Metabolic Rewiring for Palmitoleic Acid Biosynthesis by Co-Expressing COM25 and FAT5 in Rice

Palmitoleic acid (C16:1Δ 9 ) has high nutritional and industrial value, but enriching it in rice is hindered by low endogenous levels and poorly understood multi-gene mechanisms. We engineered rice to express castor bean ( Ricinus communis ) Δ9 desaturase gene COM25 and nematode ( Caenorhabditis elegans , C. elegans ) Δ9 desaturase gene FAT5 , individually or combined. Both genes were functionally expressed, enabling de novo palmitoleic acid synthesis for the first time in rice. Co-expression acted synergistically, boosting seed total fatty acids by up to 388% vs. wild type, with marked enrichment of monounsaturated fatty acids, especially oleic acid. This metabolic shift triggered lipid homeostasis feedback, altering palmitic acid distribution across tissues. RNA-seq revealed distinct yet complementary roles: COM25 overexpression broadly reprograms the fatty acid synthesis, central carbon metabolism, and hormone signaling; FAT5 acts more locally in the ER, primarily governing fatty acid elongation. Their combination redirected carbon flux toward monounsaturated fatty acid biosynthesis. These findings deliver nutrient-enhanced rice germplasm for breeding and systematically map the lipid homeostasis network, offering theoretical guidance for precision lipid engineering in rice.