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 therefore used metabolic modeling in conjunction with [(14)C]Cho labeling experiments and in vivo (31)P NMR analyses to define the constraints on GlyBet synthesis, and hence the processes likely to require further engineering. The [(14)C]Cho doses used were large enough to markedly perturb Cho and phosphocholine pool sizes, which enabled development and testing of models with rates dynamically responsive to pool sizes, permitting estimation of the kinetic properties of Cho metabolism enzymes and transport systems in vivo. This 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 transport limitation corrected, additional engineering interventions would still 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 metabolism network and illustrates how computer modeling can help guide rational metabolic engineering design.

既往研究表明，经基因工程改造使其在叶绿体中表达菠菜胆碱单加氧酶（choline monooxygenase）的普通烟草（Nicotiana tabacum）植株，若不外源施加胆碱（Cho），几乎无法积累甘氨酸甜菜碱（GlyBet）。因此，本研究联合代谢建模、[碳-14标记胆碱（[(14)C]Cho）]标记实验与活体磷-31核磁共振（(31)P NMR）分析，明确了甘氨酸甜菜碱合成的限制条件，进而锁定需进一步开展工程改造的潜在途径。本次实验采用的[碳-14标记胆碱]剂量足以显著扰动胆碱与磷酸胆碱的池内浓度，这为开发并验证可响应池浓度动态变化的代谢模型提供了基础，从而可在活体状态下估算胆碱代谢酶及转运系统的动力学参数。研究结果显示，胆碱向叶绿体的转运是甘氨酸甜菜碱合成的主要限制环节，其转运速率较胆碱磷酸化以及向液泡的转运速率低约两个数量级，而液泡转运与叶绿体转运存在竞争关系。模拟研究表明，即便解除叶绿体转运的限制因素，仍需实施额外的工程改造策略，才能达到天然积累甘氨酸甜菜碱的植株的甘氨酸甜菜碱积累水平。本研究揭示了胆碱代谢网络的刚性特征，并阐明了计算机建模如何为理性代谢工程设计提供指导。