Tuesday Sept. 17 // Driving metabolic flux and avoiding carbon loss; lessons from opiate alkaloid biosynthesis

 Mehran Dastmalchi
Department of Biological Sciences,
Brock University, St. Catharines, ON

Opium poppy has been in use as a medicinal plant since before the dawn of civilization. Today, it remains the only source for natural opiate analgesics, codeine and morphine, and precursors used to synthesize derivatives such as the overdose antidote, naloxone. Total chemical synthesis of opiates is not commercially feasible due to complex stereochemistry. Engineering microbes with alkaloid biosynthetic machinery from opium poppy has the potential of providing a sustainable global supply of opiate pharmaceuticals. However, microbial bioproduction often suffers from carbon loss to alternative pathways. In the latter stages of opiate biosynthesis, thebaine is converted, via codeine, to morphine. When this pathway is simulated in vitro or in microbial systems, two undesirable isomers accumulate: neopine and neomorphine. We addressed this aberrant pathway in two studies: 1) In the penultimate step of morphine biosynthesis COR reduces codeinone to codeine. However, codeinone exists in an apparent equilibrium with its isomeric form, neopinone. We showed that COR irreversibly reduces neopinone to neopine. Using natural and synthetic protein variation we also identified four residues that can confer COR with higher protein stability and performance, improving metabolic flux. 2) We sought an explanation for why the plant does not accumulate the isomeric byproducts. Using a proteomics approach, we found a novel enzyme, neopinone isomerase (NISO) that catalyzes the assumed spontaneous conversion of neopinone to codeinone. NISO provides the substrate for COR to produce codeine and precludes the formation of neopine. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products and avoids carbon loss.