Team:CCA San Diego/Part Collection

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Part Collection

An Overview

While the focus of our project was to degrade the Polycyclic Aromatic Hydrocarbons (PAHs) Chrysene, Naphthalene, Phenanthrene, and Fluorene, two additional PAHs named Salicylate and Phthalate were extremely important in carrying out the degradation. Being directly involved in the pathways of our focus PAHs, both salicylate and phthalate functioned together to help degrade the target PAHs into intermediates usable by the hydrogen synthesis construct . In such, they played a crucial role in both the PAH degradation and the biohydrogen synthesis parts of our project, while also contributing to the overall collection of parts that contribute to the degradation of PAHs. More information about each of Salicylate and Phthalate is described below, accompanied by detailed codon maps.

Salicylate

Circular Map
Description

Our Salicylate construct degrades salicylate to tricarboxylic acid cycle intermediates (pyruvate and acetyl coenzyme A) through the meta-cleavage pathway. Nucleotide sequences coding for the upper pathway have been determined in several Pseudomonas species, most notably Pseudomonas sp. strain C18, which is where the genes were derived. The strain was isolated from a Western Mediterranean region and sequenced. The specific genes coding for the degradation of salicylate were isolated and utilized in the degradation pathway into the tricarboxylic acid cycle intermediates.

Our salicylate construct furthers our ability to degrade PAHs, as it is a common intermediate within multiple PAH degradation pathways. Indeed, the degradation of naphthalene, which is known to be the most abundant PAH in crude oil, most commonly degrades into salicylate. The proposed construct, which degrades salicylate, will further the degradation of naphthalene and allow for it to be fully degraded into useable cellular energy. Furthermore, it contributes to the overall collection of parts that contribute to the degradation of PAHs.

The specific genes involved in this degradation are NahAc and NahAd, which both function to degrade salicylate. nahAc is a component of the naphthalene dioxygenase (NDO) multicomponent enzyme system which catalyzes the incorporation of both atoms of molecular oxygen into salicylate. The alpha subunit has a catalytic role in the holoenzyme. Also able to catalyze the cis-dihydroxylation of biphenyl and phenanthrene. nahAd is the beta subunit that seems to have a structural role in the holoenzyme.

Phtalate

Circular Map
Description

Our Phthalate construct degrades phtalate to tricarboxylic acid cycle intermediates (succinate and acetyl coenzyme A) through the meta-cleavage pathway. Nucleotide sequences coding for the upper pathway have been determined in several Pseudomonas species, most notably Pseudomonas sp. strain PTH10, which is where the genes were derived. The strain was isolated from a Western Mediterranean region and sequenced. The specific genes coding for the degradation of phthalate were isolated and utilized in the degradation pathway into the tricarboxylic acid cycle intermediates.

Our phthalate construct furthers our ability to degrade PAHs, as it is a common intermediate within multiple PAH degradation pathways. Indeed, the degradation of two of the most common PAHs in crude oil, Phenanthrene and Fluorine, often require the degradation of Phthalate in their pathways. The proposed construct, which degrades phthalate, will further the degradation of phenanthrene and fluorene, and allow for them to be fully degraded into useable cellular energy. Furthermore, Phthalate contributes to the overall collection of parts that contribute to the degradation of PAHs.

The specific genes involved in this degradation are PHT2, PHT3, PHT4, and PHT5, all of which function to degrade phthalate. In the overall process, PHT2 functions as a Phthalate 4,5-dioxygenase oxygenase reductase subunit, while PHT3 acts as a Phthalate 4,5-dioxygenase oxygenase subunit. Continuing, PHT4 acts as a Putative 4,5-dihydroxyphthalate dehydrogenase, while PHT5 functions as a 4,5-dihydroxyphthalate decarboxylase. This subpathway is part of the pathway phthalate degradation, which is itself part of Xenobiotic degradation. Furthermore, each of these four are believed to be repressed by glucose.