Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli

• Published in: Nucleic acids research Vol. 36; no. 16; p. e102
• Main Authors: Lee, Ju Young, Sung, Bong Hyun, Yu, Byung Jo, Lee, Jun Hyoung, Lee, Sang Hee, Kim, Mi Sun, Koob, Michael D, Kim, Sun Chang
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.09.2008; Oxford Publishing Limited (England)
• Subjects: Cyclic AMP Receptor Protein - chemistry; Cyclic AMP Receptor Protein - genetics; DNA-Binding Proteins - chemistry; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Gene Expression Regulation, Bacterial; Gene Library; Genes, Bacterial; Genetic Engineering - methods; Humans; Industrial Microbiology - methods; Methods Online; Phenotype; Protein Engineering; Protein Structure, Tertiary; Recombinant Fusion Proteins - chemistry; Temperature; Transcription, Genetic; Zinc Fingers
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkn449

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 10⁴ 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.