Broad Range Shuttle Vector Construction and Promoter Evaluation for the Use of Lactobacillus Plantarum WCFS1 As a Microbial Engineering Platform.

As the field of synthetic biology grows, efforts to deploy complex genetic circuits in nonlaboratory strains of bacteria will continue to be a focus of research laboratories. Members of the Lactobacillus genus are good targets for synthetic biology research as several species are already used in many foods and as probiotics. Additionally, Lactobacilli offer a relatively safe vehicle for microbiological treatment of various health issues considering these commensals are often minor constituents of the gut microbial community and maintain allochthonous behavior. In order to generate a foundation for engineering, we developed a shuttle vector for subcloning in Escherichia coli and used it to characterize the transcriptional and translational activities of a number of promoters native to Lactobacillus plantarum WCFS1. Additionally, we demonstrated the use of this vector system in multiple Lactobacillus species, and provided examples of non-native promoter recognition by both L. plantarum and E. coli strains that might allow a shortcut assessment of circuit outputs. A variety of promoter activities were observed covering a range of protein expression levels peaking at various times throughout growth, and subsequent directed mutations were demonstrated and suggested to further increase the degree of output tuning. We believe these data show the potential for L. plantarum WCFS1 to be used as a nontraditional synthetic biology chassis and provide evidence that our system can be transitioned to other probiotic Lactobacillus species as well.