Team:CCA-San Diego/Design

Horizon starts with the idea of solving the issue of degrading the carcinogenic and toxic parts of crude oil called PAHs (CDC PAH-degrading isolated systems are the most important for the future of integrity and strength in crude-oil degrading microorganisms (Ghosal et. al The incorporation of four major PAH degradation constructs into the Horizon project, serve as the fuel source for the bacteria that later produce clean energy. The design of these constructs closely follows research conducted this current year, as well as those that were discussed last year in our project “imPAHct.”


Fluorene is a major component of crude oils, and its degradation is highly impactful. egraded into pyruvate and acetaldehyde via an operon pathway derived from .

Chrysene is degraded into acetyl-CoA via a novel operon pathway derived from Pseudomonas fluorescent. Previous research involving the catabolic degradation of chrysene has been done, and multiple pathways have the capacity to

Out of the PAHs found in crude oil, naphthalene is present at the highest concentration [11]. The Eawag Biocatalysis/Biodegradation Database details degradation pathways derived from several organisms; previous research shows that the dox operon from Pseudomonas sp. strain C18 aligns with this degradation.

Figure 1: Degradation of naphthalene from genes encoded by the Dox operon.

Biohydrogen was a portion of our project that allows for the degradation of crude oil to be repurposed into clean energy via bacteria fermentation.

In certain species of bacteria, including Escherichia coli, the anaerobic process of mixed acid fermentation converts formate to hydrogen and carbon dioxide via the formate hydrogen lyase (FHL) complex. The FHL complex is regulated by fhlA, which activates transcription of the fdhF, hyp, hyc, and hydN-hypF operons [9].

hyfR is a homolog of fhlA that transcribes the hyf operon (hydrogenase-4) and has also been found to enhance expression of fdhF [10, 4]. Hydrogenase-4, along with hydrogenase-3 (hyc operon), is the hydrogen-producing unit of the FHL complex [9].  

The fnr and arc systems work in concert to regulate anaerobic respiration [3]. In the absence of oxygen, both ArcA and FNR repress aerobic processes (ETC “dampers”) and promote the transcription of anaerobic enzymes [1,5], including fhlA. FNR is activated by its oxygen-sensitive [4Fe-4S] clusters and appears to be specific to anaerobic respiration and fermentation pathways. ArcA is phosphorylated by its counterpart ArcB and exerts a broader range of control over various intracellular redox conditions [6]. Both systems are sensitive to different levels of oxygen in the environment, meaning the level of expression of target operons varies depending on the degree of air saturation [8].

In anaerobic conditions, both FNR and ArcA induce the transcription of pyruvate formate-lyase, the enzyme responsible for converting pyruvate into formate [2,7]. Thus, overexpression of the fnr and arc systems may increase formate concentrations during fermentation and consequently increase biohydrogen production via the FHL complex.

There is a lack of experimental evidence for the combined effects of FNR and ArcA on fhlA or hyfR expression. However, it is possible that the binding of the FNR enzyme nearby the hyf sequence may minimize the ability of the larger hyfR protein to bind to its upstream target sequence [4]. This possibility may be tested in our experiments.

Our goal with the beyond constructs was to target more hydrocarbons in crude oil for the purpose of expanding the Horizon project. To decide which hydrocarbons we wanted to target we analyze which hydrocarbons were possible to degrade, but also are harmful to the environment. We decided to focus on three of the most prevalent hydrocarbons in crude oil: the long chained hydrocarbons including alkanes, alkenes, and alkynes.

Alkanes are the most simple type of hydrocarbon, following the formula CnH2n+2. By reading literature that studied bacterial degradation, we determined the genes and enzymes necessary for the degradation of these compounds. One article that looked in n-alkane biodegradation mechanisms highlighted the bacteria Pseudomonas Putida (Ji et. al); specifically, we looked at the Alk gene system of Pseudomonas putida. This operon encodes seven proteins, of which three are involved in alkane degradation (Kok et. al). To determine which exact genes were necessary we looked at the degradation pathway for octane, a type of alkane. The necessary enzymes were alkane monooxygenase (coded for by the gene AlkB), rubredoxin-2 (coded for by the gene AlkG), and rubredoxin-Nad(+) reductase (coded for by the gene AlkT). These enzymes all work in conjunction to degrade octane into 1-octanol. The enzymes already present in E. coli strain K12 can then degrade the new product of 1-octanol all the way down to Octanoyl-CoA, which the bacteria can use as an energy source. The functions and names of these three enzymes present in E. coli can be seen in the degradation diagram below.

Alkenes are a type of hydrocarbon that follows the formula CnH2n. During our initial research, we discovered an article that examined the bacteria Xanthobacter Strain Py2 and the enzyme Alkene Monooxygenase (Zhou et. al). The Xamo operon present in this bacteria plays an integral role in the degradation of alkenes. From the Eawag Biocatalysis/Biodegradation Database, we obtained a degradation pathway for propylene. The first enzyme in the pathway was alkene monooxygenase, which degrades propylene (also known as 1-propene) into 1,2-epoxypropane. This enzyme is coded for by XamoA-F. The enzymes

XecA, XecD, and XecC, all from Xanthobacter, work separately to degrade 1,2-epoxypropane all the way down to acetoacetate. Acetoacetate can be broken down by E. coli into 2 acetyl-CoA molecules, which can be directly inserted into the Krebs Cycle for ATP production. Therefore, by using a combination of enzymes present in Xanthobacter, in conjunction with enzymes already present in E. coli, an alkene can be broken down into a compound that eventually the bacteria can use to produce energy.

Alkynes are a hydrocarbon which follows the formula CnH2 n‐‐2.

Figure 2: Degradation of Alkane into intermediate compound using recombinant gene.

Figure 3: Conversion of intermediate compound into usable energy form within the E Coli genome.

 (Ji et. al) (Kok et. al) (Zhou et. al)