title: Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the rhaBAD Promoter.
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 analogues 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. Rescreening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterized 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.
authors.affiliation: Centre for Synthetic Biology and Innovation, Department of Life Sciences, Imperial College London , South Kensington Campus, London SW7 2AZ, U.K.
authors.forename: Ciarán L
authors.initials: CL
authors.lastname: Kelly
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford , Oxford, OX1 3TA, U.K.
authors.forename: Zilei
authors.initials: Z
authors.lastname: Liu
authors.affiliation: International Institute of Rare Sugar Research and Education, Kagawa University , Miki, Kagawa 761-0795, Japan.
authors.forename: Akihide
authors.initials: A
authors.lastname: Yoshihara
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford , Oxford, OX1 3TA, U.K.
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Jenkinson
authors.affiliation: Glycobiology Institute, Department of Biochemistry, University of Oxford , Oxford, OX1 3QU, U.K.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Wormald
authors.affiliation: Departamento de Química Orgánica and Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares, Universidade de Santiago de Compostela , 15782 Santiago de Compostela, Spain.
authors.forename: Jose
authors.initials: J
authors.lastname: Otero
authors.affiliation: Departamento de Química Orgánica and Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares, Universidade de Santiago de Compostela , 15782 Santiago de Compostela, Spain.
authors.forename: Amalia
authors.initials: A
authors.lastname: Estévez
authors.affiliation: Department of Hospital Pharmacy, University of Toyama , Toyama 930-0194, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kato
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford , Oxford, OX1 3TA, U.K.
authors.forename: Mikkel H S
authors.initials: MH
authors.lastname: Marqvorsen
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford , Oxford, OX1 3TA, U.K.
authors.forename: George W J
authors.initials: GW
authors.lastname: Fleet
authors.affiliation: Departamento de Química Orgánica and Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares, Universidade de Santiago de Compostela , 15782 Santiago de Compostela, Spain.
authors.forename: Ramón J
authors.initials: RJ
authors.lastname: Estévez
authors.affiliation: International Institute of Rare Sugar Research and Education, Kagawa University , Miki, Kagawa 761-0795, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Izumori
authors.affiliation: Centre for Synthetic Biology and Innovation, Department of Life Sciences, Imperial College London , South Kensington Campus, London SW7 2AZ, U.K.
authors.forename: John T
authors.initials: JT
authors.lastname: Heap
mesh_terms: D023381:Endpoint Determination; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012210:Rhamnose / Q000378:metabolism; D014157:Transcription Factors
chemical_list: D029968:Escherichia coli Proteins; D014157:Transcription Factors; D012210:Rhamnose
keywords: expression system; inducible promoter; mannose; nonmetabolized inducer; rhaBAD; rhamnose