Characterization of a Novel Acyl-ACP Δ9 Desaturase Gene Responsible for Palmitoleic Acid Accumulation in a Diatom Phaeodactylum tricornutum
Baoling Liu, Yan Sun, Hang Wei, Xiaodan Wang, Jinai Xue, Ruiyan Ma, Xiaoyun Jia, Runzhi Li
Abstract
Palmitoleic acid (16:1Δ 9 ) possesses a double bond at the seventh carbon atom from methyl end of the acyl chain and belongs to unusual ω-7 monounsaturated fatty acids with broad applications in food, pharmaceuticals, cosmetics, biofuel, and other industries. This high-value fatty acid accumulates up to >40% of total lipid in the marine diatom Phaeodactylum tricornutum. The present study was conducted to determine the key gene responsible for 16:1Δ 9 biosynthesis in this unicellular alga. A new full-length cDNA and genomic DNA encoding acyl-ACP Δ 9 desaturase (PtAAD) were isolated from P. tricornutum cells. Expression levels of PtAAD gene under normal and stress culture conditions were both positively correlated with 16:1Δ 9 accumulation, implying its potential role for fatty acid determination. Functional complementation assay of a yeast mutant strain BY4839 evidenced that PtAAD could restore the synthesis of unsaturated fatty acid, especially generating high levels of 16:1Δ 9 . Further transient expression of PtAAD gene in Nicotiana benthamiana leaves was accompanied by the accumulation of 16:1Δ 9 , which was absent from control groups. Three-dimensional structure modeling studies showed that functional domain of PtAAD contained three variant amino acids (F160, A223, and L156), which may narrow the space shape of substrate-binding cavity to ensure the entry of 16:0-ACP. Consistent with this prediction, the mutated version of PtAAD gene (F160L, A223T, and L156M) in N. benthamiana systems failed to accumulate 16:1Δ 9 , but increased levels of 18:1Δ 9 . Taken together, PtAAD exhibits a strong enzymatic activity and substrate preference for 16:0-ACP, acting as the key player for high biosynthesis and accumulation of 16:1Δ 9 in this alga. These findings provide new insights for better understanding the palmitoleic acid and oil biosynthetic mechanism in P. tricornutum , indicating that PtAAD gene may have practical applications for enriching palmitoleic acid and oil yield in other commercial oleaginous algae and crops.