Team:Calgary/Glycolysis

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Glycolysis

Aim

In many species of bacteria, acetyl-CoA results from the breakdown of glucose through glycolysis and the breakdown of fatty acids through beta-oxidation. In Ralstonia eutropha, excess acetyl-coA is converted into poly(3-hydroxybuturate) (PHB) and stored as a carbon energy source (Hiroe et al., 2012). R. eutropha accomplishes this feat with the use of three genes:

• phaA, which codes for 3-ketothiolase and converts acetyl-CoA to acetoacetyl-CoA;
• phaB, which codes for acetoacetyl-CoA reductase, which converts acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA, and;
• phaC, which codes for pha synthase and converts (R)-3-hydroxybutyryl-CoA, to PHB.

Our goal was to express these genes in E. coli to produce PHB using glucose as an initial substrate.

Operon rearrangement

In R. eutropha, the aforementioned genes exist in the order phaCAB. However, literature has shown that the rearrangement of the operon to phaCBA results in higher production of PHB (Hiroe et al., 2012). Therefore, we decided to change the order of our operon.

Figure 1. Naturally existing phaCAB operon in R. eutropha on top and our rearranged operon phaCBA on bottom.

Hiroe et al. (2012) showed that the content of PHB is dependent on the expression of phaB. This rearrangement from phaCAB to phaCBA will lead to higher expression of phaB compared to that of the native operon, resulting in more PHB produced. However, the molecular weight of PHB was not affected by the different expression of phaB, indicating the chemical structure of the polymer remained consistent.

To ensure efficient protein expression, we placed all three genes downstream of a strong RBS, BBa_B0034. We also optimized the codons of all three genes for expression in E. coli and were sure to remove all illegal restriction sites, making our part compatible with all iGEM RFC standards.

You can find our phaCBA operon (BBa_K2260000) on the iGEM Registry here.

Media Composition

The phaCBA operon can acetyl-CoA produced from the breakdown of glucose and volatile fatty acids (VFAs) present in fermented synthetic feces supernatant (which is referred to as "syn poo" supernatant). In order to test our gene construct, the operon was inserted into pET29(B)+ under the control of an IPTG-inducible promoter and transformed into E. coli BL21(DE3). Various media were used to test the ability of the recombinant bacteria to use glucose and VFAss to produce PHB. Differing combinations of these initial substrates were compared to demonstrate our operon’s working efficiency.

Results

We were able to conclude that our phaCBA operon produced PHB!

Detailed results of these experiments can be found on our Results page and this part's Registry page.

References

Hiroe A, Tsuge K, Nomura CT, Itaya M, Tsuge T. 2012. Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli. Appl. Environ. Microbiol. 78:3177–3184. 10.1128/AEM.07715-11.