Bioengineering space-compatible yeast strains.
Our goal is to create Saccharomyces cerevisiae strains that are tolerant to microgravity-induced stress for bio manufacturing applications in space. To achieve this we used the AstroBio database to select promoters whose expression is highly and consistently upregulated in microgravity. These will be used as the promoter for a GFP-based reporter system that will be used to track the changes in expression of these genes due to microgravity stress. The reporter will be constructed within the yeast using homologous recombination and inserted using the CRISPR-Cas9 system. We will use a microgravity simulator as a way to apply the microgravity stress to the yeast. This will be used as the stressor for adaptive evolution experiments that will lead to the eventual resistance to microgravity. As a proof of concept, the genes necessary for vitamin A production will be inserted into the resistant yeast to see if it has an effect on the production of this biomolecule.
1. We selected promoters with upregulated expression in microgravity
We developed and used the AstroBio database to be able to easily compare microgravity-induced promoter expression changes in Saccharomyces cerevisiae across previous research publications. Promoter expression was considered to be significantly altered by microgravity when the adjusted p-value was less than 0.05. We selected an initial set of promoter candidates whose activity was significantly upregulated under microgravity conditions (positive Fold Change) when compared to control conditions.
2. We refined our selection criteria to promoters with expression upregulation that is specific to microgravity
To construct functional reporter strains, we narrowed down our initial selection of promoter candidates to those that show increased activity specifically under microgravity conditions when compared to their activity under other stressor conditions such high salt concentration, heat shock, hypoosmotic shock conditions, to name a few. We have accomplished this by using meta-analysis results available in AstroBio, as well as researching yStreX, and examining published studies.
3. We further refined our selection criteria to promoters in pathways which have been shown to be significantly affected by microgravity
HOG (High-Osmolarity-Glycerol): required for cell adaptation to multiple stress conditions (Hohmann, 2009)
Cell Wall Integrity: regulates the cell’s structural architecture including cell wall membrane and the cytoskeleton (Hohmann, 2009)
Heat Shock: regulates protein folding at elevated temperatures. Notably, HSP30 is the most highly upregulating gene in microgravity versus ground controls at 4.20 log2 Fold Change as reported by Kate McInnery of Montana State University (Sheehan, McInnerney, Purevdorj-Gage, Altenburg, & Hyman, 2007)
Oxidative Stress Response: Defense mechanism against reactive oxygen species (Moradas-Ferreira & Costa, 2000)
4. We added a promoter for Vitamin A
In partnership with iGEM Toulouse, we included pGAL10 as a proof of concept for Vitamin A production in yeast under microgravity conditions.
Preliminary Promoter List:
Human Practices Feedback on our Promoter List
Dr. Corey Nislow
Affiliation: NASA, University of British Columbia, Genetic Networks LLC
"The Osmoregularity pathway, the HOG pathway- it comes up in many of our experiments. This is a both a regulatory pathway and a stress responsive pathway. I think it'd be useful. The genes involved in managing oxidative stress in yeast, SOD1, SOD2, Glutathione are relevant in microgravity. I would also consider the cell wall integrity pathway."Interview with Dr. Nislow
Building the Reporter
A reporter could be used to measure the level of response that a biological system has to a change in the environment. For AstroYeast, the environmental change is microgravity. To measure this, we will insert a fluorescent reporter for genes that are highly and consistently upregulated in microgravity. To do this, we will cotransform a plasmid encoding Cas9 and a guide RNA targeting the genomic locus of choice with our PCR-amplified promoters Envy GFP and terminators. Sufficient upstream and downstream homology is used to enable assembly of the transcriptional unit by yeast homology-directed repair.
This figure shows our construct design for OXR1
The first step in building a reporter strain is identifying cellular processes that are affected by this environmental change. In the case of AstroYeast, we identified 11 promoters that are upregulated when yeast is exposed to microgravity. These promoters will be used to drive the expression of the reporter signal. Secondly, the type of reporter signal must be determined, which could be fluorescent, colorimetric, or of another kind. It is important to know whether one is looking for something that is turned on or off qualitatively, or something that produces a signal that must be quantified. For AstroYeast, fluorescence was selected as it is a time-tested way to produce a quantifiable signal. This being said, an on/off signal is not ideal as it would be on as long as the yeast are alive. Therefore, the only option available is a quantifiable signal that needs to be baseline corrected. Given these considerations, we chose a yeast GFP variant called Envy as a signal for our reporter strain. For AstroYeast, the CRISPR-Cas9 system will be used in order to insert the genes into the yeast genome to allow for stable expression of the construct. Genome target sites that have a limited effect on the native gene expression were identified and will be used as the insertion site.
Considering that AstroYeast is using extra copies of native promoters to drive the expression of a fluorescent protein, it is important to note that there will be some basal expression of the reporter due to the activity of these promoters. Once this part of the design is complete, one must figure out how to introduce their reporter into the biological system. This includes targeting specific sites for insertion, if necessary, and seeing what kind of system should be used for the insertion, be it CRISPR-Cas9-directed genomic integrations, or plasmid-based expression systems. The CRISPR-Cas9 system was chosen due to its efficiency compared to other insertion methods and its ability to target specific sites for insertion.
