Targeted Repression of Essential Genes To Arrest Growth and Increase Carbon Partitioning and Biofuel Titers in Cyanobacteria

Photoautotrophic production of fuels and chemicals by cyanobacteria typically gives lower volumetric productivities and titers than heterotrophic production. Cyanobacteria cultures become light limited above an optimal cell density, so that this substrate is not supplied to all cells sufficiently. Here, we investigate genetic strategies for a two-phase cultivation, where biofuel-producing <i>Synechocystis</i> cultures are limited to an optimal cell density through inducible CRISPR interference (CRISPRi) repression of cell growth. Fixed CO₂ is diverted to ethanol or <i>n</i>-butanol. Among the most successful strategies was partial repression of citrate synthase <i>gltA.</i> Strong repression (>90%) of <i>gltA</i> at low culture densities increased carbon partitioning to <i>n</i>-butanol 5-fold relative to a nonrepression strain, but sacrificed volumetric productivity due to severe growth restriction. CO₂ fixation continued for at least 3 days after growth was arrested. By targeting sgRNAs to different regions of the <i>gltA</i> gene, we could modulate GltA expression and carbon partitioning between growth and product to increase both specific and volumetric productivity. These growth arrest strategies can be useful for improving performance of other photoautotrophic processes