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Combinatorial Engineering of Intergenic Regions to Tune Expression of Multiple Genes in Operons

Brian Pfleger1, Doug Pitera1, Christina Smolke2, and Jay D. Keasling1. (1) Chemical Engineering, University of California, Berkeley, Berkeley, CA 94720, (2) Chemistry & Chemical Engineering, California Institute of Technology, 1200 E. California Blvd, MC 210-41, Pasadena, CA 91125

Many applications of synthetic biology require the balanced expression of multiple genes, and operons are convenient for coordinating the expression of two or more genes in both prokaryotes and eukaryotes. The relative expression levels of genes encoded in operons is largely controlled by the competing processes of transcription termination, mRNA degradation, and translation initiation in intergenic regions, making it difficult to design the relative expression levels of genes encoded in the operon. We describe here a novel method for simultaneously tuning the expression of multiple genes within operons by generating libraries of Tunable InterGenic Regions (TIGRs) by recombining oligonucleotides containing various post-transcriptional control elements (mRNA secondary structures, RNase cleavage sites, ribosome binding site sequestering sequences, etc.) and screening for the desired relative expression levels. The TIGRs enabled us to vary the relative expression of two reporter genes in an operon over a 100-fold range. Analysis of samples from the libraries identified the three underlying mechanisms of gene expression control: differential mRNA processing, premature transcription termination, and ribosome binding site sequestration. Application of this method to a three-gene, heterologous, mevalonate biosynthetic pathway in Escherichia coli resulted in a seven-fold increase in mevalonate production. Interestingly, the operon variants that produced the highest levels of mevalonate expressed two of the three enzymes at lower levels than the original construct rather than at increased levels as would be expected to improve flux through a metabolic pathway. We anticipate that this technology will be useful for tuning expression of multiple genes in synthetic operons, both in prokaryotes and eukaryotes. Portions of this work have recently been published in Nature Biotechnology and Metabolic Engineering. http://www.nature.com/nbt/journal/v24/n8/abs/nbt1226.html