Finally, the Cas9 repair template must be designed. For an insert to be expressed, it must have a promoter, a gene of interest, and a terminator, assuming that homologous knockouts are not being performed. For AstroYeast, the promoter used was an additional copy of the promoter being investigated, the gene of interest was for the Envy GFP, and the terminator was that of the CPS1 gene, due to its efficiency.
Inserting into yeast
Yeast is an ideal chassis for synthetic biologists due to its diverse set of applications, ranging from being a model organism for human studies to operating as a mini factory for the biomanufacturing of nutrients. Another benefit to using yeast comes from the fact that unlike bacteria, yeast does not uptake external genetic material via horizontal gene transfer. However, gene insertion into yeast can have several complications, mostly involving the gene introduction and cloning processes (Amen & Kaganovich, 2017).
One challenge in our decision for insertion method was the process of reproduction. Bacterial binary fission allows for daughter cells to retain the genetic insert by copying the plasmid with both daughter cells receiving a copy. Bacteria also undergo conjugation, which can result in bacteria which do not have a plasmid to uptake a copy from another bacterial cell, which is known as horizontal gene transfer, allowing for more cells to carry the gene of interest. Yeast, on the other hand, are eukaryotic, meaning that the gene to be inserted must be in a form that is compatible with mitosis. In other words, it must have a centromere. If a gene were to be integrated into the native yeast genome, centromeres would already be present, however, this is no longer an issue thanks to advancements in biotechnology. A centromere must be introduced on the DNA fragment to ensure that the inserted DNA is properly segregated during mitosis (Meluh & Koshland, 1997).
Another major issue is the copy number, which has to be carefully controlled in reporter systems as it can be a significant source of error. While plasmids can be used, they offer limited control over the copy number in comparison to directly integrating the gene into the yeast genome itself (Da Silva & Srikrishnan, 2012). This is particularly important when introducing fluorescent reporters, as this variation in copy number leads to artificial changes in signal intensity, and therefore the potential emergence of false positives. Now with advancements such as the CRISPR-Cas9 system, the DNA fragment can be inserted directly into the yeast’s chromosome, making it so that centromeres are not needed for the insert, as the chromosomes themselves have the necessary molecular machinery. Additionally, this tool for insertions allows for better control over the copy number.
For these reasons, we have selected the CRISPR-Cas9 system which has proven to be one of the major go-to tools when it comes to eukaryotic gene editing as exemplified by this year's Nobel Prize in Chemistry awarded for its development. The CRISPR-Cas9 system can target specific sequences and integrate the gene of interest with the progenature continuing to carry the integrated gene. Yeast also has the ability to assemble DNA fragments together using homologous recombination. This allows for the DNA fragments with overlapping regions, which makes more traditional construct-building techniques, such as the use of restriction enzymes, redundant (Dicarlo et al., 2013). CRISPR-Cas9 paired with Flagfeldt sequences allows for the insertion of our constructs with limited interruptions to the expression of the native proteins. Flagfeldt sequences are integration sites at which the expression of inserted constructs has been characterized and has shown to be a good insertion site for studies on heterologous proteins (Flagfeldt et al., 2009).
The CRISPR-cas9 system has the ability to target specific sequences and integrate the gene of interest, allowing for progenature to continue to have this gene. Yeast also has the ability to stitch together oligonucleotides with overlapping regions, making it so that more traditional construct-building techniques, such as the use of restriction enzymes, redundant (Gibson, 2009). This paired with Flagfeldt sequences allows for the insertion of a construct with limited interruptions to the expression of the native proteins. Flagfeldt sequences are integration sites at which the expression of inserted constructs has been characterized and has shown itself to be a good insertion site for studies on heterologous proteins (Bai Flagfeldt et al., 2009).
Evolution is a process that occurs over a very long time period, but this process can be accelerated in a laboratory through evolutionary experiments. These experiments involve exposing cells to a new environment that they will adapt to as they experience selection pressure (McDonald, 2019). This process can be accelerated by the addition of a mutagen. Applied before the evolutionary pressure, the mutagen will cause random mutations in the genome, creating a genetically diverse population of cells (Springman, Keller, Molineux, & Bull, 2010). After many generations which have gone through natural selection, the final result will be a population of cells that have adapted to the new environment. There are two types of evolutionary experiments, the first of which is adaptive evolution, which involves evolving the organism as a whole to the new environment (LaCroix, Palsson, & Feist, 2017). The second type of evolutionary experiments is directed evolution, which involves targeting specific enzymes and pathways to adapt to new environments rather than the organism as a whole (Johannes & Zhao, 2006).
For AstroYeast, we plan on using adaptive evolution in order to create a yeast strain that is tolerant to the microgravity-induced stress response. To accomplish this, we will add a mutagen to the yeast cells in order to create a genetically diverse population. The yeast cells will then be exposed to simulated microgravity for a period of 2-3 weeks, allowing the cells to adapt to and evolve in a microgravity environment. To create this microgravity environment, we will use a microgravity simulator that we are planning to build.
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