Identifying and investigating metabolic pathways activated in high-producing California poppy suspension cultures.

摘要

Plant cell culture offers an alternative production system capable of providing consistent, high yields of pharmaceutical compounds. Currently, 9 pharmaceutical compounds have been produced through plant cell culture [1]. However, further increases in productivity for other compounds from plant cell cultures will require a greater understanding of metabolic pathways which limit production. Proteins and pathways with changes in abundance in high-producing cell lines represent potential targets for further increases in production. In this thesis, Eschscholzia californica cultures which produce benzophenanthridine alkaloids, including the anti-microbial and anticancer drug sanguinarine, were used as a model system to study metabolic bottlenecks using proteomics.;Enhancements in BPA production was explored using elicitation, in situ product extraction, and medium composition. Cultures treated with a purified yeast elicitor (PYE) enhanced BPA production to greater than a 20-fold. In situ extraction combined with PYE-treatment increased production 63-fold, i.e. up to 85 mg BPAs / g dry weight. Medium optimization (hormones, sugar, nitrogen, and phosphate) did not increase production in elicited cultures.;A proteomic comparison of unelicited and PYE-elicited cultures was performed to assess differences in global metabolism associated with enhanced BPA production. Approximately 650 proteins were identified using a liquid chromatography / mass spectrometric method. Three proteins related to (S)-adenosylmethionine (SAM) biosynthesis and metabolism were significantly induced in elicited cultures. SAM is a universal methyl donor in biological reactions including 6 steps in sanguinarine biosynthesis. The results suggest a coordination between primary metabolic pathways and increased BPA production.;The role of the SAM biosynthetic pathway in supporting enhanced BPA synthesis was investigated further by adding methionine, ethylene, and the ethylene biosynthesis inhibitor cobalt chloride to E. californica suspension cultures. Methionine did not affect production in elicited cultures suggesting that methionine is not limiting BPA production. Amino acid analysis of culture media from elicited cultures reflected increased methionine biosynthesis with elicitation. Both ethephon (soluble ethylene) and cobalt chloride did not affect production suggesting that ethylene is not required for elicitor-induced BPA production.;Recommendations include enhancing production using cyclodextrin for in situ extraction, improved protein identification using fractionation by differential centrifugation, and investigation of the role of DNA methylation in supporting increased production. These proposed experiments will provide insight into pathways which can be manipulated by genetic engineering to enhance production.