Using a transcriptomic approach to understand enhanced lipid droplet and alkane production in Nostoc punctiforme

Nostoc punctiforme is a filamentous cyanobacterium capable of producing cytoplasmic lipid droplets (LDs) that contain triacylglycerides, alpha-tocopherol (vitamin E), and alkanes. Triacylglycerides are a feedstock for biodiesel, and alkanes found in N. punctiforme are of the length typically found in jet fuel. Synthesis of these compounds by photosynthetic microorganisms could potentially steer away from the use of fossil fuels. It is important to understand how the cell makes LDs or responds to LD production in order to alter metabolic pathways to improve and increase the efficiency of LD production. Previous work from our lab has shown that a N. punctiforme strain that overexpresses two N. punctiforme genes involved in alkane biosynthesis, aar and adc (2- gene expression strain), overproduces heptadecane and has an increased number of LDs in exponential phase compared to the wild-type strain. Proteomic analysis conducted on proteins associated with isolated LDs identified a putative lipase that when overexpressed alone increased alkane production. This putative lipase was confirmed to be associated with isolated LDs by Western blotting. Overexpression of this lipase in conjunction with aar and adc (3-gene expression strain) further increased LDs and heptadecanes compared to the 2-gene expression strain. Comparative transcriptomic analysis of wild-type and these LD/alkane overproducer strains were conducted to illuminate the function and formation of LDs and alkanes. Genes exhibiting a statistically significant >1.4 fold increase in the 2- and 3-gene overexpression strains relative to the plasmid-only control were hypothesized to enhance or cope with overproduction of LDs and alkanes. Thirteen genes showing up-regulation were cloned on a multi-copy plasmid and transformed into wild-type either alone and/or in an operon, or in conjunction with the 3-genes mentioned above to test the hypothesis that overexpression due to increased copy number enhanced, or mimicked, the increased LD or alkane production phenotype. A total of 17 overexpression strains were made. LDs from each of these strains were stained with BODIPY and viewed using fluorescence microscopy. Two overexpressor strains expressed structural alterations in LDs including one which did not express any visible neutral lipids. Alterations in alkane and lipid fatty acid composition were also analyzed using gas chromatography-mass spectrometry (GC-MS). Only one strain showed an enhanced heptadecane production relative to its control strain while three strains exhibited a decrease in heptadecane which could be indicative of a stress response mechanism. These data will ultimately help us develop strains capable of producing LDs enriched in biofuels or other high value compounds that can be utilized for biotechnological applications.