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Synthesis

Overview

The overarching goal for the synthesis component of the project was to produce poly-3-hydroxybutyrate (PHB) by utilizing the nutrients present in human waste. To accomplish efficiently convert organic feedstocks into PHB, we genetically engineered bacteria to produce PHB by manipulating two metabolic systems within E. coli as follows:

Beta-oxidation pathway
Glycolysis pathway

Genes and Choice of Pathways

The genes that we manipulated and the choice of our pathway was informed by the types of organic compounds that are able to serve as substrates for PHB synthesis. We searched through literature and found several articles that analyzed solid human waste. We discovered that human fecal waste contains volatile fatty acids (short-chain fatty acids such as acetic acid, propionic acid, and butyric acid), long chain fatty acids, and glucose which can be used as substrates for PHB production (Rose et al., 2015). We wanted our system to be able to make use of a wide range of carbon sources and transform them into our desired product, PHB. Therefore, we decided to manipulate pathways and genes that use these substrates to synthesize PHB. (Hiroe et al., 2012; Balck & DiRusso, 1994; DAvid et al., 2008; Tsuge et al., 2011)

Beta-oxidation Pathway

VFAs and long-chain fatty acids can be broken down through the fatty acid β-oxidation pathway to synthesize PHB (Davis et al., 2008). We manipulated this pathway by transforming the bacteria with the genes phaC and phaJ.

Glycolysis Pathway

Glucose can be broken down into acetyl-CoA, which is then converted to PHB by genes involved in PHB synthesis: phaC, phaA and phaB (Hiroe et al. 2012). This pathway can also indirectly use VFAs as its source for PHB production.

Chassis and vector

We chose to use E. coliBL21(DE3) as our chassis because our genetic constructs were placed under a T7 IPTG-inducible promoter in the pET29(B)+ vector. The genome of E. coli BL21(DE3) contains the sequence for T7 RNA polymerase to allow for transcription of our constructs. An advantage to using this bacterium is that E. coli is known to adapt well to both aerobic and anaerobic conditions and grow quickly given an adequate carbon source. Being a popular model organism, its metabolic pathways are well studied and thus provided us with an extensive array of identified molecules and structural components to manipulate (Black & DiRusso, 1994).

WORKS CITED

Black, P.N. & DiRusso, C.C. (1994). Molecular and biochemical analyses of fatty acid transport, metabolism, and gene regulation in Escherichia coli. Biochimica et Biophysica Acta. 1210: 123-145

Davis, R., Anilkumar, P.K., Chandrashekar, A. & Shamala, T.R. (2008). Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus. Antonie Van Leeuwenhoek. 94: 207-216

Hiroe, A., Tsuge, K., Nomura, C.T., 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. Applied and Environmental Microbiology. 78: 3177–3184.

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

Sato, S., Kanazawa H. & Tsuge, T. (2011). Expression and characterization of (R)-specific enoyl coenzyme A hydratases making a channeling route to polyhydroxyalkanoate biosynthesis in Pseudomonas putida. Applied Microbiology Biotechnology. 90: 951-959