(594a) Engineering Medium Chain Fatty Acid Production in Synechococcus Sp. PCC7002

Sustainable production of target chemicals in microbes is limited by the need for plant-derived sugar feedstocks and freshwater requirements. These processes compete for arable land and resources that could otherwise be used for food production. The model cyanobacterium Synechococcus sp. PCC 7002 (henceforth PCC7002) is an attractive photoautotrophic host that grows in brackish waters, removing the requirement for an external sugar feedstock and freshwater. Medium chain fatty acids (MCFAs) are an important class of chemical precursors for fuels, pharmaceuticals, detergents, and personal care products but are typically derived from unsustainable coconut and palm oils. Competitive production of MCFAs in heterotrophic organisms like Escherichia coli has been enabled by protein engineering of thioesterase enzymes that terminate the cycle of fatty acid biosynthesis (FAB).

In this project, we generated analogous strains of PCC7002 that overexpress these thioesterase enzymes and produce up to 860 mg/L of octanoic acid (C8). We also characterize growth effects of various organic overlays on PCC7002 and demonstrate their functionality to partition free fatty acids from aqueous media and subsequently increase titers. Like other cyanobacteria, PCC7002 lacks functional β-oxidation and has a naturally low flux through its lipid pathways, making oleochemical pathway engineering in PCC7002 unique from other microbes. To enhance titers further, we seek to identify the flux and genetic bottlenecks of our FAB-enhanced strains using untargeted metabolomics, ¹³C-based isotope nonstationary metabolic flux analysis (INST-MFA), and CRISPR interference. This work provides a foundation for engineering cyanobacterial lipid metabolism and can be extended to produce oleochemicals with other functionalities, such as fatty alcohols and triacylglycerols.