Gabant, P. | Search bioRxiv

bioRxiv2023

Bacteriocin peer selection for the production of antibiotic selection free biotherapeutic pDNA

Plasmid vectors are well established tools used to genetically engineer bacteria both in the laboratory and at industrial scale. The past few decades have seen a rising interest in the use of plasmid DNA (pDNA) for biotherapeutic applications. This interest is a strong driver for the development of technologies to increase pDNA production at biopharmaceutical scale in terms of decreasing production costs and meeting regulatory requirements. Although cell free technologies are emerging, pDNA vectors are still produced by fermentation in Escherichia coli strains. As plasmids are extra-chromosomic molecules there is a probability of losing a certain ratio within the E. coli population during the fermentation process leading to a decrease of DNA production efficiency. Maintaining pDNA in the population is thus a key element to reach efficient and robust production. Traditionally, antibiotic resistance genes and antibiotics have been used to generate a selective pressure to ensure pDNA stability in the microbial population during the production process. Nowadays, having an antibiotic resistance gene in the pDNA coding sequence represents a limitation both for safety and legal requirements and in terms of production yield. For this reason, we have developed a pDNA antibiotic-free bacteriocin-based selection system, based on the genes involved in the production, processing, secretion and immunity of the bacteriocin microcin V. Our approach is based on the peer pressure exerted by the bacteriocin and does not rely on the addition of any selective agent in the medium to limit population drift and ensure plasmid stability. This novel antibiotic-free approach may be applied to any pDNA vector in different E. coli strains and expands their potential applications in both animal and human health as delivery vectors for biotherapeutics.

synthetic biology↗

bioRxiv2023

Enhancing the antibacterial function of probiotic Escherichia coli Nissle: when less is more

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 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. IMPORTRANCEBacteria that resist killing by antibiotics are a major risk to modern medicine. The use of beneficial probiotic bacteria as chassis to make antibiotic-like compounds at the site of infection in the body is emerging as a popular alternative to the use of conventional antibiotics. A potential drawback of engineering probiotic bacteria in this way is that producing antibiotic-like compounds could impart undesired side-effects on the performance of such bacteria and thereby compromise their intended use. This study highlights considerations researchers may take into account when engineering probiotic bacteria for therapeutic, prophylactic or diagnostic applications.

microbiology↗