Glycolysis

Aim

In order to utilize acetyl-coA, which is a product downstream of the glycolysis pathway, a naturally occurring operon in R. eutropha was used. The operon exists in the order phaC, phaA, and phaB. The bacteria uses this operon to convert its excess carbon source into poly[(R)-3-hydroxybutyrate] (PHB) . R. eutropha stores PHB as an energy source (Hiroe et al., 2012). Transcription of the phaCAB operon leads to expression of the following enzymes in the order: pha synthase, acetoacetyl-CoA reductase, and 3-ketothiolase. The expression of phaA leads to expression of 3-ketothiolase that converts acetyl-coA to acetoacetyl-CoA. The acetoacetyl-CoA reductase enzyme resulting from the expression of phaB leads to conversion of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. Finally, pha synthase leads to synthesis of PHB from (R)-3-hydroxybutyryl-CoA (Hiroe et al., 2012).

Operon rearrangement

The naturally existing operon phaC, phaA, and phaB in R. eutropha is known to produce PHB. However, literature has shown that the rearrangement of the operon to phaC, phaB, and phaA results in higher production of PHB (Hiroe et al., 2012). Hence, we decided to change the gene order from phaCAB to phaCBA. The operon rearrangement to phaCBA will lead to relatively higher expression of phaB compared to expression in phaCAB. Hiroe et al. showed that the content of PHB is dependent on expression of phaB. However, the molecular weight of PHB was not affected by the different levels of expression of phaB. The following image shows the rearrangement of the phaCAB operon to phaCBA.

Media/Culture composition

The phaCBA operon can utilize acetic acid 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)+. Competent E. coli (BL21) was then transformed with the plasmid. The overnights of successfully transformed cells was then used for our experiments. The different media composition used were glucose only and fermented syn poo supernatant. The glucose only condition will show the ability of our construct to use glucose as a feedstock (positive control) and the syn poo supernatant will be used to test whether our construct can synthesize PHB from synthetic feces.

Results

The results of the experiments for our phaCBA construct is given on results page and our parts registry.

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.