ACP Activation Enhances Differential Fatty Acid Accumulation in Engineered Thraustochytrids via Optimized Transformation

Thraustochytrids are marine protists valued for producing docosahexaenoic acid (DHA) and saturated fatty acids (SFAs), but limited genetic tools have hindered pathway studies and optimization. Here, we developed an optimized DNA transformation protocol for Aurantiochytrium strains to overexpress the Sfp-type phosphopantetheine transferase gene (ppt) and evaluate its role in fatty acid synthesis. Zeocin proved the most effective selection marker, while optimized electroporation parameters (0.50 kV/mm, 1 pulse, 50 μF, 200 Ω) enabled efficient transformation of Aurantiochytrium sp. PKU#Sw8. The overexpression of ppt significantly increased fatty acid yields SFAs rose 2.86-fold, DHA 2.06-fold, and total fatty acids 2.02-fold. Palmitic acid production increased 7.13-fold, shifting the PA/DHA ratio from 0.41 to 1.02. Transcriptomics revealed enhanced precursor supply for FAS and PKS pathways, reduced fatty acid oxidation, and flux redirection toward SFAs via FAS. These results clarify ppt-driven fatty acid enhancement and provide a framework for engineering thraustochytrids for SFAs production.