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  • Introduction
  • Effects of Varying Rates and Pool Sizes - A Sample Program
  • Consideration of Multiple Compartments
  • Consideration of Cycles - The GS/GOGAT Cycle
  • Compounds Receiving Several 13C Atoms from 13CO2
  • Isotopomers of the Citric Acid Cycle Supplied with 3-13C-Pyruvate
  • Modeling Radioactive Precursor Uptake Kinetics
  • Simulation of The Pathway of DMSP Biosynthesis in Enteromorpha intestinalis
  • Simulation of The Pathway of Synthesis of DMSP in Spartina alterniflora
  • Making Rates Linearly or Hyperbolically Responsive to Pool Size Changes
  • Metabolic Engineering of Glycine Betaine Synthesis - Metabolism of 14C-Choline in Transgenic Tobacco Expressing Choline Monooxygenase in the Chloroplast
  • Considering Feedback Inhibition
  • Modeling Allosteric Behavior - Cooperative Substrate Binding
  • Links to Other Metabolic Modeling Resources on the www
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  • Computer Simulation of Metabolism

    Metabolic Engineering of Glycine Betaine Synthesis - Metabolism of [14C]-Choline in Transgenic Tobacco Expressing Choline Monooxygenase in the Chloroplast

    McNeil, S.D., Rhodes, D., Russell, B.L., Nuccio, M.L., Shachar-Hill, Y., Hanson, A.D. 2000. Metabolic modeling identifies key constraints on an engineered glycine betaine synthesis pathway in tobacco. Plant Physiol. 124: 153-162.

    Glycine betaine (GlyBet) is a potent osmoprotectant that is a well established target for metabolic engineering of osmotic stress resistance in plants (Nuccio et al, 1999). In the GlyBet-accumulating plant, spinach, GlyBet is synthesized from choline (Cho) via the intermediate betaine aldehyde (BetAld) in a two-step oxidation, catalyzed by ferredoxin-dependent choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). Because BADH is present in non-GlyBet-accumulating plant species, the introduction of the gene encoding CMO is recognized as an important step in the engineering of this pathway in plants that lack the ability to synthesize and accumulate this osmoprotectant (Nuccio et al, 1998; 1999). However, when spinach CMO was expressed in tobacco and successfully targeted to the chloroplast, the resulting transgenic plants accumulated only modest levels of GlyBet unless the plants were supplied with exogenous Cho (Nuccio et al, 1998; 1999). To investigate the metabolic constraints limiting flux to GlyBet in transgenic tobacco expressing CMO, the metabolic fate of [14C]-choline was examined and analyzed by computer simulation (McNeil et al, 2000).

    The following figure illustrates a Michaelis-Menten model developed for interpreting the metabolism of [14C]-choline in non-salinized transgenic tobacco expressing spinach choline monooxygenase (CMO) in the chloroplast. Model goals have been to quantitatively account for the observed labeling kinetics (open circles) of the total endogenous pool of choline [green], phosphoryl-choline (P-Cho) [red], phosphatidyl-choline (Ptd-Cho) [magenta], and glycine betaine (GlyBet) [dark blue], in tobacco leaf disks supplied with a 170 nmol.g-1 Fw dose (P2) of [14C]-choline of specific activity 54 nCi.nmol-1 (P1). The initial specific activities of the endogenous pools of metabolites (A1, A5, B1, C1, D1, E1, F1) are all assumed to be 0 nCi.nmol-1. The initial pool sizes (A2 + A6 + F2, B2, C2, D2, E2) were experimentally measured. The total measured endogenous choline pool of ~72 nmol g-1 Fw is partitioned into cytosolic (A2), vacuolar (A6) and chloroplastic (F2) pools. The chloroplastic choline pool serves as substrate for the chloroplast localized transgene product, CMO. BetAld synthesized via the action of CMO on chloroplastic Cho at rate B3 is then metabolized to GlyBet via the catalytic action of BADH at rate C3. The unknown kinetic parameters of the transport systems and enzymes of the network (Km1 - Km11, and Vm1 - Vm11) were progressively adjusted until the observed data was satisfactorily accommodated by mimimizing mean absolute deviations between observed and simulated values. Model variables are defined in both the JavaScript and Java Applet versions of the program provided.

    | Code | Download VB 5.0 program | JavaScript | Java Applet |

    The major conclusion from this model was that flux to GlyBet in transgenic tobacco expressing CMO is primarily constrained by the low capacity [i.e. high apparent Km and/or low Vmax] of the chloroplast Cho transport system (F3; Km11, Vm11), which does not compete effectively with choline kinase (rate D3) or transport of cytosolic Cho to the vacuole (rate A4) for available cytosolic Cho (McNeil et al, 2000). This can be verified by downloading and running the Visual Basic 5.0 model [if you have Visual Basic 5.0 installed on your computer] or running the JavaScript or Java Applet versions of the program on your JavaScript and/or Java-enabled browsers. The simulated radiolabeling kinetics of GlyBet is critically dependent on the values of the kinetic parameters assumed for the chloroplast Cho transport system (Km11, Vm11).

    References

    McNeil, S.D., Rhodes, D., Russell, B.L., Nuccio, M.L., Shachar-Hill, Y., Hanson, A.D. 2000. Metabolic modeling identifies key constraints on an engineered glycine betaine synthesis pathway in tobacco. Plant Physiol. 124: 153-162.

    Nuccio, M.L., Rhodes, D., McNeil, S.D., Hanson, A.D. 1999. Metabolic engineering of plants for osmotic stress resistance. Cur. Opin. Plant. Biol. 2: 128-134.

    Nuccio, M.L., Russell, B.L., Nolte, K.D., Rathinasabapathi, B., Gage, D.A., Hanson, A.D. 1998. The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase. Plant J. 16: 487-496.

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    David Rhodes
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    Last Update: 8/20/03