RESUMO
Oleaginous microalgae are gaining great attention as feedstock for biofuels because of their substantial accumulation capacity for neutral lipids in the cytosolic compartment called the lipid droplet (LD). Understanding the regulatory 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 are waiting to be characterized in many microalgae. Stramenopile-type LD protein (StLDP) was previously identified as a primary LD protein in the marine diatom Phaeodactylum tricornutum. We produced a knockout mutant of StLDP by clustered regularly interspaced short palindromic repeat (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:enhanced green fluorescent protein was localized to both LDs and the outer chloroplast-endoplasmic reticulum. The decrease in the LD number per cell, increase in 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 LDs per cell increased in the complemented strain compared to wild-type (WT) 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 WT cells, suggesting that the low surface-to-volume ratio of fused LD decreased the efficiency of LD hydrolysis during the initial growth phase.
