Practical 4 Structural Analysis of the Woods_Ljungdahl Pathway

Introduction

The goal of this practical is to use what we have learned so far about structural analysis to extend the analysis of this pathway in Acetobacter woodii that was briefly described in Lecture 5.

The organism is capable of assimilating CO₂, CO and H₂ in various combinations, but each of these gases can also potentially appear as a product under different circumstances as many of the reactions are potentially reversible. The biotechnological interest is the extent to which this organism (and others that contain this pathway) can be used to capture CO₂ and CO emissions. The major product of A woodii is acetate, though ethanol can be formed, and ethanol can be a major product of some WLP organisms.

ATP synthesis is linked to unusual electron-bifurcating redox reactions, and the yield is low as the energy available in the gas-assimilating reactions is low, so the organism survives close to the threshold of life. Calculating the potential pathways and ATP yield is challenging by inspection of the network diagram, so elementary modes is a useful tool in this context.

The questions we therefore want to answer are:

1. What are the potential routes of gas uptake?
2. Which of these produces ATP?
3. What fraction of the gaseous carbon taken up is conserved in acetate or ethanol?
4. How does the utilisation of hydrogen affect ATP production?

Instructions

(a) Create and load the WLP model as you learned in the previous practical.

(b) Make sure that you can relate the reactions and metabolites in the model to those in the diagrams and reaction key in lecture 4.

(c) Calculate the enzyme subsets. Check your result against the diagram in the lecture

(d) Determine the sets of conserved moieties in the scheme using m.ConsMoieties(). Why do you think that, in the Pi-related conserved set, ATP only has a coefficient of 1 and ADP does not appear at all?

(e) Calculate the elementary modes of the model using ems = m.ElModes(). The output from print(ems.Stos()) will not be conveniently ordered as in the table in the lecture though it should show all the corresponding conversions. The modes can be classified and counted as follows:

(f) Extract the set of modes that consume CO2 by emco2 = ems.Consumes(“x_co2”). The number of these modes is given by len(emco2), and their reactions by the Stos() method as above.

(g) Partition the modes as in the table by the gas mixture consumed. ‘dir(ems)’ will list the methods available to do this. Similarly, divide the modes by different products (method ‘Produces()’. To compare the stoichiometries in the table, you may need to apply a scaling factor. ?Easy way to do this.

(h) Calculation of ATP stoichiometry for each mode. The ATP formed per C atom assimilated is the rate of the 'ADENOSINETRIPHOSPHATASE-RXN' divided by the net C incorporated, which is the sum of the CO2 and CO transport rates ('CO2_sf_tx' and 'CO_s_tx') Instructions to follow. For modes that consume H2, the amount of ATP per H2 can be found in a similar way ('H2_sf_tx').