Team:Calgary/BetaOxidation

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Beta-Oxidation

Aim

The objective of this part of our project was to genetically engineer E. coli to produce PHB from volatile fatty acids (VFAs) found in human fecal waste. VFAs (acetic acid, propionic acid, butyric acid, and lactic acid) serve as precursors for the synthesis of PHB via the beta-oxidation pathway native to bacterial cells (Reyhanitash, 2017).

Manipulation of the beta-oxidation pathway

In order to synthesize PHB from fatty acids found in human fecal waste, we manipulated the fatty acid beta-oxidation pathway within E. coli. We designed a construct that contains the phaJ4 gene from Pseudomonas putida, encoding for enoyl-CoA hydratase that converts the enoyl-CoA into (R)-hydroxybutyrate (Lu, 2003). Our construct also includes the phaC1 gene from Pseudomonas aeruginosa, encoding the PHA synthase, which converts the (R)-hydroxyacyl-CoA into polyhydroxybutyrate (PHB). To our advantage, this pathway not only uses VFAs but can also use undigested long-chain fatty acids in human fecal waste, thus maximizing the substrates available for PHB synthesis.

Genetic construct

Figure 1: Genetic construct for phaC1J4, including the ribosomal binding sites (BBa_B0034) and the phaJ and phaC genes

Our construct has been designed for translation in E. coli, as it contains the common strong RBS BBa_B0034 in front of each gene, and codons have been optimized. We also ensured that our BioBrick was compatible with all iGEM RFC standards by removing illegal restriction sites. Our construct can be placed under the control of an inducible or constitutive promoter, resulting in controlled or continuous production of PHB.

You can find our phaC1J4 operon (BBa_K2260001) on the iGEM Registry here.

Experiments

Vector Experiments

We ordered linear DNA inserts from IDT. The inserts are listed as follows:

• phaC1_{P. aeruginosa}J4_{P. putida}
• fadE
• fadD

We performed restriction enzyme digestion to ligate the inserts listed above 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.

Protein analysis

SDS-PAGE was performed on the pET29b(+)-phaCJ to confirm the expression of the gene after induction with IPTG because our construct was under a T7 inducible promoter.

Detailed results of the protocols for our experiments can be found on our Experiments page.

Results

Vector Experiments

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

PHB Production

The E. coli transformed with the ligated pET29b(+)-phaCJ 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(+)-phaCJ

Protein analysis

SDS-PAGE indicated that phaCJ was being expressed by the transformed E. coli.

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

Future Directions

Due to time restraints, we were not able to explore the effect of fadE or fadD upregulation on PHB production via the β-oxidation pathway. We would like to maximize the amount of PHB produced with our construct by adding fadE and fadD to the our phaCJ construct.This may produce more substrates for phaJ and phaC to convert into PHB.

We would also like to test out the effects of using constitutive promoters and compare them to the use of our IPTG-inducible T7 promoter by comparing the amount of PHB produced.

Works Cited

Lu, X., Zhang, J., Wu, Q. & Chen, G.Q. (2003) Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) via manipulating the fatty acid beta-oxidation pathway in E. coli. FEMS Microbiol Lett. 221: 97-101

Reyhanitash, E., Kersten, S. & Schuur, B. (2017) Recovery of volatile fatty acids from fermented wastewater by adsorption. ACS sustainable Chemical Engineering. 5: 9176-9184

Rose, C., Parker, A., Jefferson, B. & Cartmell, E. (2015). The characterization of feces and urine: a review of the literature to informed advanced treatment technology. Critical Reviews in Environmental Science Technology. 45: 1827-1879