Team:Calgary/Biocontainment Engineering
Overview
Understanding the Problem
Research and Ideate Solutions
Experimental Design
Results
Shifting to Y. lipolytica
Future Directions
References
OVERVIEW
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Our initial goal is to create a proof of concept in the lab. To do this, we followed the engineering design cycle to build our project:
- Understand the Problem
- Research and Ideate Solutions: Identifying a biocontainment strategy
- Design Solutions: Part design
- Design Solutions: Experimental design
- Create: Results and Troubleshooting
It is important to note that there will be circular action in this process, particularly between steps 4-5 (i.e the results of step 5 informing iterations of step 4). However, we are currently presenting one turn of the engineering design cycle by working towards a lab proof of concept. In reality, all of these steps will repeat once we have accomplished our goals/tested our product in the lab and look towards community implementation. We highlight our plans beyond iGEM in the Future Directions section.
UNDERSTANDING THE PROBLEM
Why is biocontainment important?
Synthetic biology is an emerging field that promises scalable and low-cost solutions to global health, agricultural, and environmental problems in the form of self-replicating biological devices. As these solutions increasingly become a reality, they migrate from tightly regulated laboratories to private spaces, such as households. This raises significant safety concerns regarding biocontainment, as the introduction of non-native organisms into the environment can result in unintended adverse effects on the ecosystem. Oviita is a community product that is meant to be cultivated outside of the laboratory by the people in the community, as such ensuring high levels of biosafety was a crucial part of our project. In order to ensure safety, iGEM Calgary set out to design a robust, yet sustainable biocontainment system.
Previous strategies
When we began our journey towards finding an effective biocontainment strategy, we considered many different options including auxotrophy and a kill switch system, before eventually settling on our final strategy of a complementary auxotrophy co-culture system.
Auxotrophy prevents the production of a vital molecule by an organism, thereby forcing the organism to become reliant on the external supplementation of the molecule of interest for survival (ND1). A positive side to auxotrophy is the low mutation rate of reverting the auxotrophy back to prototrophy. Find reference that says this. However, such a system introduces high costs, and is ultimately unsustainable because of the need for supplementation.
An alternative biocontainment strategy is the introduction of a kill switch system. Such a system comes with its own positive aspects and limitations. Kill switch systems are genetic circuits that lead to the production of a toxin or inhibitory molecule following exposure to a certain stimulus, resulting in the death of the organism (1). For example, a kill switch system could involve the production of a nuclease in response to blue light. However, some limitations for this method is that the system relies upon the availability of the stimulus in order to be effective. In addition, when we talked to Dr. Robert Mayall, he explained how there is a higher rate of mutating out the kill switch system due to the fitness cost instilled in an organism.
RESEARCH AND IDEATE SOLUTIONS
Our Goals
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Initial ideas
Light-induced kill switch system
The first biocontainment strategy we considered was a light-induced kill switch system. We reasoned that since light is readily available in the environment, and we don’t want our yeast cells to survive outside of the growth vessel in the environment, light would be a viable option to induce the death of our yeast cells. When we were exploring this option, we first came across an inducible kill switch system where exposure to blue light results in the expression of Cas9. Specifically, we wanted Cas9 to target essential genes of the yeast, as cleavage of these genes would result in the death of the organism. We found a system by Kuwana et al. (1), which involves a photoreceptor system of two magnets (pMag and nMag) that each are linked to one-half of the Cas9 gene. In response to blue light, the two magnets join together, allowing Cas9 to join together and cleave the target gene.
However, upon speaking to Dr. Marija Drikic, a metabolomics research associate, we decided to pivot from this for our biocontainment strategy. Her concerns included the fact that in her experience with research, the whole process in order to result in the cleavage of the gene of interest had numerous areas that could go wrong. For example, the cas9 protein might take a lengthy time to be expressed--too long of a time to be suitable for a biocontainment system.
