Sustainable 2-Phenylethanol Production: Co-Cultivation of <i>Yarrowia lipolytica</i> Strains in Mixed Agro-Industrial By-Products

The bioproduction of 2-phenylethanol (2-PE), a high-value aromatic compound widely used in the fragrance, cosmetic, food and beverage, and pharmaceutical industries, through yeast fermentation offers a sustainable alternative to chemical synthesis and rose extraction. This study explores the fermentation of <i>Yarrowia lipolytica</i> strains using mixed agro-industrial by-products as substrates to produce 2-PE via de novo synthesis, without supplementation with the costly precursor L-phenylalanine. <i>Y. lipolytica</i> strains were genetically engineered to enhance flux through the shikimate pathway and enable the hydrolysis of a broader range of substrates. The culture media consisted solely of a mixture of agro-industrial by-products: sugar beet molasses (SBM), brewer’s spent grain (BSG) pressing extract, and chicory root (CR) pressing extract, serving as the primary carbon and nitrogen sources without the addition of nutrients, minerals, synthetic, complex ingredients, or costly additives. The co-culture approach enhanced substrate utilization, leading to an increase in 2-PE titers, reaching approximately 2.5 g/L 2-PE production after 240 h of fermentation. This study demonstrates the feasibility of integrating co-culture fermentation and agro-industrial waste valorization for sustainable 2-PE production, offering a scalable bioprocess for industrial applications.