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Odd-chain fatty acids (OCFA) have been studied for their therapeutic and nutritional properties, as well as for their potential use in the chemical industry for the production of biofuel. Genetic modification strategies have demonstrated an improved production of OCFA by oleaginous microorganisms. In this study, the production of OCFA-enriched lipids by fermentation using a genetically engineered <i>Yarrowia lipolytica</i> strain was investigated. The major fatty acid produced by this strain was the <i>cis</i>-9-heptadecenoic acid (C17:1). Its biosynthesis was optimized using a design of experiment strategy involving a central composite design. The optimal responses maximizing the cell density (optical density at 600 nm) and the C17:1 content (%) in lipids were found using 52.4 g/L sucrose, 26.9 g/L glycerol, 10.4 g/L sodium acetate, 5 g/L sodium propionate, and 4 g/L yeast extract. Under these conditions, in a 5 L scale bioreactor, the respective contents of lipids and C17:1 in culture medium were 2.52 ± 0.05 and 0.82 ± 0.01 g/L after 96 h fermentation. The results obtained in this work pave the way toward the process upscale of C17:1 and encourage its industrial production.