Expanding the Toolbox of Broad Host-Range Transcriptional Terminators for Proteobacteria through Metagenomics

Abstract

As the field of synthetic biology moves toward the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacterium Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria, for instance Pseudomonas putida. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-range plasmid, we identified 20 clones with potential terminator activity in P. putida. Further characterization allowed the identification of 4 unique sequences ranging from 58 to 181 bp long that efficiently terminate transcription in P. putida, E. coli, Burkholderia phymatum, and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

Keywords: Proteobacteria; biological parts; functional metagenomics; synthetic biology; transcriptional terminators.