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.