Refactoring Ehrlich Pathway for High-Yield 2-Phenylethanol Production in Yarrowia lipolytica

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Efficient microbial synthesis relies on coordinated precursor and cofactor supply for cell growth and product formation.
• Substrate cofeeding can bypass native metabolism for higher carbon conversion rates.
• The Ehrlich pathway is crucial for producing high-value aromatics like 2-phenylethanol (2-PE).

Purpose of the Study:

• To engineer *Y. lipolytica* for efficient microbial synthesis of 2-phenylethanol (2-PE).
• To overcome the limitations of the Ehrlich pathway and 2-oxoglutarate (aKG) availability.
• To enhance the production of high-value aromatic compounds in oleaginous yeast.

Main Methods:

• Refactoring the Ehrlich pathway to identify optimal catalytic modules for 2-PE synthesis.
• Engineering a cytosolic 2-oxoglutarate (aKG) source pathway by combining bacterial aconitase and native isocitrate dehydrogenase.
• Modifying dicarboxylic acid transporters to improve aKG flux and blocking competing pathways.

Main Results:

• Identified optimal catalytic modules for the Ehrlich pathway, including l-phenylalanine permease and phenylacetaldehyde reductase.
• Successfully constructed a cytosolic aKG source pathway and engineered transporter activity, alleviating mitochondrial bottlenecks.
• Achieved a 4.16-fold increase in 2-PE production (2669.54 mg/L) with a high carbon conversion yield (0.702 g/g).

Conclusions:

• Metabolic engineering strategies can overcome Ehrlich pathway limitations and enhance 2-PE production in *Y. lipolytica*.
• Improving 2-oxoglutarate (aKG) availability and trafficking is critical for efficient l-phenylalanine assimilation.
• This work expands the utility of the Ehrlich pathway for producing high-value aromatics in yeast.