title: Metabolic flux analysis of halogenated monoterpene biosynthesis in microplantlets of the macrophytic red alga Ochtodes secundiramea.
abstract: The bioprocess engineering of marine macroalgae (i.e. seaweeds) for the production of secondary metabolites is an emerging area of marine biotechnology. One novel system is the biosynthesis of halogenated monoterpenes by "microplantlet" suspension cultures derived from the red alga Ochtodes secundiramea. This biosynthetic platform has three principal components: elaboration of myrcene from geranyl diphosphate (GPP); bromonium-ion promoted halogenation of myrcene to 10E-bromomyrcene, 3-chloro-10E-bromo-alpha-myrcene, and 3,10E-dibromomyrcene; bromonium-ion promoted cyclization of myrcene to Apakaochtodene B. In this study, a metabolic flux analysis on halogenated monoterpene biosynthesis was performed. To facilitate this effort, a "bromine free" cell line of O. secundiramea microplantlets was developed where biohalogenation was temporarily disabled but myrcene biosynthesis was still enabled. This cell line was cultivated within an airlift photobioreactor under nutrient medium perfusion. Halogenated monoterpene biosynthesis was "turned on" by coordinated addition of bromide and vanadate (a co-factor for vanadium bromoperoxidase) to the perfusion medium. From these experiments, the effects of bromide and vanadate delivery on the metabolic flux of each metabolite were determined. Bromination of myrcene at its Delta(6-10) olefinic bond was the dominant branch of the bioreaction network, whereas chlorination steps in the pathway were "weakly rigid". This study represents the first application of metabolic engineering principles to the analysis and manipulation of secondary metabolism in macrophytic marine organisms.
authors.affiliation: Department of Chemical Engineering, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Polzin
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Rorrer
authors.forename: Donald P
authors.initials: DP
authors.lastname: Cheney
mesh_terms: D019149:Bioreactors / Q000382:microbiology*; D001965:Bromides / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D003198:Computer Simulation; D004734:Energy Metabolism / Q000502:physiology; D005818:Genetic Engineering / Q000379:methods*; D006846:Hydrocarbons, Halogenated / Q000378:metabolism*; D008954:Models, Biological; D039821:Monoterpenes / Q000378:metabolism*; D000461:Rhodophyta / Q000166:cytology / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*
chemical_list: D001965:Bromides; D006846:Hydrocarbons, Halogenated; D039821:Monoterpenes