Decoupling growth and production by removing the origin of replication from a bacterial chromosome

Abstract

Efficient production of biochemicals and proteins in cell factories frequently benefits from a two-stage bioprocess in which growth and production phases are decoupled. Here we describe a novel growth switch based on the permanent removal of the origin of replication (oriC) from the Escherichia coli chromosome. Without oriC, cells cannot initiate a new round of replication and they stop growing while their metabolism remains active. Our system relies on a serine recombinase from bacteriophage phiC31 whose expression is controlled by the temperature-sensitive cI857 repressor from phage lambda. Reporter protein expression in switched cells continues after cessation of growth, leading to protein levels up to five times higher compared to non-switching cells. Switching induces a unique physiological state that is different from both normal exponential and stationary phases. Switched cells remain in this state even when not growing, retain their protein synthesis capacity, and do not induce proteins associated with the stationary phase. Our switcher technology is potentially useful for a range of products and applicable in many bacterial species for decoupling growth and production.

Bacillus subtilis remains translationally active after CRISPRi-mediated replication initiation arrest

“…Enhanced translation upon replication arrest is a possible mechanism to increase protein and metabolite production and has been studied in E. coli (8, 9, 28). To observe whether the levels of translation-related proteins were increased upon replication arrest in B. subtilis , we made individual profile plots of translation-related proteins as a function of the z-score (Fig.…”

“…Studies in several bacteria have suggested that protein synthesis continues during growth arrest and that maintaining functional translation machinery may be required for their viability (10–12, 38). Additionally, several studies in E. coli demonstrated enhanced protein and biochemical production of proteins and metabolites during replication arrest (8, 9, 28). We further confirmed that non-replicating cells maintained a higher rate of protein expression than WT cells (Fig.…”

“…Notably, metabolically active states in non-growing bacteria have previously been reported, with some displaying enhanced protein production (10–13). The methods to induce these non-replicating states include, for example: i) the use of toxins from toxin-antitoxin systems to induce a persister-like phenotype (14, 15), ii) exploiting repressible promoters to downregulate the RNA polymerase (10), iii) the elimination of chromosomal content with endonucleases such as I-CeuI (11), iv) the removal of the oriC with serine integrases (16), v) mimicking nutrient depletion, the addition of growth inhibitors, and the use of CRISPRi to knockdown genes related to growth or replication (8, 9, 17, 18). In this study, we constructed a strain with a chromosomally integrated CRISPRi system specifically targeting DnaA boxes 6 and 7, which have been previously reported to be the most important to promote DNA unwinding during replication initiation in B. subtilis (19).…”