Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes


Many applications of synthetic biology require the balanced expression of multiple genes. Although operons facilitate coordinated expression of multiple genes in prokaryotes and eukaryotes, coordinating the many post-transcriptional processes that determine the relative levels of gene expression in operons by a priori design remains a challenge. We describe a method for tuning the expression of multiple genes within operons by generating libraries of tunable intergenic regions (TIGRs), recombining various post-transcriptional control elements and screening for the desired relative expression levels. TIGRs can vary the relative expression of two reporter genes over a 100-fold range and balance expression of three genes in an operon that encodes a heterologous mevalonate biosynthetic pathway, resulting in a sevenfold increase in mevalonate production. This technology should be useful for optimizing the expression of multiple genes in synthetic operons, both in prokaryotes and eukaryotes.

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Figure 1: TIGR assembly and reporter operon.
Figure 2: Expression from TIGR library in the original operon vector p70RG.
Figure 3: TIGR effects on expression.
Figure 4: Optimization of mevalonate production in E. coli using the TIGR method.


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The authors would like to acknowledge Vincent J.J. Martin, Jack D. Newman, Katherine D. McMahon, Sydnor T. Withers and Wesley D. Marner II for their constructive comments, Hector Nolla for performing FACS experiments, and Chris J. Paddon for performing the HMGR assays. This research was conducted under the sponsorship of the Institute for OneWorld Health, through the generous support of The Bill and Melinda Gates Foundation and by National Science Foundation Grant no. BES 9906405. D.J.P. is the recipient of a National Science Foundation graduate fellowship.

Author information




B.F.P. designed and conducted experiments; D.J.P. designed and conducted experiments; C.D.S. designed experiments; J.D.K. designed experiments.

Corresponding author

Correspondence to Jay D Keasling.

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Competing interests

J.D.K. is a founder of Amyris Biotechnologies, a company that may eventually use the optimized mevalonate operons and engineered bacterium described in this publication, to produce artemisinin. However, neither Amyris Biotechnologies nor the University of California will make any profit from the production and sale of artemisinin, the anti-malarial drug.

Supplementary information

Supplementary Fig. 1

Effect of TIGR on individual gene expression. (PDF 164 kb)

Supplementary Fig. 2

Effect of gene location on expression in the TIGR library. (PDF 210 kb)

Supplementary Fig. 3

Assembly of larger operons using megaprimers. (PDF 183 kb)

Supplementary Table 1

Oligonucleotides used in the study. (PDF 133 kb)

Supplementary Table 2

Features of TIGR Oligonucleotides. (PDF 136 kb)

Supplementary Table 3

Sequences of TIGRs from library samples. (PDF 142 kb)

Supplementary Table 4

Strains and plasmids used in this study. (PDF 108 kb)

Supplementary Methods (PDF 196 kb)

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Pfleger, B., Pitera, D., Smolke, C. et al. Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat Biotechnol 24, 1027–1032 (2006).

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