Team:KULeuven/Modelling/Vanillin Production

=Vanillin Production=

overview


The vanillin synthesis consists of a five step process starting from tyrosine, which is used quite regulary in the biosynthesis of vanillin in E. Coli.

The behaviour of this subsystem should be a production unit of vanillin which can be regulated by the instantaneous amount of RIBOKEY present in the cell.

E. coli controls its own tyrosine production which is an non essential amino acid. However,should this be insufficient, we can always add extra tyrosine. The production process will be tested in two steps. First, from tyrosine to ferulic acid; then from ferrulic acid to vanillin.

By locking both the transcription of the Sam 8 and COMT enzyme we prevent vanillin synthesis without the presence of RIBOKEY. Because we want the production of vanillin proportional to the amount of input key, it is essential that the regulated enzymes that catalyse the production of (intracellular) vanillin degrade as fast as possible. As proteins are normally very stable in the intracellular environment the SAM8 and COMT where labelled with a LVA-tag, which insures relatively fast half times. Due to the non zero half life time of the regulating enzymes, the production depends on the integrated amount of RIBOKEY over several half lives.

Biological model


Sequences used to test the vanillin production, the pathway from ferulic acid to vanillin in E. Coli is better investigated than the first. [7 ]



Simulation
Because COMT and SAM8 are regulated by the amount of mRNA key its worthy to investigate the change of COMT and SAM8 production in function of the input amount of mRNA key. Because Sam8 and COMT are located behind the same lock they are both translated at the same rate, therefore only the concentration of COMT is shown, the translation rate of both RNA strands is considered equal.

The time scale on which COMT and Sam8 reaches equilibrium conditions is about 2 magnitudes faster than the time scale on which vanillin reaches equilibrium. Therefore only the steady state levels of COMT and Sam8 are relevant for further investigation regulatory role of both enzymes in the production of vanillin.

Because we expect only to encounter small amount of free RIBOKEY (because of the fast decay times of untranslated RNA), the steady state levels of COMT and Sam8 are relatively linearly controlled by the RIBOKEY.

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Due to the complicated non linear (Michaelis-Menten kinetics) pathway from tyrosine to vanillin, the steady state level of vanillin well not be linearly depend on the concentration of RIBOKEY, also it takes a relatively large amount of time (hours) to reach steady state conditions.

The following figure shows that the steady state level of vanillin concentration is (non linearly) regulated by the amount of mRNA input key. Thought not linearly, the most important fact remains that the concentration of vanillin is efficiently regulated by the amount of RIBOKEY, and the leak of the RIBOLOCK is sufficiently low to obtain relatively low levels of vanillin.