(472f) Measuring Photosynthetic Productivity Rates and Regulation in Picosynechococcus sp. PCC 7002 to Enhance Medium Chain Fatty Acid Production

Cyanobacteria are an intriguing microbial host for sustainable bioconversion owing to their photosynthetic ability to convert carbon dioxide to high-value chemicals. Specifically, the species Picosynechococcus sp. PCC 7002 (henceforth PCC7002) is high light and halotolerant, allowing it a range of conditions for industrial growth. PCC7002 also has a well-established and growing genetic toolkit. These advancements have led to progress in metabolically engineering the organism to produce a suite of relevant chemicals and oleochemicals. Here, we are interested enhancing in the production of medium chain fatty acids (MCFAs, such as octanoic acid, C8-FA). MCFAs are a valuable but naturally scarce class of chemicals used in personal care products, lubricants, pharmaceuticals, and more. Towards this effort, we employ two strategies: metabolic engineering of fatty acid biosynthesis (FAB) and investigation of FAB regulation using a CRISPR-interference (CRISPRi) screen.

Firstly, we show that PCC7002 can produce over 800 mg L^-1 C8-FA using an engineered thioesterase specific to C8-ACP cleavage under given conditions. Moreover, we seek to examine how kinetic control of FAB, mediated by relative expression of FAB initiation, elongation, and termination enzymes affects total C8-FA production. Using a set of orthogonal promoters, driving three integrated cassettes of different FAB enzyme homologs, we demonstrate how expression of specific homologs increase the C8-FA linear productivity rate (LPR) by over 50%. Additionally, we explore bioprocess optimizations such as induction time and amount to enhance LPR further.

One downside of a metabolic engineering approach is the limited knowledge the field has regarding lipid metabolism and FAB in PCC7002 specifically. PCC7002 lacks functional β-oxidation and many common transcriptional regulators of FAB. This raises uncertainty about potential targets for further engineering of cyanobacterial lipid metabolism. We seek to apply a modified CRISPRi screen with barcoded expression reporter sequencing (CiBER-seq) to investigate native regulation of FAB and identify novel control points that are then modified for enhanced C8-FA productivity. This work provides a foundation for lipid engineering in PCC7002 and uncovering other unique aspects of this industrially relevant cyanobacteria.