Expanding the toolbox for gene editing and expression in Lactococcus lactis
Date
2022-12
Authors
Sen, Swastik
Major Professor
Advisor
Mansell, Thomas J
Jarboe, Laura
Phillips, Gregory
Lamsal, Buddhi
Sashital, Dipali
Committee Member
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Department
Chemical and Biological Engineering
Abstract
Lactic acid bacteria are an order of generally regarded as safe microbes that are often found as commensals in mammalian microbiomes as residents of mucosal surfaces. They have attracted interest in recent decades as potential probiotics due to these characteristics, and several pre-clinical and clinical studies have been undertaken for this purpose for therapeutic applications for numerous indications. Lactococcus lactis, one of the better characterized members of this group was the first bacterium to be used for secretion of cytokines in clinical studies. However, as with many potential probiotics, observed efficacies were low in these trials. Some predominant reason for low efficacies in clinical trials have been attributed to the lack of versatility of native strains, a scarecity of heterologous protein expression strategies and poor survival and/or colonization potential in the host. In this dissertation, we aim to address some of these challenges in developing L. Lactis as an effective probiotic. Firstly, we expand the repertoire for inducible gene expression systems in L. lactis NZ9000 by repurposing the two-component regulation system of the xylose metabolism pathway from L. lactis KW2. Additionally, we also explore the potential use of T7 RNA Polymerase as a driver of gene expression based on it’s robust activity in other organisms. In an attempt to address the low efficacy of current gene editing strategies in L. lactis, we developed a Cas12a based platform for dsDNA recombineering and regulation of gene expression and demonstrate improved efficacy and flexibility of this system. Finally, we engineer L. lactis NZ9000 with the capability to metabolize levoglucosan in order to design a novel synbiotic pair with enhanced growth attributes with higher survival potential.