Dual Pathway Enables High-Level Production of Very Long-Chain Fatty Alcohol in <i>Rhodotorula toruloides</i>

Very long-chain fatty alcohols (VLCFOHs) are crucial components for the production of lubricants, cleaners, and cosmetics. Microbial production of these fatty alcohols has emerged as a more sustainable and potentially cost-effective alternative to traditional manufacturing methods; however, the production levels require significant optimization. Oleaginous microorganisms have received special attention for their ability to produce fatty acid derivatives due to their high lipid content. In this study, we report for the first time the expression and optimization of the biosynthetic pathway for VLCFOHs in the oleaginous yeast <i>Rhodotorula toruloides</i>, targeting both the cytoplasm and peroxisome. Using the cytoplasmic pathway, 601.3 mg/L fatty alcohols and 215.9 mg/L VLCFOHs were obtained. Furthermore, two peroxisomal targeting signals were characterized and employed for VLCFOHs production. By implementing a dual-pathway approach involving both the cytoplasm and peroxisome, the fatty alcohol titer increased to 871.9 mg/L, with 320.5 mg/L VLCFOHs. In a 1-L bioreactor fed-batch fermentation, the strain expressing the dual pathway achieved titers of 5.4 g/L fatty alcohols and 2.1 g/L VLCFOHs, representing the highest reported titer of microbially produced VLCFOHs. The results expand the genetic toolboxes for <i>R. toruloides</i> and demonstrate its potential as a next-generation host to produce fatty acid derivatives.