Contribution
Paving the way for metabolic engineering in E.coli and more!
Contribution
Our contribution to future iGEM teams lies in the implementation and expression of an ectopic Beta-Oxidation pathway. The goal is to create a new, independent metabolic pathway that enforces a reliance in bacteria on metabolizing fluorinated compounds.
Objective 1: Defluorinating Enzyme Library
To facilitate the expression of multiple enzymatic inserts at the same time, we decided to create an optogenetically controlled backbone that is set up for Golden Gate. The design uses BbsI as a driver for goldengate. The original plasmid is set up with an mScarlet insert between the BbsI sites. This is to ensure that one may quickly choose successful golden gates after transforming, without needing to sequence the plasmid at every step. This modular method for Golden Gate cloning greatly facilitated our efforts. We hope that this will be a tool used by future iGEM teams in their cloning efforts.
- BBa_K4895203: mScarlet/pDusk GoldenGate Backbone (No His Tags)
- BBa_K4895202: mScarlet/pDusk GoldenGate Backbone (N-terminally His Tagged)
- BBa_K4895201: GoldenGate Insert Template
Throughout thorough literature review and rigorous testing, we came down to 5 main defluorinating enzymes with the best chance of inserting onto our endogenous metabolic pathway. The goal for this section is to aggregate our efforts in investigation and demonstrate the solutions we came up with.
Objective 2: A Modular Beta-Oxidation Cassette for E.Coli
With the defluorination out of the way, we had to find homologous enzymes that would prove more catabolically active and less substrate specific. However, to begin with, we needed to understand the endogenous metabolic pathways of E.coli. This proved to be useful, as it would lead us to our third obective over time.
Regulation of Enzymes
In E.coli, the regulation of Beta-Oxidative enzymes follows a somewhat convoluted, but also easy to understand path. The family of genes that encode for beta-oxidative enzymes are called the "fad" family. In the family, most of them share a singular regulator; fadR. fadR encodes an apoprotein, which binds to the promoter regions of the fad genes. Interestingly, fadR is a double regulator, and when it inhibits the transcription of the fad genes, it also promotes the transcription of the fab genes, as demonstrated in the at-a-glance image above. fadR, however, will no longer inhibit fad transcription in the presence of acyl-coAs. However, this is the tricky part:
acyl-coAs are synthesized in E.Coli by fadD or fadK, both members of the fad family. And while fadK is less regulated than fadD, it is also only expressed in anaerobic conditions. Therefore, to actively pursue beta-oxidation in E.coli, the best solution is to constitutively express them; using a plasmid.
Enzyme Activity/Pathway
Fatty acids, initially recognized by FadL in E. coli through a low-affinity binding site, move to a higher affinity site, leading to the displacement of a plug in FadL and subsequent release into the outer membrane. The process of crossing from the outer to the inner membrane is currently unknown. Once inside the inner membrane, fatty acids are activated via a two-step reaction by FadD through vectorial thioesterification, which is energetically costly. The Beta-Oxidation cycle involves four enzymatic steps, progressively shortening acyl-CoA by two carbons and generating acetyl-CoA. This cycle also yields FADH2 and NADH at each turn through specific enzymatic steps. More specific information can be seen on our parts pages here.
After all of that, we decided to strengthen E.Coli's beta-oxidation with homologous enymes from Salmonella Enterica. The benefit of Salmonella's Enzymes is that, with everything else the same, it is mostly more catabolically active and less substrate-specific. This advantage is likely due to the nature of Salmonella to reside in low sugar content environments. The relationship between Salmonella's fadBAE genes and E.Coli's fadBAE genes can be seen here.
With the documentation and allocation of knowledge of Beta-Oxidation on this page, we hope to continue the conversation of augmented metabolic systems for E.Coli. The hope for this page is to create a good stepping stone on which future teams can use for their metabolic engineering efforts. Specifically, our curation and design of BBa_K4895004 represents a complete beta-oxidation cassette for E.Coli, using Salmonella Enterica enzymes. The use cases for this part are mostly in the context of beta-oxidation and bioremediation, but there is literature to suggest that fadB also helps in biosynthesis and maintenance of membrane stability. Overall, our hope is to bolster the efforts of future teams with our concentrated knowledge.
Parts and Documentation
The following are the parts used in this cycle of iGEM, from the rationale provided above about the metabolic chassis construction.
Objective 3: Establishing Protocol to Create Micro Knockout Plasmid (MiKOP)
Due to our project's shift in attention from pure beta-oxidation, and a look at alternate metabolic cascades, we decided to test the activity of Salmonella's fadBA against the naturally-occuring anaerobic defluorinating enzymes of E.coli: fadIJ. Interestingly enough, the aerobic and anaerobic cycles share the same acyl-coA dehydrogenase: fadE. As such, we moved forward in knocking out the fadIJ genes of E.coli. Thankfully enough, the two enzymes shared one promoter genomically, and fadJ was directly downstream of fadI. This makes sense, as fadIJ also tetramerize, similarly to fadBA. As such, the strategy moving forward was to simply insert a second, putative terminator in front of the fadI promoter. However, we would also require a selection mechanism, so it was determined that an antibiotic resistance needed to be added.
This novel technique of designing and directly synthesizing inserts to create a small KO plasmid is entirely different from existing methods. Our new method is better in that it is more accessible, easier to design, as well as being relatively simple. At the same time, this technique demonstrates that circularized inserts ~2kb in length are able to enter bacteria, regardless of origin of replication or not. Furthermore, this technique requires only the ability to design and order linear inserts, as well as the ability to ligate and replicate DNA. The possibilities for genomic expression are truly endless, and have only become easier with the advent of this technique. For a more detailed workflow of this technique, click here.
Parts and Documentation
The following contain links to the documentation of and more specific ideas of the construction of this part.