Probiotic bacteria confer multiple health benefits, including preventing the growth, colonisation, or carriage of harmful bacteria in the gut. Bacteriocins are antibacterial peptides produced by diverse bacteria and their production is tightly regulated and coordinated at the transcriptional level. A popular strategy for enhancing the antibacterial properties of probiotic bacteria is to retrofit them with the ability to overproduce heterologous bacteriocins. This is often achieved from non-native constitutive promoters or in response to host or pathogen signal from synthetic promoters. How the dysregulated overproduction of heterologous bacteriocins affects the fitness and antibacterial efficacy of the retrofitted probiotic bacteria is often overlooked. We have conferred the prototypical probiotic Escherichia coli strain Nissle (EcN) the ability to produce microcin C (McC) from the wild- type promoter and two mutant promoters that allow, relative to the wild-type promoter, high and low amounts of McC production. This was done by introducing specific changes to the sequence of the wild-type promoter driving transcription of the McC operon, whilst ensuring that the modified promoters respond to native regulation. By studying the transcriptomic responses and antibacterial efficacy of the retrofitted EcN bacteria in a Galleria mellonella infection model of enterohemorrhagic E. coli, we show that EcN bacteria that produce the lowest amount of McC display the highest antibacterial efficacy with little to none undesired collateral impact on their fitness. The results highlight considerations researchers may take into account when retrofitting probiotic bacteria with heterogenous gene products for therapeutic, prophylactic or diagnostic applications
