Escherichia coli couples the initiation of DNA replication with cell size by modulating the activity of the replication initiator protein DnaA. The activity of DnaA is regulated both by its interconversion between an active and inactive form and by its titration on chromosomal binding sites. Whereas its interconversion has been thoroughly studied, the extent to which DnaA titration can control replication initiation is poorly understood. Here, we describe the control of E. coli DNA replication via titration by modulating the expression of a constitutively active DnaA variant in four growth conditions. While we obtained stable cell cycles during slow growth, faster growth associated with overlapping replication forks led to initiation instability and replicative stress. Overall, our results provide insights into the limits of titration-based systems in the control of genome replication and their potential role in the evolutionary trajectory of E. coli. Finally, this study provides design principles for a simplified, titration-only regulatory mechanism for DNA replication in synthetic cells.