Fine-tuning Transcription Level: Differential Expression of Genes in an Operon

Source: Pfleger et al (2006). Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat Biotech 24(8):1027-1032.

Coordinated expression of genes active in a pathway or a protein complex is very important with respect to maximizing output and minimizing toxic intermediate compunds. In prokaryotes, related genes are clustered in operons and further fine-tuned through intergenic sequences (e.g. premature termination or RBS masking). While evolution has taken care of endogenous pathways, we have a hard time achieving such an optimality with engineered pathways. In this paper, the authors generate a library of random intergenic regions (which they call TIGRs). These TIGRs may include random hair-pin sites or RNase E sites. They initially test these TIGR libraries on an operon with RFP and GFP genes (see the figure below from the original paper). Screening this library shows a range of both absolute and relative expression levels for these two genes. For example, they show that in regions where the RBS for the second gene is captured in a stem-loop structure the expression of the second gene is drastically reduced. The same holds for the cases where the hair-pin structures prematurely terminate transcription.


Upon making the case for the applicability of their method, they employ it for optimizing an exogenousy introduced mevalonate pathway in E. coli which includes an operon of three genes. Upon screening for higher mevalonate production, the authors identify constructs with upto a seven-fold increase in production.