In our Engineering Cycle 1, before performing PEX10 knockout, we would like to first test by simple chemical treatment whether impaired peroxisomal function will lead to an increase in lipid accumulation as we expected. We identified several inhibitors that can affect the yeast peroxisomal function, among which the common catalase inhibitor ,2,4-triazole (3-AT, Cas:61-82-5) was finally chosen for this proof-of-concept test. 3-AT inhibits peroxisomal function by dysfunctioning the catalase activity and leads to its accumulation in peroxisomes[1] [2]. It can achieve a similar effect with the PEX10 knockout cassette which inhibits peroxisomal function by impairing peroxisome biogenesis.
For this experiment, we inoculated the Yarrowia lipolytica strain Po1f in standard YPD as control, and in YPD containing 10 mM of 3-AT obtained from Yingxin Laboratory Equipment Co.Ltd, Shanghai. The cells were cultured for 16 hours before harvested for lipid staining with Nile Red to test the level of cellular lipid accumulation. Please refer to the Lipid Staining page for more detail. 3 biological replicates are tested and measured 3 times for fluorescence in each trial.
Lipid staining result of 3-AT treated Yarrowia lipolytica and control
From our experimental results, we clearly observed that the existence of 3-AT leads to an increased lipid accumulation, with a difference exceeding the statistical error range, which indicates that impairing peroxisomal function is indeed an effective strategy in Yarrowia lipolytica. This provides strong evidence and a solid basis for future engineering of constructing the PEX10 knockout strain to achieve a similar but stronger and prolonged effect.