AUTHOR=Liu Baoling , Sun Yan , Hang Wei , Wang Xiaodan , Xue Jinai , Ma Ruiyan , Jia Xiaoyun , Li Runzhi 

TITLE=Characterization of a Novel Acyl-ACP Δ9 Desaturase Gene Responsible for Palmitoleic Acid Accumulation in a Diatom Phaeodactylum tricornutum

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.584589

DOI=10.3389/fmicb.2020.584589

ISSN=1664-302X

ABSTRACT=<p>Palmitoleic acid (16:1Δ<sup>9</sup>) 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 &gt;40% of total lipid in the marine diatom <italic>Phaeodactylum tricornutum.</italic> The present study was conducted to determine the key gene responsible for 16:1Δ<sup>9</sup> biosynthesis in this unicellular alga. A new full-length cDNA and genomic DNA encoding acyl-ACP Δ<sup>9</sup> desaturase (PtAAD) were isolated from <italic>P. tricornutum</italic> cells. Expression levels of <italic>PtAAD</italic> gene under normal and stress culture conditions were both positively correlated with 16:1Δ<sup>9</sup> accumulation, implying its potential role for fatty acid determination. Functional complementation assay of a yeast mutant strain <italic>BY4839</italic> evidenced that <italic>PtAAD</italic> could restore the synthesis of unsaturated fatty acid, especially generating high levels of 16:1Δ<sup>9</sup>. Further transient expression of <italic>PtAAD</italic> gene in <italic>Nicotiana benthamiana</italic> leaves was accompanied by the accumulation of 16:1Δ<sup>9</sup>, 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 <italic>PtAAD</italic> gene (F160L, A223T, and L156M) in <italic>N. benthamiana</italic> systems failed to accumulate 16:1Δ<sup>9</sup>, but increased levels of 18:1Δ<sup>9</sup>. 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<sup>9</sup> in this alga. These findings provide new insights for better understanding the palmitoleic acid and oil biosynthetic mechanism in <italic>P. tricornutum</italic>, indicating that <italic>PtAAD</italic> gene may have practical applications for enriching palmitoleic acid and oil yield in other commercial oleaginous algae and crops.</p>