Usefulness of Rnase III Cleavage Sites for Tuning Relative Gene Expression

Wesley D. Marner II1, Brian Pfleger1, and Jay D. Keasling2. (1) University of California, Berkeley, 201 Gilman Hall, Berkeley, CA 94720, (2) Department of Chemical Engineering, University of California, Berkeley, 201 Gilman Hall, Berkeley, CA 94720

With the advent of metabolic engineering, more and more small molecules used in a variety of medical and industrial applications are targeted for production in microbial hosts. These molecules often require simultaneous expression of multi-gene exogenous pathways to affect each of the necessary enzymatic steps. The intracellular concentration of each enzyme must be coordinated to balance flux through production pathways and make efficient use of metabolic resources. One method for modulating intracellular concentrations of pathway enzymes is to engineer the stability of mRNA secondary transcripts produced from a multi-gene operon. Previous work has demonstrated that engineering the stability of each mRNA transcribed from the operon can modulate gene expression levels. Using a reporter system consisting of two fluorescent genes with an interchangeable intergenic region, we investigated the usefulness of a variable RNase III cleavage site in modulating the relative expression level of each gene. RNase III is an endonuclease that cleaves double-stranded RNA hairpins, and work by other groups indicates that RNase III sites with an asymmetric bulge in the hairpin will be cut in a single-stranded manner. A combinatorial library of RNase III cleavage sites was engineered to include the consensus site and a variety of asymmetric bulges inserted between the two fluorescent genes of the reporter plasmid. We hypothesize that single-strand cleavage at such a bulge will leave a hairpin on one of the resulting secondary transcripts, thus affecting the stability of the upstream and downstream genes. Here, we report the utility of RNase III in the processing of mRNA transcripts in the context of metabolic pathway engineering.