This is the third and final installment of the series on this paper. If you missed it, you can check the previous posts, Part 1 and Part 2. Here, we continue our examination of the steps taken by the authors by diving into Step 5:
- Design a pathway
- Screen for optimal acetyl-CoA pathway
- Test affect of pathway on growth
- Test the effect of feed mixes on IBA production
- Test pathway with isotope tracing
- Demonstrate technique to increase glucose consumption
In this step, the authors confirm that carbon is flowing as expected and as drawn out in Fig. 1. They accomplish this with the magic of isotope tracing.
How did they test their hypothesis?
It is now time for the grand confirmation: is glucose and acetate being used the way we think it is? We can actually trace where the molecules are going in a chemical reaction by using isotopes and instruments like GC-MS. Gas Chromatography-Mass Spectrometry works by first separating out the chemical components of a sample (the GC part) and then weighing the m/z values of the components (the MS part). M/z is the mass-to-charge ratio, which changes if you change the isotopic components of a molecule. In this case, we are working with carbon. The typical isotope for carbon is C-12, but we can get glucose or acetate spiked with C-13 isotopes to change the m/z values and analyze the GC-MS data to note where the carbon is going.
Fig. 4A is your key to understanding and interpreting the GC-MS data. At the top, we have a complete isobutyl acetate molecule, divided into red and blue depending on whether it should be coming from isobutanol (the glucose) or acetyl-CoA (the acetate). The left and right columns show how the m/z values change for different fragments of IBA change as you alter isotopic sources of glucose and acetate. The middle column shows the m/z values for isobutanol when the isotopes are changed.
So for example, in normal glucose and acetate, m/z = 56 for isobutanol, and 73 for the right fragment from IBA.
If you spike the acetate but use regular, C-12 glucose, the isobutanol’s m/z value does not change (no acetate is going into isobutanol production). There are two carbon atoms in the right IBA fragment, however, and if they are indeed coming from acetate, the m/z value will increase from 73 to 75. There are similar predictions made from spiking the glucose instead of the acetate with C-13.You can trace out similar patterns and changes in the rest of the diagram.
A quick glance over Fig. 4B-G shows that this is all, in fact, confirmed. The m/z measurements confirm the reaction drawn out in Fig. 1. What is further interesting is that Fig. 4B-D give the data for Strain 7, and Fig. 4E-G for Strain 8. What is the difference for these two strains? They both contain the ackA/pta pathway, but are built into the different hosts (JCL260 & AL2045). That means that even if the PDHC enzyme is present, this new pathway for acetate → acetyl-CoA production is the favored enzymatic pathway to take for acetyl-CoA, and then IBA, production.
The authors used two general approaches to improve carbon yield:
- Prevent carbon loss.
- Maintain a redox balance.
By avoiding PDHC enzymatic reactions, both of these strategies could be employed. No more redox build-up, and no more carbon loss with CO2 by-products.
Then, in choosing a new pathway to insert into the E. coli genome, these rules were applied again. Using acetate to produce acetyl-CoA provided an alternative to carbon-loss pathways to acetyl-CoA previously used, and the pathway options were selected for (1) no carbon loss and (2) redox balance. Once an ideal reaction was identified, optimal glucose:acetate feeding ratios could be identified.
The authors confirmed their new strains built IBA using the glucose → isobutanol and acetate → acetyl-CoA pathways using C-13 isotopes in carbon sources and GC-MS analysis. They further confirmed that although increasing initial acetate concentrations were unfavorable for E. coli growth, one could optimize the glucose:acetate consumption ratio by adding acetate over time as the carbon sources were consumed.
This is, in short, a success story in biotech. Increased production, ultimately by reducing the need for sugar and adding in a carbon source gleaned from waste water? It’s an environmentally sustainable production method that’s more efficient and less costly.
Tashiro, Y., Desai, S. H., & Atsumi, S. (2015). Two-dimensional isobutyl acetate production pathways to improve carbon yield. Nature Communications, 6(May), 1–9. https://doi.org/10.1038/ncomms8488