Development of High Palmitoleic (16:1 n-7) Acid Oil by Fermentation of Microalgae

Palmitoleic acid (POA; 16:1 n-7 or cis-9 16:1) is a bioactive monounsaturated fatty acid (FA) with emerging metabolic and skin-health relevance, yet conventional botanical and animal sources provide limited and variable levels. Here we report on the development of a high-yield POA product platform in the heterotrophic microalga Prototheca moriformis through targeted genetic engineering. A Δ9-fatty acid desaturase from Macadamia integrifolia (MiSAD1618) was integrated using a phosphite-based selection system. Primary screening identified stable transformants producing up to 54% POA of total fatty acids, compared to 0.8% in the parental strain. In 1 L shake-flask cultivation, POA reached up to 58.2% of total fatty acids. In a 1 L fed-batch fermentation, the engineered strain accumulated 47.8 g/L of lipids with 43.5% POA after 96 h of fermentation, corresponding to 20.8 g/L of POA. GC–MS analysis of 4,4-dimethyloxazoline (DMOX) derivatives confirmed that the major 16:1 isomer was 16:1 n-7 (Δ9). Together, these results establish P. moriformis as a scalable fermentation platform for producing POA-rich oil and highlight its potential as an efficient alternative source of POA, providing a foundation for further strain and process optimization toward commercial production.