The Cattlelyst’s biofilter is based on a co-culture of two engineered microorganisms. The biofilter has to be implemented in cattle stalls, therefore it is important to ensure it is safe. This is why the safety is one of the pillars of our project. The iGEM PIPE was used in the context of the biosafety for the generation of auxotrophic microorganisms
The biosafety aspect of our project consists of multiple levels of safety. We designed a kill switch that makes sure that the organisms in our biofilter die in the absence of methane (formaldehyde). Additionally, our biosafety design also includes auxotrophies for both engineered organisms in our coculture. These auxotrophies make them co-dependent on each other for carbon sources and required amino acids. The full explanation of our three-layered safety system is described in the Biosafety page. For Cattelyst, we have created a co-culture of methantrophic Escherichia coli and ammoniatrophic Pseudomonas putida. More specifically, we engineered E. coli to grow on methane and secrete acetate, while P. putida needed to be able to use acetate as its carbon source. Additionally, the microorganisms must be auxotrophic for one amino acid each. From literature tryptophan (Trp) and arginine (Arg) were found to be the most promising pair of amino acids for generating a mutual dependency. Therefore, with this information, we used the iGEM PIPE and the COBRApy toolbox  to answer the following questions:
- Once models of the two microbes of our project are made auxotrophic, can we simulate their growth on the proposed carbon sources?
- Can we test if the models of the auxotrophic microorganisms can produce the amino acids needed for their co-dependency?
In particular, we want E. coli to grow on methane and P. putida to grow on acetate. The auxotrophy for both amino acids was simulated in the models of both organisms.
In this page you will find a description of the procedure used to perform the analysis for the metabolic models of E. coli and P. putida and the results we found. Experimental verification of our models can be found here.
Check our the experimental results on this topic
Functions of the iGEM PIPE have been used together with other existing toolboxes (CobraPy)  and were run in Jupyter notebooks that are available in our GitLab repository within the directory called “tutorial”. To generate auxotrophic strains we followed these steps:
- The first or second reaction in the amino acid biosynthesis pathways was manually removed from the model.
- The amino acid for which the model was made auxotrophic was added back to the media in silico.
- Successively, we checked whether the auxotrophic microbes could grow on the selected substrate and produce the compounds needed for co-dependency.
This should make the model unable to grow without an external amino acid.
This allows the model to restore growth.
Modelling auxotrophy in methanotrophic E. coli
The genome-scale metabolic model iML1515 of E. coli was used. Firstly, we removed the reactions corresponding to the first step in the biosynthetic pathway. The pathways accessible from databases such as KEGG  give information on the gene and/or enzyme that catalyzes the reaction. The code corresponding to the BiGG reaction was manually found. The knock-out (KO) strategies chosen for the two amino acids are explained below:
- For Trp auxotrophy we decided to remove the gene trpC encoding for Indole-3-glycerol-phosphate synthase.
- For auxotrophy using Arg, the gene argA encoding the N-acetylglutamate synthase was selected for the KO.
This corresponds to the reactions IGPS and PRAli (different reactions independently associated to trpC). The removal of the reaction IGPS from the model of E. coli was sufficient for obtaining an auxotrophic strain unable to grow on glucose.
The gene corresponds to the reaction ACGS, which if deleted from the model led to the desired auxotrophy.
The amino acid Arg or Trp was added to the medium of the respective auxotrophic model. The flux of the added amino acid corresponded to the amount consumed by the wild type (WT) model when growing on glucose, so that the same growth rate was restored. In the auxotrophic strain for Trp the WT growth rate (0.87 1/h) could be restored supplying 0.05 mmol/gDW/h of Trp in the medium. In the strain auxotrophic for Arg, growth rate (0.87 1/h) can be restored by supplying 0.26 mmol/gDW/h of Arg in the in silico medium.
It is already known that E. coli cannot grow on methane thus the addition of reaction(s) was expected to be needed. Thus, we used the functions of the iGEM PIPE to look for which reactions to add to the auxotrophic models for making them able to consume methane and produce the respective AA.