Based on this feedback, we went back to literature review to find an alternative strategy. Eventually, we came across a different light inducible system by Xu et al. (2), which instead involves the expression of a transcription factor that is sensitive to light. In response to light, the transcription factor binds to its binding region, and causes the expression of the GOI. We decided to explore whether the GOI could be toxic protein that could thus be expressed in response to light. However, in speaking to Dr. Zaramberg, a professor specializing in yeast research, we learned that there were a number of difficulties with the successful implementation of this idea. For example, whatever toxin we chose had to be non-toxin to humans or denatured by heat. As well, leaky expression of the protein has to be controlled for, as any premature expression would result in the killing of our yeast cells. Though we did extensive literature review, we unfortunately could not find a toxin that had previously been heterologously expressed with sufficient characterization or testing. We received advice, once again by Dr. Marija Drikic, who suggested we research further into proteins or enzymes that could target components of the yeast cell wall, protein inhibitors and nucleases. After considering several options we decided upon nucleases as our GOI to be expressed by our cells in response to light. We reasoned that this would be the simplest means for the yeast to be killed.
Nucleases as the GOI
Anika add stuff
However, upon introduction of our finalized biocontainment strategy to faculty members, we received a number of comments about our system that we decided to completely switch gears. For instance, if our cells were ever released into the environment, we were questioned on whether light would be able to penetrate soil or deep water bodies in order to reach the escaped organisms. As well, light can be a very variable environmental condition. Thus, there were concerns on whether light could be relied upon as our inducible agent on a consistent basis to activate our kill switch system. Based on these concerns, we decided to go back to the drawing board.
Toxin-Antitoxin System:
One of the other biocontainment strategies we started exploring was the toxin-antitoxin system. These systems usually comprise two expression systems; a toxin expression system and an antitoxin expression system. The toxin would have a lethal effect on the cell upon expression but the antitoxin would be able to diffuse the toxin effect on the cell. The antitoxin would be under the control of a weak constitutive promoter while the toxin would be under the control of an environmentally inducible promoter. Low expression of the antitoxin would buffer out any leaky expression of the toxin but once a certain environmental cue activates the toxin expression system, the toxin would be produced in significant quantities compared to the antitoxin and would be able to exert its lethal effect in the cell.
This biocontainment strategy has a major advantage over the typical kill switch system. Kill switch systems are most often evolutionary unstable. The leaky expression of the toxin in these systems even when the system is turned off would result in reduced fitness of the cells containing the kill switch system. As such, there is an evolutionary pressure for these switches to be selected out of the population. The toxin-antitoxin system, however, would reduce the fitness cost associated with the toxin expression system as an antitoxin would be expressed to buffer out leaky expressions of the toxin. Therefore, the toxin-antitoxin systems tend to be more evolutionary stable.
Despite some of the advantages of this biocontainment method, there are still serious shortcomings associated with this strategy. Our discussion with Dr. Robert Mayall, helped us identify some of the challenges of the toxin-antitoxin system. One of the major downsides of using such a system is the extra stress we would put the cells under by engineering two unnecessary pathways into the cells. In particular, the antitoxin expression pathway needs to always be active which would significantly reduce the fitness and growth rate of the host cell. Although some improvements have been made in recent years in this field, these systems are not yet close to the robust and viable biocontainment strategy we are looking for. Second, such systems have shown to have mild levels of success only in bacteria since they are native to those organisms. The introduction of these exogenous systems in yeast would be challenging and have not shown to be effective to the best of our knowledge. Third, reliance on a specific environmental signal to activate the toxin system is not always viable, especially since our yeast is designed to be used in all kinds of communities with varying environmental conditions. As such, we decided to look for a more viable biocontainment strategy.
Finalized strategy
Benefits of Auxotrophy
Syntrophic Co-culutre of Auxotrophs
We wanted to create a biocontainment system that took advantage of the robustness of an auxotrophy system but one that did not have the sustainability issues most associated with an auxotrophic system. Our discussion with Dr. Robert Mayall led us to the idea of using a cross-feeding co-culture of auxotrophic organisms. In this system, each strain will be auxotrophic for the molecule the other strain can produce, resulting in cross-feeding between the two strains. This eliminates the need to continuously add amino acid supplements to the culture and at the same time, it ensures tight regulation of the organisms as they would be mandated to stay together in the bioreactor to obtain their required amino acid. In case microorganisms escape the bioreactor, they would not be able to survive as they require their partner in sufficient quantity to grow, a condition only met inside the bioreactor. Thus this allows us to design a robust yet sustainable biocontainment strategy.
