Advancing Yarrowia lipolytica sub-organelle engineering with endogenous mitochondrial targeting sequence

Area of Science:

• Biochemistry
• Molecular Biology
• Yeast Genetics

Background:

• Mitochondrial engineering requires precise protein localization within yeast cells.
• Existing mitochondrial targeting signals (MTS) often necessitate codon optimization for heterologous expression.
• Yarrowia lipolytica is a key organism for industrial biotechnology and metabolic engineering.

Purpose of the Study:

• To identify and validate an endogenous mitochondrial targeting signal (MTS) from Yarrowia lipolytica.
• To enable efficient compartmentalization of target proteins within the mitochondria of Y. lipolytica.
• To provide an alternative to heterologous MTS sequences, avoiding codon optimization.

Main Methods:

• Identification and isolation of the MTS sequence from Yarrowia lipolytica citrate synthase.
• Fusion of the identified MTS (YlCISY-MTS) with Green Fluorescent Protein (GFP).
• Comparison of YlCISY-MTS efficiency with the MTS from Saccharomyces cerevisiae cytochrome oxidase subunit IV using confocal microscopy.

Main Results:

• The endogenous YlCISY-MTS successfully directed GFP to the mitochondrial matrix in Y. lipolytica.
• Localization efficiency of YlCISY-MTS was confirmed to be significant.
• This endogenous MTS eliminates the need for codon optimization of S. cerevisiae MTS in Y. lipolytica.

Conclusions:

• An endogenous MTS from Y. lipolytica has been successfully identified and validated.
• This finding facilitates targeted protein delivery to mitochondria, crucial for metabolic engineering.
• Leveraging Y. lipolytica's unique mitochondrial environment opens new avenues for biotechnological applications.