Published online 18 July 2008 Nucleic Acids Research, 2008, Vol. 36, No. 16 e102 doi:10.1093/nar/gkn449 Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli 1 1 1 1 1 Ju Young Lee , Bong Hyun Sung , Byung Jo Yu , