(236e) A Crispri Platform for Investigating Metabolic Regulation in the Cyanobacterium Synechococcus Sp. PCC 7002

The requirement for sugar feedstocks and freshwater in the microbial production of chemicals can lead to competition for arable land and other resources. Photoautotrophic cyanobacteria are attractive, alternative hosts for sustainable bioconversion of CO₂ to high-value chemicals. The model cyanobacterium Synechococcus sp. PCC 7002 (henceforth PCC7002) is high light and halotolerant and has an established genetic toolkit. PCC7002 has been engineered to produce a variety of industrially relevant chemicals, such as fatty acids and other lipid derivatives. However, metabolic engineering efforts in PCC7002 are hindered by our limited understanding of native regulatory processes. PCC7002 lacks functional β-oxidation and homologs to many common regulators to fatty acid biosynthesis (FAB), posing questions about how this cyanobacterium regulates flux to essential membrane and phospholipid compounds.

In this project, we apply a synthetic biology technology dubbed CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq) to explore native regulation within PCC7002. CiBER-seq was previously used as a knock-down screen for detecting transcriptional, translational, and post-translational regulators for the integrated stress response in yeast. Here, we describe the implementation of the CiBER-seq system to screen for novel regulators of FAB in PCC7002. We aim to use knowledge about PCC7002’s unique lipid physiology to enhance production of medium chain fatty acids (MCFAs), an important class of chemical precursors for fuels, pharmaceuticals, detergents, and personal care products. This work provides a foundation for uncovering other unique aspects of PCC7002 regulation to inform future metabolic engineering and synthetic biology efforts in cyanobacteria.