Team:NRP-UEA-Norwich/Collaborations/Manchester
Manchester-Graz Collaboration
The Manchester-Graz team have developed an expression system designed to regulate single and multi-gene pathways for an intestine expression. For controlling a wide range of pathways it is designed in a flexible and modular manner. They tested the production of butyrate in the gut. The pathway was incorporated into the expression system model to observe the expression of butyrate under the control of the developed system. The model generated helped us to understand how the system is dealing with pathways that consist of several enzymes at an intestine level.
The system consists of two quorum sensing (QS) systems EsaR/I and CepR/I. The EsaR/I system belongs to the plant pathogen Pantoea stewartii. The second QS-System, CepR/I, belongs to the opportunistic pathogen Burkholderia cenocepacia. For details about the system and the model please look into: https://2015.igem.org/Team:Manchester-Graz/modelling.
Figure 1: Overview of the reduced pathway. Coenzyme A (Co A) is recycled in the pathway by the butyryl CoA-acetyl CoA-tranferase.
Butyrate is converted starting from two Acetyl CoAs over several steps to Butyryl CoA. In the last step the Coenzyme A is transferred to Acetate, producing Acetyl CoA and Butyrate1. For simplicity, the pathway was reduced to some essential parts and steps in the pathway. 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 (Figure 1).
The pathway is controlled by three enzymes whose expression is controlled by the two quorum sensing systems.
If we simulate the model, in the first two minutes the acetate gets first converted into Butyryl CoA and then the Butyrate production starts (Figure. 2). The following time butyrate gets produced constantly by the cells. The butyrate is transported out of the cells through diffusion by a rate of 207,6 µmol/h/L or 18 mg/h/L.
Figure 2: Butyrate production in the first two minutes.
Figure 3: Overview of the Simbiology model. For simplification the model was reduced to essential components.
Differential equations
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. Acetate is also 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.References
1. Susan E et al (2002) The microbiology of butyrate formation in the human colon.
