Characterization of Delta-7 Alkenone Desaturase in Haptophyte Gephyrocapsa huxleyi Through Heterologous Expression in Tisochrysis lutea

The marine haptophyte Gephyrocapsa huxleyi is an ecologically and geochemically important phytoplankton due to its contribution to the global carbon cycle and its ability to biosynthesize certain alkenones. These alkenones are long-chain alkyl ketones with two to four trans-type double bonds. The genes encoding alkenone desaturase in G. huxleyi have not been experimentally characterized so far, partly due to the difficulty of inducing genetic transformation in G. huxleyi. Therefore, we introduced the putative alkenone delta-7 desaturase of G. huxleyi (designated "DesT") to the transformable and alkenone-producing haptophyte Tisochrysis lutea. We found two types of coding sequences for DesT, which are probably derived from the expression products of different alleles, and designated them "DesT-1" and "DesT-2." The ratio of C_37:3 to C_37:2 methyl alkenone in the DesT-1 transformant was significantly higher than that in the mock strain that expressed only the hygromycin resistance gene, suggesting that DesT-1 was an alkenone delta-7 desaturase in G. huxleyi. In the protein structure, a tunnel where a substrate alkenone penetrates was predicted to be located around the histidine box of DesT, and hydrophilic and hydrophobic amino acids were respectively located at the proximal (near side to the histidine box) and distal ends of the tunnel. This is the first study to conduct experimental characterization of the alkenone metabolism-related gene in G. huxleyi. The heterologous expression system using T. lutea paves the way for further characterization of the alkenone metabolism-related genes in less transformable haptophytes.