Enhanced protein and biochemical production using CRISPRi-based growth switches

Production of proteins and biochemicals in microbial cell factories is often limited by carbon and energy spent on excess biomass formation. To address this issue, we developed several genetic growth switches based on CRISPR interference technology. We demonstrate that growth of Escherichia coli can be controlled by repressing the DNA replication machinery, by targeting dnaA and oriC, or by blocking nucleotide synthesis through pyrF or thyA. This way, total GFP-protein production could be increased by up to 2.2-fold. Single-cell dynamic tracking in microfluidic systems was used to confirm functionality of the growth switches. Decoupling of growth from production of biochemicals was demonstrated for mevalonate, a precursor for isoprenoid compounds. Mass yield of mevalonate was increased by 41%, and production was maintained for more than 45h after activation of the pyrF-based growth switch. The developed methods represent a promising approach for increasing production yield and titer for proteins and biochemicals. [Display omitted] •E. coli growth and protein/biochemical production both require carbon and energy.•CRISPRi inhibition of replication or nucleotide synthesis arrests E. coli growth.•Arrested biomass accumulation enhances both protein and biochemical production.•Production yield of mevalonate was increased by 41% through growth inhibition.•Cell activity was maintained for 40h after decoupling growth from production.