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Plant Physiol, September 2000, Vol. 124, pp. 153-162
Metabolic Modeling Identifies Key Constraints on an Engineered
Glycine Betaine Synthesis Pathway in Tobacco1
Scott D.
McNeil,
David
Rhodes,
Brenda L.
Russell,2
Michael L.
Nuccio,
Yair
Shachar-Hill, and
Andrew D.
Hanson*
Horticultural Sciences Department, University of Florida,
Gainesville, Florida 32611 (S.D.M., B.L.R., M.L.N., A.D.H.); Center for
Plant Environmental Stress Physiology, Department of Horticulture and
Landscape Architecture, Purdue University, West Lafayette, Indiana
47907 (D.R.); and Department of Chemistry and Biochemistry, New Mexico
State University, Las Cruces, New Mexico 88003 (Y.S.-H.)
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 [14C]Cho labeling experiments and in
vivo 31P NMR analyses to define the constraints on GlyBet
synthesis, and hence the processes likely to require further
engineering. The [14C]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.
1
This work was supported in part by the U.S.
Department of Agriculture National Research Initiative-Competitive
Grants Program (grant no. 98-35100-6149 to A.D.H.), by the National
Science Foundation (grant no. IBN-9813999 to A.D.H.), by the
Department of Energy (grant no. DE-FG02-99ER20344 to D.R.), by a
grant from the National Institute of Science and Technology (to
Y.S.-H.), by an endowment from the C.V. Griffin, Sr., Foundation, and
by the Florida Agricultural Experiment Station. This is journal series
paper no. R-07426.
2
Present address: Corning Community College, Corning,
NY 14830.
*
Corresponding author; e-mail adha{at}gnv.ifas.ufl.edu; fax
352-392-6479.
© 2000 American Society of Plant Physiologists
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