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Beta Oxidation

Aim

The objective 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 a precursors for the synthesis of PHB (Reyhanitash, 2017).

In order to synthesize PHB from fatty acids found in human fecal waste, we manipulated the fatty acid beta-oxidation pathway within E. coli. By designing a construct that contains the gene phaJ, encoding for an enoyl-CoA hydratase that converts the enoyl-CoA from the beta-oxidation cycle into (R)-hydroxybutyrate (Lu, 2003). Our construct also includes the gene phaC, encoding for 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.

Volatile fatty acids & beta-oxidation pathway

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Genetic construct

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

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Results

The O/Ns were grown in the respective media for ~24 hours. The flasks containing different media was inoculated with the O/Ns after adjusting the OD₆₀₀. The composition of each of the replicate in the flasks is shown below:

Glucose (Positive control)	pET29B in BL21 (Negative control)
10 ml O/Ns in LB+Kan 7 ml 20% Glucose 100 uL MgSO₄ 5 uL CaCl₂ 10 ml M9 salts 5 uL 1M IPTG 20 ml dH₂O	10 ml O/Ns in LB+Kan "Syn poo" fermented supernatant 100 uL MgSO₄ 5 uL CaCl₂ 10 ml M9 salts 5 uL 1M IPTG
Fermented "syn poo" supernatant	Pure VFAs
10 ml O/Ns in LB+Kan 10 ml "Syn poo" fermented supernatant 100 uL MgSO₄ 5 uL CaCl₂ 10 ml M9 salts 5 uL 1M IPTG 23 ml dH₂O	10 ml O/Ns in LB+Kan VFAs 410 uL propionic acid 118 uL acetic acid 55 uL butyric acid 100 uL MgSO₄ 5 uL CaCl₂ 10 ml M9 salts 5 uL 1M IPTG 30 ml dH₂O

The OD₆₀₀ readings of .....???? were taken and recorded:

Condition	OD₆₀₀ of replicate 1	OD₆₀₀ of replicate 2	OD₆₀₀ of replicate 3
pET29b in BL21 (Negative control)	0.571	0.531	0.487
Glucose (Positive control)	0.190	0.195	0.139
Pure VFAs	0.140	0.134	0.146
Fermented "syn poo" supernatant	0.135	0.107	0.144

After spinning down the culture in flasks, the cells were resuspended in 1x PBS. The OD₆₀₀ readings were taken:

Condition	OD₆₀₀ of replicate 1	OD₆₀₀ of replicate 2	OD₆₀₀ of replicate 3
pET29b in BL21 (Negative control)	2.659	2.001	2.899
Glucose (Positive control)	1.934	1.887	1.919
Pure VFAs	0.510	0.571	0.532
Fermented "syn poo" supernatant	2.533	2.559	2.349

Future Directions

We would like to maximize the amount of PHB produced with our construct by upregulating the genes fadE and fadD upstream of the beta-oxidation cycle. This may produce more intermediates for phaJ and phaC to convert into PHB.

We would also test out the effects of using a constitutive promoter.

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.