proof of concept of this novel ET production strategy a codon optimized NRPS encoding gene that has been identified within the ET pathway EtuA 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 EtuA 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 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 pathway enzymes thereby providing the foundation for Phase II genetic engineering efforts aimed at pursuing the transfer of the entire set of ET biosynthetic genes for heterologous production of this clinically valuable anti cancer natural product When completed this Randamp D program is expected to result in a powerful biotechnology tool that can be applied toward the sustainable bio production of ET as well as novel structural analogs not readily accessible through chemical synthesis PUBLIC HEALTH RELEVANCE Cancer represents a significant global human health concern that justifies substantial research investments for the discovery and development of novel treatments ET 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