 Hi, I'm Ann Ruffing from Sandia National Laboratories, and in this video I'll be discussing our recent publication in biotechnology and bioengineering, entitled Physiological Effects of Pre-Patiacin Production in Genetically-Engineered Cinecococcus Elongatus 7942. Cyanobacteria like Cinecococcus are among the types of microalgae under consideration for biofuel production. Like other microalgae that can fix carbon dioxide into organic molecules like fuels and fuel precursors, using photosynthesis as the energy source. Unlike eukaryotic algae, however, cyanobacteria do not accumulate liquids in the form of triosolblissarol. However, there are some distinct advantages to using cyanobacteria. First, they're very easy to genetically manipulate. So this allows us to introduce fuel-producing pathways and use other common metabolic engineering techniques to boost fuel production. Secondly, they're mentioned to excrete fuel precursors like free fatty acids. This enables a continuous production system, which would reduce the requirements for nutrients like nitrogen and phosphates, and also reduce the time required for microalgae growth. In this work, we engineer a model cyanobacterium, Cinecococcus elongatus 7942, to produce and excrete free fatty acids. We show free fatty acid excretion for two mutants. SC01, which has gene knockout of the acyl-ACP synthetase involved in free fatty acid recycling. And SC02, which has the same gene knockout, but also expresses a thioesterase for release of the free fatty acid. While the engineered strains excrete free fatty acids, they have reduced final cell concentrations compared to the wild type, as well as reduced photosynthetic yields. Accompanying these changes, there is also a change in the photosynthetic pigments with a selective degradation of chlorophyll A. Using hyperspectral imaging and MCR analysis, we show changes in the subcellular localization of photosynthetic pigments for the free fatty acid-producing strains. Specifically, the phycabilia proteins phycocyanin and alofycocyanin are shown to aggregate at the cell poles. This is in contrast to the wild type, which shows these pigments being evenly dispersed throughout the thylakoi membrane. We identify two possible causes for these physiological effects. First, unsaturated free fatty acids, like linoleic acid, may oxidize into a variety of products which can be toxic to the cell. Exogenous linoleic acid addition was shown to inhibit cell growth and lead to the complete degradation of all photosynthetic pigments, including the phycabilia proteins and chlorophyll A. The second potential cause is that manipulating the fatty acid biosynthesis pathway may lead to changes in the chemical composition of membranes, specifically the photosynthetic membranes. In turn, this may affect the membrane structure, the activities of enzymes embedded within the membrane, or the attachment of light-harvesting pigments to the photosynthetic membrane. Changes in the degree of saturation of membrane fatty acids provides preliminary evidence to support this mechanism. The physiological changes described in the study will have a significant impact on large-scale bio-filled productivities. Therefore, further investigation is necessary to identify ways to prevent or overcome these detrimental effects of free fatty acid production.