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. In order to accomplish this, we:

1. analyzed human waste and chose organic compounds to use as feedstocks for our bacteria, and
2. researched and optimized efficient pathways to turn relevant components of human waste (acetyl-CoA and volatile fatty acids) into PHB

Chassis and vector

Our genetic constructs were placed under a T7 IPTG-inducible promoter in the pET29(B)+ vector, which contains the gene for kanamycin resistance. The chassis used for our experiments was E. coli BL21(DE3), whose genome contains the sequence for T7 RNA polymerase to allow for transcription of our constructs. The bacterium, 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).

Genes and Choice of pathways

The glycolysis pathway includes genes involved in PHB synthesis: phaC, phaA and phaB. This pathway uses short-chain fatty acids as its source. From several analyses of solid human waste in literature, we found that short-chain fatty acids including acetic acid, propionic acid, and butyric acid, were present in our feedstock (Rose et al., 2015). We chose to manipulate the beta-oxidation pathway and its fad genes because this process of fatty acid degradation feeds on both long-chain and medium-chain fatty acids (Black & DiRusso, 1994). 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.

Hiroe et al. (2012) investigated the effect that gene order of the phaCAB operon had on the PHB molecular yield. They found that the highest PHB yield was found when the operon's genes were ordered as phaCBA. (More details can be found here.) Therefore, we decided to use this gene order to maximize PHB production.

In addition, Davis et al. (2008) showed that expression of phaC1 and phaJ from PHA-producing bacteria such as Pseudomonas aeruginosa and putida produced PHB and medium chain length PHAs. The phaC1 gene from P. aeruginosa encodes PHA synthase, which regulates the final step in both the beta-oxidation and PHA synthesis pathways. This function of PHA synthase makes it one of two important enzymes in our pathway as it accepts 3-hydroxybutyryl-CoA molecules, cleaves off the CoA segment, and polymerizes the 3-hydroxybutyrate into PHB. The other key enzyme in our pathway is encoded for by phaJ4, which we selected from P. putida based on findings from Tsuge et al. (2011). Our research showed that the enzyme encoded for by phaJ4, enoyl-CoA hydratase, channels products of the beta-oxidation pathway into the glycolysis pathway.