Auxotrophy for triptophan
Four strategies involving the addition of reactions to E. coli model were found. Figure 1 shows the variants of the models simulating a Trp auxotrophic strain growing on methane. Production of Arg (when set as an objective for the model) was already possible in all the model variants without the addition of any other reactions. The rate of uptake of Trp for restoring growth and the rates of production of A rg are indicated in Figure 1. Production of Arg is needed to complement the ammoniatroph P. putida when auxotrophic for Arg.
Auxotrophy for arginine
For growth on methane and production of Trp six strategies were found by the iGEM PIPE. Figure 2 shows which reactions the PIPE suggested to add to the model of E. coli auxotrophic for Arg. Production of Trp is possible in the first three model variants without the addition of any other reactions. The last three strategies required the addition of two extra reactions to consume methane and produce Trp. The addition of reactions for succinate (i.e. SUCCt2r and SUCCt2b) and malate production (i.e. MALtpp) suggest that co-production of these two compounds might go hand in hand with Trp secretion.
Modelling auxotrophy in ammoniatrophic P. putida
P. putida needs to be able to grow on acetate according to our biosafety design. The model iJN1463 of P. putida can simulate growth on acetate when the compound is present in the in silico medium and glucose uptake is blocked. This was known from literature research  and the replication of these conditions in the model did not require the addition of any reaction for allowing growth on acetate.
Again, we removed the reactions corresponding to the first step in the biosynthetic pathway:
- To make P. putida auxotrophic for trp, either of the following genes needs to be knocked-out:
- To make P. putida auxotrophic for Arg one of the following genes has to be knocked out according to literature :
trpA, trpC, trpD or trpE . trpE encodes for Anthranilate synthase, is represented by reaction ANS. Knocking it out did not lead to auxotrophy and ANS did not carry flux when simulating growth of the WT model. So ANPRT , the next reaction in the biosynthetic pathway, was removed to simulate the knock-out of trpD.
argH, argG, argA, argD, argB or argF . argA gene correspond to reaction ACGS and its removal from the model was sufficient for simulating auxotrophy.
To restore growth one of the two amino acids was added to the medium. The model auxotrophic for Trp could grow on acetate with a very little supplement of Trp (uptake flux of 0.0455 mmol/gDW/h ). On the other hand, auxotrophy for Arg could be complemented by supplying Arg with a higher uptake rate of 0.22 mmol/gDW/h.
Auxotrophy for arginine while growing on acetate
The Arg-auxotrophic model of P. putida growing on acetate allowed the production of Trp with a rate of 1.105 mmol/gDW/h without the need of any reaction knock-in.
Auxotrophy for tryptophan while growing on acetate
At first the auxotrophic model capable of growing on acetate could not produce arginine and no reaction could be found by the PIPE for providing this functionality.
We wondered if the model was missing efflux transporters for Arg. Studying the model we found no co-transport mechanisms of Arg with protons from the cytosol to the periplasm. Therefore, a reaction for that transporter has been added to the reaction database and the PIPE identified it as the solution that allows Arg production.The production of Arg in P. putida had then a flux equal to 2.18 mmol/gDW/h.
Figure 3 summarizes the results obtained for the two auxotrophic strains of P. putida.
The iGEM PIPE was used to validate predictions based on the literature and to guide the experimental choices for the construction of amino acid co-dependency.
We could draw three main conclusions from this application of the iGEM PIPE to our own project:
- Arg might not be the most optimal amino acid to choose for the auxotrophic co-dependency.
- Wild-type P. putida is expected to be able to use acetate as its carbon source even when auxotrophic for an amino acid.
- Engineered E. coli is expected to be able to consume methane as a carbon source while producing the amino acid necessary for co-dependency.
This is because since for both organisms it should be supplied in a high amount to the media for re-establishing the wild type growth rate . This was evident when the flow rates of amino acid uptake by the auxotrophic models were compared : 0.05 mmol/gDW/h for Trp complementation for both E. coli and P. putida vs 0.26 and 0.22 for Arg complementation of E. coli and P. putida respectively.
In our project we were able to experimentally confirm point 1: higher concentrations of arginine should be supplied to the medium of an Arg auxotrophic E. coli compared to other amino acid-auxotrophic pairs. This is why we chose to base the auxotrophic co-dependency on Try and histidine in our final design. Time constraints made it hard to test the last two points. Nonetheless, they indicate the feasibility of our approach.
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