Polzin Jason J, Rorrer Gregory L, Cheney Donald P
Department of Chemical Engineering, Oregon State University, Corvallis, OR 97331, USA.
Biomol Eng. 2003 Jul;20(4-6):205-15. doi: 10.1016/s1389-0344(03)00054-6.
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.
利用海洋大型藻类（即海藻）进行生物加工工程以生产次生代谢产物，是海洋生物技术中一个新兴的领域。一种新型系统是通过源自红藻Ochtodes secundiramea的“微型植株”悬浮培养物来生物合成卤代单萜。这个生物合成平台有三个主要组成部分：从香叶基二磷酸（GPP）合成月桂烯；溴鎓离子促进月桂烯卤化为10E-溴月桂烯、3-氯-10E-溴-α-月桂烯和3,10E-二溴月桂烯；溴鎓离子促进月桂烯环化生成阿帕考托烯B。在本研究中，对卤代单萜生物合成进行了代谢通量分析。为便于开展这项工作，开发了一种O. secundiramea微型植株的“无溴”细胞系，在该细胞系中生物卤化作用暂时被禁用，但月桂烯生物合成仍可进行。该细胞系在气升式光生物反应器中于营养培养基灌注条件下培养。通过向灌注培养基中协同添加溴化物和钒酸盐（溴过氧化物酶的一种辅助因子）来“开启”卤代单萜的生物合成。从这些实验中，确定了溴化物和钒酸盐添加对每种代谢产物代谢通量的影响。月桂烯在其Δ(6-10)烯键处的溴化是生物反应网络的主要分支，而该途径中的氯化步骤则“刚性较弱”。本研究代表了代谢工程原理在大型海洋生物次生代谢分析和操纵中的首次应用。
