Identification of small droplets of photosynthetic squalene in engineered Synechococcus elongatus PCC 7942 using TEM and selective fluorescent Nile red analysis

Abstract

To identify microbial squalene that has been widely used in various industrial applications, intracellular formation of photosynthetic squalene was investigated using the previously engineered Synechococcus elongatus PCC 7942 strain. Unlike the proposed localization of squalene in the membrane bilayer, small droplets were identified in the cytoplasm of S. elongatusPCC 7942 as squalene using transmission electron microscopy analysis. Determination of the diameters of the squalene droplets with manual examination of 1016 droplets in different squalene-producing strains indicated larger squalene droplets in larger cells. Based on the observation of a sole droplet of squalene in a cyanobacterium, fluorescent Nile red was used for the selective staining of squalene. The fluorescent intensities were correlated with squalene contents determined using gas chromatography-mass spectrometry. Photosynthetic squalene was identified as a small droplet in S. elongatusPCC 7942, and this noninvasive quantitative method could be useful to promote high-throughput strain development for squalene production.