| As of 2007, 63% of small molecule drugs developed to battle cancer and 54% of drugs against infectious diseases were of natural origin (Newman and Cragg 2007). The recent use of heterologous hosts to produce natural products has shown significant potential, although limitations still exist regarding optimal production titers. In this study, we utilize micro-scale cultures and well-defined screening methods to identify key medium components that influence the heterologous production of the complex polyketide 6-deoxyerythronolide B (6dEB), the parent macrolactone precursor of erythromycin, through E. coli.; As a preliminary step, a thorough evaluation of a mass spectrometer (MS), an evaporative light scattering detector (ELSD), and a charged aerosol detector (CAD) were used to analyze erythromycin and 6-dEB. The work highlights the capabilities of each detector to analyze polyketide compounds that do not possess a natural chromophore. Each detector was evaluated based upon limit of detection (LOD), dynamic range, and precision in the context of polyketide analysis. Due to its low LOD, wide dynamic range, and ability to provide molecular weight information, the MS was deemed the best detection option for analysis of low-concentration, poorly identified polyketide compounds. Alternatively, both the CAD and ELSD systems studied showed better precision and accuracy. The ELSD demonstrated the best precision at 3%, but its LOD was limited to concentrations primarily greater than or equal to 1 mg/L-1. The Corona CAD demonstrated a LOD (0.012 mg/L-1) and dynamic range comparable to mass spectroscopy and, therefore, serves as a more cost efficient alternative for polyketide production schemes with low titers.; Using an ELSD with an online HPLC separation, an 8-run Plackett-Burman influenced screening method was conducted using micro-scale cultures. It was determined that tryptone had a significant effect on 6dEB production and could supplement substrate requirements and improve recombinant protein levels, including the essential deoxyerythronolide B synthase proteins which catalyze 6dEB conversion. As a result, we demonstrate the use of micro-scale experiments to effectively model larger-scale cultures and describe an enhanced medium which generated over 160 mg/L-1 6dEB (a 22-fold improvement over current culture media). Additional feeding experiments provided new insight and understanding related to the heterologous production of 6dEB from E. coli. These results will allow further studies to explore alternate pathways to 6dEB production. Long term goals are to sufficiently produce therapeutic natural products using these newly developed techniques. |
