A novel biosynthetic strategy for the production of a key cryptophycin precursor

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DESCRIPTION (provided by applicant): The cryptophycins are a structurally diverse class of polyketide/non-ribosomal peptide natural products that possess potent anticancer activity. In fact, a cryptophycin synthetic analogue has demonstrated promise in treating platinum resistant ovarian cancer. Despite this impressive activity, the development of the cryptophycins into a beneficial cancer chemotherapeutic agent has suffered due to clinically significant peripheral neuropathy that correlates with treatment. However, the promising therapeutic spectrum of these natural products has motivated Alluvium Biosciences to pursue the continued discovery and development of a cryptophycin compound that that can enter clinical evaluation. To this end, Alluvium is currently developing a high throughput, solid-phase chemoenzymatic production technology to enable access to structurally diverse cryptophycin compounds that can be readily screened for desirable pharmacological properties. Key to this effort is the availability of four chemical fragments, or units, which are assembled on solid-phase and subsequently biocatalytically transformed into mature cryptophycin compounds. Three of the required units are either commercially available or can be readily obtained by simple synthetic methods, while the fourth unit, 4- hydroxy phenyloctenoic acid, must currently be generated by custom synthetic strategies that result in low overall yields and high cost per compound. To add value to its cryptophycin chemoenzymatic production technology, Alluvium Biosciences is motivated to develop a low-cost, high yielding solution for the production of 4-hydroxy phenyloctenoic acid. Accordingly, in this Phase I SBIR proposal, Alluvium Biosciences will develop a novel biosynthetic technology that employs bacterial fermentation to produce this critical cryptophycin polyketide intermediate. Specifically, the proposed strategy aims to design and engineer a biosynthetic pathway, comprised of known cryptophycin biosynthetic enzymes, which will direct the biosynthesis of 4- hydroxy phenyloctenoic acid within an E. coli bacterial host. Subsequent to the construction and transfer of the engineered biosynthetic pathway into the bacterial host, fermentation conditions will be established for the production of the desired cryptophycin intermediate. Once proof-of-concept is demonstrated, Phase II research efforts will focus on further refinement of the genetically engineered bacterial strain and transfer of the fermentation technology toward a large-scale production system for the low-cost generation of 4-hydroxy phenyloctenoic acid. Efforts in Phase II will also develop strategies for the biosynthetic production of chemically diverse 4-hydroxy phenyloctenoic acid analogues. Purified 4-hydroxy phenyloctenoic acid, and structural analogues, produced by this fermentation technology will serve as starting material for the scalable production of desirable cryptophycin compounds via Alluvium's solid-phase chemoenzymatic technology. PUBLIC HEALTH RELEVANCE: Cancer represents a significant global human health concern that justifies substantial research investments for the discovery and development of novel treatments. Cryptophycin is a known, potent anti-cancer compound that has been dropped from clinical testing due to intolerable side-effects. This proposed research seeks to aid in the development of a novel technology for the rapid generation of cryptophycin analogues that may display fewer side effects, thereby enabling cryptophycin to be utilized by physicians in the battle against this oft-deadly disease.