Reliable and predictable gene expression tools for Pseudomonas putida

Pseudomonas putida has emerged as a promising host in metabolic engineering because it is a fast growing, genetically tractable gram-negative bacteria with high solvent tolerance.  Being gram-negative, the previously held assumption has been that any gene expression tools commonly used in Escherichia coli will work as equally well in P. putida.  However, through an E. coli-P. putida comparative gene expression characterization study, we found that many expression systems normally used in E. coli, including some of the Anderson library of constitutive promoters and several different inducible expression systems, often behave in a strikingly different manner when used in P. putida. 

Considering how critical it is to have reliable and predictable gene expression for metabolic engineering purpose in any host of choice, we aim to build and characterize a library of tools for P. putida consisting of (1) a set of constitutive promoters whose strength spans three orders of magnitude and (2) a set of IPTG inducible promoters with low basal expression levels (i.e., leakiness) and high fold induction ratio.  Additionally, we also look into the relationship between predicted ribosome binding site (RBS) strength and actual expression profile of model proteins in the host.

These libraries, together with the comparison of predicted vs. actual RBS strength, are expected to facilitate smoother design of reliable genetic pathways and increase the utility of P. putida as an industrial chemical production platform.