In order to bring our new biocontainment strategy to fruition, we looked in the literature for the mechanism of microorganism cross-feeding in co-cultures. We found many successful previous cross-feeding experiments done using different bacteria species such as S. indica, B. subtilis, and E. coli. In fact, such cooperative interaction in bacterial communities is thought to be prevalent in nature as such bacteria already have some of the mechanisms required for setting up successful cooperative relationships with other members of the community.
Yeast, however, are not naturally very good at exchanging metabolites with one another. A study by Shou et al. found that metabolite exchange among co-growing yeast cells are not occurring at growth-relevant quantities. Thus, two complementary auxotrophs would not be able to supports each other in a co-culture. However, they also found that a small set of genetic modifications in the biosynthesis of the amino acids could help establish cooperation between the two auxotrophic strain. These modifications would allow the yeast strains to overproduce the amino acid required by the other co-culture and thus would allow them to establish a cooperative community. In particular, they engineered an adenine auxotrophic strain of S. cerevisiae to overproduce lysine for a lysine auxotroph strain of S. cerevisiae that would overproduce adenine in return. The engineered complementary strains were able to form stable communities where a minimum cell density of each strain was required to support the growth of the co-culture. Another study by Hoek et al. performed similar experiments but with a tryptophan auxotrophic, leucine overproducing strain, and a leucine auxotroph, tryptophan overproducing strain of S. cerevisiae. They found that not only the two strains were able to establish a viable cooperative community but also over time the two strains established an obligate mutualism relationship where each strain required its partner for proliferation. The mentioned studies highlight the ability of yeast to take part in synthetic cross-feeding communities where they become completely dependent on their partner strain for growth.
EXPERIMENTAL DESIGN
Proof of concept
In order to provide a sustainable, community-based solution, we plan to genetically modify Rhodosporidium toruloides, an oleaginous yeast that naturally produces beta-carotene and lipids, to be more robust and resource-efficient. By modifying the yeast to produce cellulase, it can then use common agricultural waste products as an energy source for synthesizing its oil. It can then be eaten as a vitamin A supplement. The yeast strain, while naturally safe and non-pathogenic, will also be genetically modified to include a kill switch for bio-containment, and optimized for oil production.
Auxotrophy
We designed some simple experiments to confirm that the W303 engineered strains, ynn60 and ynn65, were indeed auxotrophic for tryptophan and leucine respectively. Both engineered strains along with the control wildtype W303 strain were each plated on three types of media:
Growth of the strains at 30 degrees Celsius on different plates were be observed for 48 hours.
Amino acid overproduction
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RESULTS
Auxotrophy
Figure 1. Auxotrophy Test. The image on the left shows that the ynn60, ynn65, and wildtype W303 strain did not grow on media lacking the required amino acid. Right image showing all three strains showing growth when supplemented with the required amino acids.
We successfully characterized the auxotrophic natures of our engineered strains. The ynn60 strain (tryptophan auxotroph) grew on all the plates except for the tryptophan deficient plate and the ynn65 strain (leucine auxotroph) grew on all plates except for the tryptophan deficient plate as expected. This confirms the auxotrophy of our engineered strains as the strains were not able to grow on the media lacking the amino acid they are auxotrophic for but they showed growth on media supplemented with the required amino acids. The wildtype W303 strain did not grow on either the leucine or tryptophan deficient media. This is expected as wildtype W303 strain is auxotrophic for both leucine and tryptophan.
Amino acid overproduction
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SHIFTING TO Y. LIPOLYTICA
Design considerations
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Part design: Genetic Constructs
In order to implement our biocontainment strategy in Y. lipolytica , we need to engineer two strains of yeast; one that is auxotrophic for tryptophan and overproduces leucine, and one that is auxotrophic for leucine and overproduces tryptophan.
