In collaboration with the iGEM Team NRP-UEA-Norwich we modeled the Butyrate pathway expressed by our expression system:
Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression .
Figure 1 Schematic representation of pathways for carbohydrate fermentation in the large intestine. 1 = Methanogenesis, 2 = reductive acetogenesis, 3 = butyryl CoA:acetate CoA transferase, 4 = phosphotransbutyrylase/butyrate kinase, 5 = phosphotransacetylase/acetate kinase, 6 = lactate dehydrogenase, 7 = acrylate pathway, 8 = succinate decarboxylation .
Butyrate is central in the cell metabolism (Fig. 1). It is converted starting from two Acetyl CoAs over several steps to Butyryl CoA. In the last step the Coenzyme A is transferred on a Acetate, producing Acetyl CoA and Butyrate.
For easier modeling the pathway was reduced to some essential parts and steps. Acetate is converted to Acetyl CoA. The steps to Butyryl CoA are reduced to one step. Coenzyme A is recycled in the last step to Butyrate and can be reused to produce Acetyl CoA (Fig 4).
Figure 2 Overview of the reduced pathway. Coenzyme A (Co A) is recycled in the pathway by the butyryl CoA-acetyl CoA-tranferase.
To simulate the butyryl pathway controlled by the iGEM Manchester-Graz expression system (http://2015.igem.org/Team:Manchester-Graz/Modeling ) the system had to be adjusted and several new species and parameters had to be introduced to the model (Fig. 3).
Figure 3 Overview of the Simbiology model. For simplification the model was reduced to essential compnents.
The pathway is controlled by three enzymes (and not 7) whose expression is controlled by the two quorum sensing systems.
Figure 4 Butyrate production in the first two minutes.
d(Acetate)/dt = fAcetate – (comp*Acetate) – (fAcetylCoA*Acetate*CoA*E1) -( dAcetate*Acetate+µ*Acetate)
Acetate is formed by the cell and gets either converted in competing reactions or by Enzyme 1 to AcetylCoA. Also Acetate is degraded constantly.
d(CoA)/dt = -( fAcetylCoA*Acetate*CoA*E1) + 2* fButyrate*[Butyryl CoA]*[AcetylCoA-ButyrylCoA transferase] – (dCoA*CoA + µ*CoA)
CoA is recycled in the pathway and is needed to produce AcetylCoA and becomes free in the Formation of Butyrate.
d([Acetyl CoA])/dt = fAcetylCoA*Acetate*CoA*E1 – (-2*( fButyrylCoA*[Acetyl CoA]*[Acetyl CoA]*E2)) – (comp2*[Acetyl CoA]) – (dAcCoA*[Acetyl CoA] + µ*[Acetyl CoA])
AcetylCoA is formed by Enzyme1 and two AcetylCoA are converted to one ButyrylCoA by Enzyme2. AcetylCoA takes also part in competing reactions and is degraded.
d([Butyryl CoA])/dt = fButyrylCoA*[Acetyl CoA]*[Acetyl CoA]*E2 – (fButyrate*[Butyryl CoA]*[AcetylCoA-ButyrylCoA transferase]) -( dButCoA*[Butyryl CoA]+µ*[Butyryl CoA])
ButyrylCoA is formed by Enzyme2 and gets converted by the AcetylCoA-ButyrylCoA transferase to Butyrate.
d(Butyrate)/dt = fButyrate*[Butyryl CoA]*[AcetylCoA-ButyrylCoA transferase] – (D*Butyrate) + r*D*Butyrateext - (dBut*Butyrate+µ*Butyrate)
Butyrate is formed by the AcetylCoA-ButyrylCoA transferase and is affected by diffusion through the cell membrane.
d(Butyrateext)/dt = D*Butyrate – (r*D*Butyrateext) – (dButext*Butyrateext)
Butyrateext is the Butyrate in the environment. It is affected by the diffusion.
 Pryde, E,. Duncan, H,. Hold, L,. Stewart, S,. and Flint, J,. (2002) The microbiology of butyrate formation in the human colon. FEMS microbiology letters, 217 (2), pp. 133-139.