期刊
METABOLIC ENGINEERING
卷 2, 期 4, 页码 300-311出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1006/mben.2000.0158
关键词
glycine betaine; choline; choline monooxygenase; choline oxidase; pathway modeling; Nicotiana tabacum
资金
- USDA NRI CGP [98-35100-6149]
- C.V. Griffin, Sr. Foundation
- Florida Agricultural Experiment Station
- NSF [IBN-9813999]
The biosynthesis of the osmoprotectant glycine betaine (GlyBet) is a target for metabolic engineering to enhance stress resistance in crops. Certain plants synthesize GlyBet in chloroplasts via a two-step oxidation of choline (Cho). In previous work, a chloroplastic GlyBet synthesis pathway was inserted into tobacco (which lacks GlyBet) by expressing spinach choline monooxygenase (CMO). The transformants had low CMO enzyme activity, and produced little GlyBet (<= 70 nmol g(-1) fresh wt). In this study, transformants with up to 100-fold higher CMO activity showed no further increase in GlyBet. In contrast, tobacco expressing a cytosolic GlyBet synthesis pathway accumulated significantly more GlyBet (430 nmol g(-1) fresh wt), suggesting that subcellular localization influences pathway flux. Modeling of the labeling kinetics of Cho metabolites observed when [C-14]Cho was supplied to engineered plants demonstrated that Cho import into chloroplasts indeed limits the flux to GlyBet in the chloroplastic pathway. A high-activity Cho transporter in the chloroplast envelope may therefore be an integral part of the GlyBet synthesis pathway in species that accumulate GlyBet naturally, and hence a target for future engineering. (C) 2000 Academic Press
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