Synthetic chemical inducers and genetic decoupling enable orthogonal control of the rhaBAD promoter

Ciarán L Kelly, Zilei Liu, Akihide Yoshihara, Sarah F Jenkinson, Mark R Wormald, Jose M Otero, Amalia M Estévez, Atsushi Kato, Mikkel H S Marqvorsen, George W J Fleet, Ramon J Estevez, Ken Izumori, John T Heap

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

Abstract

External control of gene expression is crucial in synthetic biology and biotechnology research and applications, and is commonly achieved using inducible promoter systems. The E. coli rhamnose-inducible rhaBAD promoter has properties superior to more commonly-used inducible expression systems, but is marred by transient expression caused by degradation of the native inducer, L-rhamnose. To address this problem, 35 analogs of L-rhamnose were screened for induction of the rhaBAD promoter, but no strong inducers were identified. In the native configuration, an inducer must bind and activate two transcriptional activators, RhaR and RhaS. Therefore, the expression system was reconfigured to decouple the rhaBAD promoter from the native rhaSR regulatory cascade so that candidate inducers need only activate the terminal transcription factor RhaS. Re-screening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterised further to determine the strength, kinetics and concentration-dependence of induction; whether the inducer was used as a carbon source by E. coli; and the modality (distribution) of induction among populations of cells. L-Mannose was found to be the most useful orthogonal inducer, providing an even greater range of induction than the native inducer L-rhamnose, and crucially, allowing sustained induction instead of transient induction. These findings address the key limitation of the rhaBAD expression system, and suggest it may now be the most suitable system for many applications.

Original languageEnglish
JournalA C S Synthetic Biology
Volume5
Issue10
Pages (from-to)1136-1145
Number of pages10
ISSN2161-5063
DOIs
Publication statusPublished - 2016
Externally publishedYes

Keywords

  • expression system
  • inducible promoter
  • mannose
  • nonmetabolized inducer
  • rhaBAD
  • rhamnose

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