Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli.
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.
Lee
Ju Young
JY
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Biomass Team, Korea Institute of Energy Research, Daejeon, Korea.
Sung
Bong Hyun
BH
Yu
Byung Jo
BJ
Lee
Jun Hyoung
JH
Lee
Sang Hee
SH
Kim
Mi Sun
MS
Koob
Michael D
MD
Kim
Sun Chang
SC
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
D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C481662:crp protein, E coli