Assessment of promoter regulation in Pseudomonas putida using GFP and flow cytometry

Sammanfattning: Lignin is found in all biomass and is an abundant natural source of aromatic compounds. It has the potential to serve as a renewable raw material for a variety of chemicals currently derived from fossil fuel sources such as muconic acid, an important platform chemical. Kraft lignin is a waste stream from the pulp and paper industry. Upon depolymerization of softwood-derived lignin, aromatic compounds such as guaiacol and vanillin are released and they can be further converted to muconic acid with the help of metabolically engineered Pseudomonas putida strain. To optimize the bioconversion pathway, it is important to be able to regulate gene expression using appropriate promoters. However, the lack of well-characterized promoter libraries on aromatic compounds in P. putida could pose limits on further engineering efforts. As a means to increase available genetic engineering tools, this project aimed to characterize a few promoters in this species. Two strong promoters, p14c and p14g, were chosen from a synthetic promoter library along a single common inducible promoter - lacIq-Ptrc. The promoters were coupled to a green fluorescent protein (GFP) gene in a plasmid construct. These plasmids were later introduced into P. putida to be evaluated in a common minimal medium. The expression was evaluated mainly through flow cytometry, but also through a simpler method relying on optical density-adjusted fluorometry. Fluorescence was followed over time to catch the variability in expression in different growth stages. The p14g promoter that displayed the highest fluorescence on glucose, was further evaluated in various media containing different combination of guaiacol and vanillin to study their potential effects on expression. The presence of vanillin in the medium caused a decrease in fluorescence (11%) and growth (20%) after six hours of cultivation but an almost equal increase was observed after 24-hours. Guaiacol showed growth inhibitory effects at concentrations of 5 mM and higher through an increased lag-phase (2-4 hours) and halving of the final optical density (OD). Its presence, however, increased the GFP expression by 20-28%, depending on media composition and time point. Additionally, guaiacol caused an increased expression variance among the cell population, relative to other media. Both fluorescence-determining methods showed the same trend in terms of relative fluorescence between cultures in exponential growth, although these differences were of different magnitude.