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From 2012.igem.org

Auxin production

Overview

The 2011 Imperial College iGEM [1] project showed that the plant hormone auxin or indole-3-acetic acid (IAA) can be synthetized in Escherichia coli through a two-step pathway from tryptophan. In their work, the genes encoding the auxin biosynthesis pathway, originally coming from Pseudomonas savastanoi, were expressed in E. coli. In our project, we aim to express this auxin biosynthesis pathway in Xenopus tropicalis . First, we plan to reengineer the auxin biosynthesis pathway in the tadpole with a constitutive and ubiquitous promoter. Once the system is functional, we intend to put the system under the control of inducible and tissue-specific promoters.

By modelling the auxin biosynthesis pathway, we will be able to determine the number of produced auxin molecules per plasmids injected in the cell, as well as, the number of diffused auxin molecules through the plasma membrane into the extracellular medium.

Model description

The plasmids containing the two genes involved in the auxin biosynthesis pathway, namely iaaM and iaaH, will be expressed in a constitutive and ubiquitous manner in a first step and in a second step in an inducible and tissue-specific manner. The iaaM gene encodes tryptophan-2-monooxygenase (IAAM) that catalyses the conversion of tryptophan (Trp) into indole-3-acetamide (IAM). The iaaH gene encodes indoleacetamide hydrolase (IAAH) that hydrolyse IAM to realease indole-3-acetic acid (IAA) [2]. At the same time, the synthesized IAM and IAA will competitively inhibit the enzyme activity of IAAM. Produced auxin will then diffuse through the plasma menbrane into the extracellular medium and finally into the blood. All the reactions involved in the auxin biosynthesis pathway are illustrated in Figure 1.

Figure 1. Kinetic squeme depicting the auxin production model in the cell.

Most reactions were modelled using mass-action kinetics. Reactions involving enzymes (i.e. IAAM, IAAH) were modelled using competitive inhibition kinetics and irreversible Michaelis-Menten.

Assumptions

1. As most of the parameters in the system are unknown because the auxin biosynthesis pathway has never been put into Xenopus, we took the average transcription, translation and degradation rate constants from other genes in Xenopus [3].
2. Transcription and translation rate constants are assumed to be the same for iaaM and iaaH genes.
3. Degradation rate constants are assumed to be the same for mRNA-IAAM and mRNA-IAAH; for the proteins IAAM and IAAH; and for the compounds Trp, IAM and IAA.
4. We neglect the short time delay due to synthesis of IAAM-Trp (enzyme-substrate (ES) complex), IAAH-IAM (ES complex), IAAM-IAM (enzyme- inhibitor (EI) complex) and IAAM-IAA (enzyme- inhibitor (EI) complex) and assume that these species reach their equilibrium almost instantaneously.
5. The initial concentration of the number of plasmids, which determines the number of auxin-biosynthesis pathway genes, depends on the outcome of the plasmid repartition model in the embryo cells. The average plasmid concentration per cell was set to 0.011 μM and assumed to be constant over time for constitutive promoter and variable for inducible promoter.
6. L-Tryptophan is an essential amino acid, which means that its concentration will depend on the uptake from the medium. The Tryptophan inicial concentration was set to 500 μM [8][9][4] and assumed to be constant over the time course.
7. All other initial concentrations were set to zero.

Equations

where:

• iaaM: open reading frame encoding the enzyme IAAM coming from P. savastanoi
• iaaH: open reading frame encoding the enzyme IAAH coming from P. savastanoi
• mRNA-IAAM: mRNA coding the enzyme IAAM
• mRNA-IAAH: mRNA coding the enzyme IAAH
• IAAM: Tryptophan 2-monooxygenase
• IAAH: Indoleacetamide hydrolase
• Trp: L-Tryptophan
• IAM: Indole-3-acetamide
• IAA: Indole-3-acetic acid or auxin
• dIAA: diffused indole-3-acetic acid or diffused auxin

Parameters

Results

Cellular auxin production under a constitutive promoter

Figure 2. Cellular auxin production under a constitutive promoter. A. Time course for IAM production. B. Time course for IAA and dIAA production. C. Time course for dIAA production.

Figure 3. Time course of proteins producing auxin.

With the auxin-biosynthesis pathway genes, regulated by constitutive promoters, the auxin production reaches steady state around 7.5 hours (450 min) due to the equilibrium reached, around 4 hours (250 min), by the transcription and translation rates and degradation rates involved in the production and consumption of proteins IAAM and IAAH (Figure 3).

On the other hand, the concentration of diffused auxin is very low, 3.5 x 10^-6 μM at 16.7 hours (Figure 2.C), however it is increasing over time.

Cellular auxin production under an inducible promoter

Figure 4.Cellular auxin production under an inducible promoter. A. Time course for IAM production. B. Time course for IAA and dIAA production. C. Time course for dIAA production.

Figure 5. Time course of proteins producing auxin.

With the auxin-biosynthesis pathway genes, regulated by inducible promoters, the auxin production shows a transient response around 1.5 hours (100 min) due to the transient responses reached, around 50 min, by the degradation rates and transcription and translation rates of the involved proteins, i.e. IAAM and IAAH (Figure 5).

Similarly, the concentration of diffused auxin is much lower with inducible promoter, 5.4 x 10^-8 μM from around 7 hours (430 min) (Figure 4.C). In contrast with the constitutive promoter, the diffused auxin concentration does not increase over time but instead stabilizes after 7 hours.

Sensitivity analysis

Cellular auxin production under a constitutive promoter

Figure 6. Sensitivity analysis for initial concentrations: 1. Trp, 2. iaaH, 3. iaaM; and parameters: 4. Km_IAAH, 5. d_compound, 6. d_mRNA, 7. d_protein, 8. k_IAAM, 9. k_IAAH, 10. Ki_IAA, 11. Ki_IAM, 12. Km_IAAM, 13. Kz, 14. p, 15. Pr.

Cellular auxin production under an inducible promoter

Figure 7. .

Conclusion