After the polyethylene plastic is decomposed by laccase, we plan to use bacteria to uptake the decomposed waste as a carbon source. As such, the second module of this project involves an alkane metabolism pathway, where the chassis organism is Shewanella Oniedensis MR-1, which can generate electricity for our project.
To generate the alkane metabolism pathway, the starting product (alkanes) must penetrate into the cytoplasm through the cell membrane. While short-chain alkanes (less than C12) are able to diffuse directly into the cell, medium to long-chain alkanes cannot due to their increased hydrophobic nature. Since the laccase fragmentizes PE into randomized lengths, it is in our interest to make sure that all ranges of alkane size can be efficiently metabolized within the cytoplasm. To this end, we have to use an outer membrane alkane channel protein to mediate the entry of the medium to long chains alkane.
According to the literature, an outer membrane channel protein called AlkL, encoded by alkL, from Pseudomonas putida GPo1 has been shown to enhance the diffusion efficiency of dodecane (C12) into the cell when expressed in Escherichia coli. It is also shown to be able to facilitate the transport of larger alkanes, which has high hydrophobicity, making it more difficult to pass through the lipopolysaccharide outer membrane of the bacteria. The mechanism behinds AlkL is that it penetrates through the lipopolysaccharide layer, where an extracellular domain with high affinity for hydrophobic molecules lies. After the alkane molecule is collected via this domain, it makes its way through the hydrophobic core of AlkL. This subsequently allows the alkane to makes its way to the cytoplasm via a small lateral opening of AlkL. From there, the alkane may make its way to the cytoplasm. The outer membrane alkane channel protein(AlkL) we plan to use is derived comes from Pseudomonas Oleovarans. However, there is one drawback with AlkL: when overexpressed, it is toxic to the host cell. As such, we will attempt to express it with different strengths of promoters and test the effect of AlkL toxicity on the host’s growth rate before proceeding. For more details, check out our parts or our results.
Figure 1. AlkL representation.
After successfully passing through the bacterial cell wall barrier, alkane can undergo degradation. Originally, it was thought that alkane degradation could only occur in the presence of oxygen. This was soon proven to be false: discovered in a bacterial species known as Desulfatibacillium alkenivorans AK-01 found originally in water contaminated with oil spills, its native alkane degradation can operate in anaerobic conditions. It is also able to metabolize the medium to long chain alkanes. This feature is key to developing the further step of electrogenicity in the MFC part of our project.
Desulfatibacillium alkenivorans AK-01 had its genome sequenced in 2012 and a theoretical model for its alkane degradation pathway involving 4 major enzymes was proposed, which we hope to use in the second part of our project. The pathway is as follows: once the alkane fragment enters the cell cytoplasm, it combines with fumarate under the action of alkylsuccinate synthase (ASS) to form 1-methylalkylsuccinate. Then, CoA Transferase adds a CoA group to it, producing 1-methylalkylsuccinyl-CoA. Afterwards, its C-skeleton is rearranged by methylmalonyl-CoA mutase to be converted it to 2-methylalkylmalonyl-CoA. Finally, it is converted to 4-Methyloctadecanoyl-CoA by Carboxyl transferase where a CO2 is removed. Upon reaching this stage of molecular rearrangement, the compound is ready to enter β-oxidation of the cell.
Figure 2. Alkane Metabolism pathway.
Our design incorporates the 4 above steps into Shewanella which is hypothesized to functionally metabolize alkanes. It is hoped that once the alkane can ultimately enter β-oxidation, it will subsequently enter the TCA cycle by the cell’s natural processes so that it can serve as a carbon source for Shewanella to generate electricity. However, some of the aforementioned proteins are actually made from subunit peptides. Therefore, to enable such a metabolic pathway, theoretically a total of 9 genes are necessary. The genes AssA1, AssB1, AssC1, AssD1 code for subunits that form alkylsuccinate synthase, assisted by AssE1 that encodes for a chaperone-like protein. AssK1 encodes for CoA transferase. McmSS and McmLS code for methylmalonyl-CoA mutase. And finally, Dalk_1740 codes for Carboxyl Transferase. The original plan was to transform all of them into Shewanella but the complexity of the genes posed a major challenge, one which we intend to solve next year.
Therefore, by engineering AlkL and the alkane metabolic pathway into Shewanella, we hope it provides a bridge between PE degradation and exo-electrogenicity.
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