Oleaginous yeast are yeast that are able to convert more than 20% of their body mass to lipids under certain environmental conditions. Their high lipid content gives them a potentially wide ranging applications in chemical productions, including the production of biodiesel, vegetable oils, and the makeup industry. However, industrial bottlenecks make using yeast impractical for many applications. Among the bottlenecks is the cost to destroy the cell wall during the lipid extraction process. Existing methods, such as ultrasonication, bead milling, microwaving, and chemical treatments have their problems. One promising and underexplored method of cell disruption is enzyme-assisted extraction. In their 2020 paper, Heshof et al. outlined a process whereby the enzymes produced by the mycoparasite Trichoderma harzianum were used against an oleaginous yeast to digest the cell wall. I plan on cloning two enzymes outlined in the paper and transforming them into the oleaginous yeast Y. Lipolytica. This will create a cell that can self destruct on command. To do so, I will be using the EasyCloneYALI expression vector set. I will build a custom bioreactor in order to collect accurate measurements. I predict that the autolysing yeast will result in a cheaper alternative to the method outlined in Heshof et al.
Y. Lipolytica is a promising organism for chemical synthesis as it naturally produces an excess of triacylglycerides in a nitrogen deficient environment On the flip side, the robust cell wall of these yeasts, made of chitin, β-glucan, and mannoproteins, is very difficult to disrupt by mechanical or chemical means. This results in expensive postprocessing in any chemical synthesis pathway. Both ultrasonication and bead milling methods achieve high recovery rates but the equipment is very expensive and energy-consuming; on the contrary, organic solvent extraction gives the highest lipid production but has safety and environmental issues [1].
Enzyme-assisted cell disruption has the potential of being a much cheaper method of cell wall disruption: Using the mycoparasite T. harzianum, Heshof et al. (2020) was able to degrade the cell wall of an oleaginous yeast and perform successful lipid extraction.. However, these methods require the growing of batch of T. harzianum alongside in a separate bioreactor, adding to the complexity of the extraction system. The solution is to develop Y. lipolytica with a self-supplied, inducible autolysis system—combining genetic control and enzymatic disruption—to streamline the process and cut costs.
3a. The implementation of a cheap system of lipid extraction from an oleaginous yeast has the potential to revolutionize the world. It would mean we are one step closer to a cheap source of biodiesel. Once other challenges are overcome, such as bringing the cost of the food source down, oleaginous yeast can be used to revolutionise many industries, from cometic oils to the food industry.
3b. This project's uniqueness relies on three factorces. The first factor is that the experiment has been designed to be able to be performed on a limited budget. The second factor is that the project involves improving on an open source bioreactor. The final factor is the particular genes that will be transformed into the yeast, which have never been done before.
3c. A primary ethical consideration is the environmental and bacterial release consequences: The engineering of a self‑lysing microorganism stands at the very top of a problem of unintentional gene flow and ecological damage. The basic principles of non-maleficence and justice promote strict containment measures and risk assessment to avoid the possibilities of accidental release or misuse.
In the process of ethical conduct, we will do several things such as (1) choose auxotrophic strains that cannot survive outside the lab, (2) implement physical biocontainment (close reactors, filtered vents), and (3) integrate kill-switch circuits activated by environmental cues. We will keep monitoring for the potential side effects I mentioned before by carrying out environmental simulacra tests. Other options we can consider are cell-free expression systems or the use of immobilized enzymes, which do not involve living GMOs at all.
The first aim is to build and implement the inducible autolysis cassette in Yarrowia lipolytica using the EasyClone YALI expression vector system and demonstrating that it is a viable alternative to existing cell wall destruction methods.
The second aim is to build out a DIY bioreactor using the pioreactor as a starting point and augmenting the system with atlas scientific sensors.
The final aim is to run a full scale experiment, where the bioreactor from step 2 will be used in conjunction with the yeast from step 1 in order to compare the oil extraction efficiency of inducible autolysis when compared to other methods.
For Aim 1, I will be engineering my yeast using the EasyCloneYALY expression vector system. I chose this system for multiple reasons. First off, it is modular, and so I could use this system for other experiments in the future, mixing and matching what genes I want to insert. Second off, it is well documented. I was able to find all the procedures online, and that went a long way to helping me make my decision. Other systems seemed very cool at first, but there was not enough documentation on them for me to feel comfortable using it. The biggest reason, though, is cost. The cost for all the materials is below $1,000.
The EasyClone YALI system allows me to insert genes at 11 different insertion sites in the yeast genome. For every insertion site, I can insert up to 2 genes as well as a promoter. I chose 2 genes from Heshof et. al, 2020 that were found in the highest concentrations. These were Proteases PKK55157.1 and PKK54770.1. In addition, I chose the bidirectional inducible promoter pEYK1 outlined in Trassaert et al., 2017 as it can be induced relatively easily using the common sugar erythritol.
The cloning system also requires the purchase of custom primers of the following format:
Gene 2 primers forward primer (GP2F): ATCTGTCAU GCCACA ATG(N)n reverse primer (GV2R): CACGCGAU TCA(N)n Promoter 1 primers forward primer (PV1F): CACGCGAU(N)n reverse primer (PG1R): ACCTGCACU(N)n Promoter 2 primers forward primer (PV2F): CGTGCGAU(N)n reverse primer (PG2R): ATGACAGAU(N)n USER overhang in bold, GCCACA – Kozak sequence, ATG – start codon, TCA – stop codon, (N)n – gene(promoter)-specific sequence.
These will be purchased from addgene.
https://media.addgene.org/cms/filer_public/39/3e/393ee754-938a-4658-b09c-54c070791f53/easycloneyali_integrative_vector_set_for_yarrowia_lipolytica_with_markers.pdf
The following is my experimental setup: