Stramenopile-type Lipid Droplet Protein Functions as a Lipid Droplet Scaffold Protein in the Marine Diatom Phaeodactylum tricornutum.

Oleaginous microalgae gain great attention as feedstock for biofuels because of their substantial accumulation capacity of neutral lipids in the cytosolic compartment called lipid droplet (LD). Understanding the regulation mechanism of neutral lipid accumulation and degradation, which is mediated by LD-associated proteins, is an important issue in improving lipid productivity. However, LD-associated proteins vary among species and wait to be characterized in many microalgae. Stramenopile-type lipid droplet protein (StLDP) was previously identified as a primary LD protein in the marine diatom Phaeodactylum tricornutum. We produced a knockout mutant of StLDP by CRISPR/Cas9 genome editing. Also, we tried to complement this mutant by expressing recognition site-modified StLDP (RSM-StLDP) which is designed to avoid an attack by Cas9 nuclease expressing in the mutant. The RSM-StLDP:EGFP was localized to both LDs and the outer chloroplast-endoplasmic reticulum. Decrease of LD number per cell, increase of LD size, and no alteration of neutral lipid content in the mutant under nitrogen deficiency clearly indicate that StLDP acts as an LD scaffold protein. The number of LD per cell increased in the complemented strain compared to wild-type cells. The LD morphology in the mutant is probably over-rescued in the complemented strain by the strong function of the nitrate reductase promoter, which is also supported by high neutral lipid content in the complemented strain. The growth of stldp mutant showed a long lag phase relative to wild-type cells, suggesting that the low surface-to-volume ratio of fused LD decreased the efficiency of LD hydrolysis during the initial growth phase.