title: Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli.
abstract: Now that many genomes have been sequenced and the products of newly identified genes have been annotated, the next goal is to engineer the desired phenotypes in organisms of interest. For the phenotypic engineering of microorganisms, we have developed novel artificial transcription factors (ATFs) capable of reprogramming innate gene expression circuits in Escherichia coli. These ATFs are composed of zinc finger (ZF) DNA-binding proteins, with distinct specificities, fused to an E. coli cyclic AMP receptor protein (CRP). By randomly assembling 40 different types of ZFs, we have constructed more than 6.4 x 10(4) ATFs that consist of 3 ZF DNA-binding domains and a CRP effector domain. Using these ATFs, we induced various phenotypic changes in E. coli and selected for industrially important traits, such as resistance to heat shock, osmotic pressure and cold shock. Genes associated with the heat-shock resistance phenotype were then characterized. These results and the general applicability of this platform clearly indicate that novel ATFs are powerful tools for the phenotypic engineering of microorganisms and can facilitate microbial functional genomic studies.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Biomass Team, Korea Institute of Energy Research, Daejeon, Korea.
authors.forename: Ju Young
authors.initials: JY
authors.lastname: Lee
authors.forename: Bong Hyun
authors.initials: BH
authors.lastname: Sung
authors.forename: Byung Jo
authors.initials: BJ
authors.lastname: Yu
authors.forename: Jun Hyoung
authors.initials: JH
authors.lastname: Lee
authors.forename: Sang Hee
authors.initials: SH
authors.lastname: Lee
authors.forename: Mi Sun
authors.initials: MS
authors.lastname: Kim
authors.forename: Michael D
authors.initials: MD
authors.lastname: Koob
authors.forename: Sun Chang
authors.initials: SC
authors.lastname: Kim
mesh_terms: D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007218:Industrial Microbiology / Q000379:methods*; D010641:Phenotype; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013696:Temperature; D014158:Transcription, Genetic; D016335:Zinc Fingers
chemical_list: D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C481662:crp protein, E coli