Multiple-Site Diversification of Regulatory Sequences Enables Interspecies Operability of Genetic Devices

Angeles Hueso-Gil, Ákos Nyerges, Csaba Pál, Belén Calles, Víctor De Lorenzo

Research output: Contribution to journalArticle

1 Citation (Scopus)


The features of the light-responsive cyanobacterial CcaSR regulatory module that determine interoperability of this optogenetic device between Escherichia coli and Pseudomonas putida have been examined. For this, all structural parts (i.e., ho1 and pcyA genes for synthesis of phycocyanobilin, the ccaS/ccaR system from Synechocystis, and its cognate downstream promoter) were maintained but their expression levels and stoichiometry diversified by (i) reassembling them together in a single broad host range, standardized vector and (ii) subjecting the noncoding regulatory sequences to multiple cycles of directed evolution with random genomic mutations (DIvERGE), a recombineering method that intensifies mutation rates within discrete DNA segments. Once passed to P. putida, various clones displayed a wide dynamic range, insignificant leakiness, and excellent capacity in response to green light. Inspection of the evolutionary intermediates pinpointed translational control as the main bottleneck for interoperability and suggested a general approach for easing the exchange of genetic cargoes between different species, i.e., optimization of relative expression levels and upturning of subcomplex stoichiometry.

Original languageEnglish
JournalACS Synthetic Biology
Publication statusAccepted/In press - Jan 1 2019


  • CcaSR
  • interoperability
  • optogenetics
  • Pseudomonas
  • recombineering

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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