Applied Microbiology and Biotechnology

, Volume 98, Issue 4, pp 1567–1581 | Cite as

Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly

  • Farnaz F. Nowroozi
  • Edward E. K. Baidoo
  • Simon Ermakov
  • Alyssa M. Redding-Johanson
  • Tanveer S. Batth
  • Christopher J. Petzold
  • Jay D. KeaslingEmail author
Biotechnological products and process engineering


The genes encoding the mevalonate-based farnesyl pyrophosphate (FPP) biosynthetic pathway were encoded in two operons and expressed in Escherichia coli to increase the production of sesquiterpenes. Inefficient translation of several pathway genes created bottlenecks and led to the accumulation of several pathway intermediates, namely, mevalonate and FPP, and suboptimal production of the sesquiterpene product, amorphadiene. Because of the difficulty in choosing ribosome binding sites (RBSs) to optimize translation efficiency, a combinatorial approach was used to choose the most appropriate RBSs for the genes of the lower half of the mevalonate pathway (mevalonate to amorphadiene). RBSs of various strengths, selected based on their theoretical strengths, were cloned 5′ of the genes encoding mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and amorphadiene synthase. Operons containing one copy of each gene and all combinations of RBSs were constructed and tested for their impact on growth, amorphadiene production, enzyme level, and accumulation of select pathway intermediates. Pathways with one or more inefficiently translated enzymes led to the accumulation of pathway intermediates, slow growth, and low product titers. Choosing the most appropriate RBS combination and carbon source, we were able to reduce the accumulation of toxic metabolic intermediates, improve growth, and improve the production of amorphadiene approximately fivefold. This work demonstrates that balancing flux through a heterologous pathway and maintaining steady growth are key determinants in optimizing isoprenoid production in microbial hosts.


Metabolic engineering Mevalonate pathway Ribosome binding site Amorphadiene FPP toxicity Escherichia coli 



This work was supported by the Joint BioEnergy Institute ( through a contract between Lawrence Berkeley National Laboratory and the US Department of Energy, Office of Science, Office of Biological and Environmental Research (DE-AC02-05CH11231) and the Synthetic Biology Engineering Research Center ( through a grant from the National Science Foundation (BES-0439124). We thank Chris Anderson (Department of Bioengineering, University of California, Berkeley, CA, USA) for the gift of pBca9145.

Supplementary material

253_2013_5361_MOESM1_ESM.pdf (53 kb)
ESM 1 (PDF 53.3 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2013

Authors and Affiliations

  • Farnaz F. Nowroozi
    • 1
    • 2
  • Edward E. K. Baidoo
    • 2
    • 3
  • Simon Ermakov
    • 4
  • Alyssa M. Redding-Johanson
    • 2
    • 3
  • Tanveer S. Batth
    • 2
    • 3
  • Christopher J. Petzold
    • 2
    • 3
  • Jay D. Keasling
    • 1
    • 2
    • 3
    • 5
    Email author
  1. 1.Department of BioengineeringUniversity of CaliforniaBerkeleyUSA
  2. 2.Joint Bioenergy InstituteEmeryvilleUSA
  3. 3.Physical Biosciences DivisionLawrence Berkeley National LaboratoryBerkeleyUSA
  4. 4.Department of Molecular Cell BiologyUniversity of CaliforniaBerkeleyUSA
  5. 5.Department of Chemical and Biomolecular EngineeringUniversity of CaliforniaBerkeleyUSA

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