Engineering amino acid overproduction:
To engineer a leucine overproducing and a tryptophan overproducing strain we designed two overexpression cassettes that contained the following components:
2-isopropylmalate synthase (LEU4) and Anthranilate synthase component 1 (TRP2) are important regulatory enzymes involved in the biosynthesis pathway of leucine and tryptophan respectively. These enzymes prevent overproduction of the amino acids as they are feedback-inhibited by the end products of the pathway (leucine and tryptophan). Overexpression of these two enzymes could help remove the bottleneck effect of the feedback inhibition and allow for the overproduction of the amino acids.
Therefore we designed two genetic constructs; one to overexprss LEU4 BBa_K3629023, and one to overxpress TRP2 BBa_K3629024. We used the stong intronic TEF promoter BBa_K3629001 in these constructs to help ensure that LEU4 and TRP2 are expressed at high levels. TEF promoter is one of the strongest native promoters of Y. lipolytica . We included the first intron of the TEF1 gene in the sequence of the promoter since the inclusion of the first intron has shown to allow for even stronger expression.
The mCherry reporter device BBa_K3629025 and the mCitrine reporter device BBa_K3629025 were included to help distinguish and quantify the leucine and tryptophan overproducing gene in the co-culture. Each strain will be engineered with one of the reporter devices and since each reporter device contains a fluorescent protein that fluoresces at a distinct emission wavelength, fluorescence spectroscopy can be used to distinguish and quantify the leucine and tryptophan overproducing strains in the co-culture.
Nourseothricin resistance expression construct BBa_K3629012, optimized for expression in Y. lipolytica , was also used in our cassettes as the selection marker.
Together, the LEU4/TRP2 overexpression constructs, the reporter devices (mCherry or mCitrine), and the Nourseothricin resistance expression construct made our overexpression cassette that could be used to engineer a leucine or a tryptophan overproducing Y. lipolytica . In order to allow for easy assembly of our cassettes, we made each of our genetic constructs compatible with Gibson assembly. Each part is flanked by a Gibson homology sequence that allows for all the parts to come together in a single Gibson reaction.
Engineering Auxotrophy
To introduce leucine auxotrophy, we plan to knock-out 3-isopropylmalate dehydrogenase (LEU2), a common gene targeted in Y. lipolytica leucine auxotrophs. Tryptophan auxotrophy, however, has not been introduced before inY. lipolytica . In order to make tryptophan auxotrophic strain of Y. lipolytica , we plan to target TRP1, a common gene targetted in S. cerevisiae tryptophan auxotrophs. Since the tryptophan biosynthesis pathway of S. cerevisiae and Y. lipolytica are almost identical, knockout of TRP1 should produce tryptophan auxotrophs in Y. lipolytica .
We wanted to engineer auxotrophy and amino acid overproduction in Y. lipolytica in one efficient step. Therefore, we included homology arms to upstream and downstream of the LEU2 or the TRP1 gene in our overexpression constructs. For example, our TRP2 overproducing cassette is flanked with homology arms to upstream and downstream of the LEU2. So that when the yeast is transformed with our cassette, through homologous recombination the TRP2 cassette would insert in the middle of the LEU2 gene, knocking out the gene. As a result, the engineered strain would become auxotrophic for leucine and overproducing for tryptophan in one engineering step.
FUTURE DIRECTIONS
Improving our system
In order to provide a sustainable, community-based solution, we plan to genetically modify Rhodosporidium toruloides, an oleaginous yeast that naturally produces beta-carotene and lipids, to be more robust and resource-efficient. By modifying the yeast to produce cellulase, it can then use common agricultural waste products as an energy source for synthesizing its oil. It can then be eaten as a vitamin A supplement. The yeast strain, while naturally safe and non-pathogenic, will also be genetically modified to include a kill switch for bio-containment, and optimized for oil production.
Engineering Yarrowia lipolytica
In order to provide a sustainable, community-based solution, we plan to genetically modify Rhodosporidium toruloides, an oleaginous yeast that naturally produces beta-carotene and lipids, to be more robust and resource-efficient. By modifying the yeast to produce cellulase, it can then use common agricultural waste products as an energy source for synthesizing its oil. It can then be eaten as a vitamin A supplement. The yeast strain, while naturally safe and non-pathogenic, will also be genetically modified to include a kill switch for bio-containment, and optimized for oil production.
REFERENCES
- ND1: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264511/
