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-hydroxybutyrate) (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

The aforementioned genes exist in the order phaCAB in R. eutropha. However, literature has shown that the rearrangement of the operon to phaCBA results in higher yield of of PHB (Hiroe et al., 2012). Therefore, we decided to change the order of our operon to maximize the PHB yield produced by this pathway.

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.

Experiments

Vector Experiments

We ordered the linear DNA insert from IDT. The constructs contained genes from R. eutropha arranged in the order phaCphaBphaA (phaCBA)

We performed restriction enzyme digestion to ligate the constructs with pET29b(+) vectors and transform into E. coli. Digestion confirmations were performed and the gel electrophoresis results were used to confirm the correct ligation of the vectors. Once the parts were confirmed via restriction enzyme digest and gel electrophoresis, we validated these parts through sequencing.

PHB Production

To synthesize PHB, we grew overnight cultures of our transformed E. coliBL21(DE3) for approximately 24 hours. These cells were then inoculated in flasks and supplemented with M9 media that contained different chemicals. A chemical extraction procedure was then performed on these cells to purify PHB from the cells.

PHB Analysis

PHB was digested with sulfuric acid and analysed by High Performance Liquid Chromatography (HPLC). The HPLC chromatograms were compared to that of industrial-grade PHB.

Results

Vector Experiments

We successfully ligated and transformed the phaC1_{P. aeruginosa}J4_{P. putida} insert into pET29b(+) to produce pET29b(+)-phaCBA.

PHB Production

The E. coli transformed with the ligated pET29b(+)-phaCBA part produced visible amounts of PHB.

PHB Analysis

The HPLC results verified the identity of the PHB produced by the genetically engineered bacteria which contained pET29b(+)-phaCBA

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.