RT Journal Article
SR Electronic
T1 Multiple-site diversification of regulatory sequences enables inter-species operability of genetic devices
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 771782
DO 10.1101/771782
A1 Angeles Hueso-Gil
A1 Ákos Nyerges
A1 Csaba Pál
A1 Belén Calles
A1 Víctor de Lorenzo
YR 2019
UL http://biorxiv.org/content/early/2019/09/16/771782.abstract
AB The features of the light-responsive cyanobacterial CcaSR regulatory node 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 phycobilin, 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 non-coding 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.