Metabolic modeling identifies key constraints on an engineered glycine betaine synthesis pathway in tobacco

Previous work has shown that tobacco (Nicotiana tabacum) plants engineered to express spinach choline monooxygenase in the chloroplast accumulate very little glycine betaine (GlyBet) unless supplied with choline (Cho). We the refore used metabolic modeling in conjunction with [C-14]Cho labeling exper iments and in vivo P-31 NMR analyses to define the constraints on GlyBet sy nthesis, and hence the processes likely to require further engineering. The [C-14]Cho doses used were large enough to markedly perturb Cho and phospho choline pool sizes, which enabled development and testing of models with ra tes dynamically responsive to pool sizes, permitting estimation of the kine tic properties of Cho metabolism enzymes and transport systems in vivo. Thi s revealed that import of Cho into the chloroplast is a major constraint on GlyBet synthesis, the import rate being approximately 100-fold lower than the rates of Cho phosphorylation and transport into the vacuole, with which import competes. Simulation studies suggested that, were the chloroplast t ransport limitation corrected, additional engineering interventions would s till be needed to achieve levels of GlyBet as high as those in plants that accumulate GlyBet naturally. This study reveals the rigidity of the Cho met abolism network and illustrates how computer modeling can help guide ration al metabolic engineering design.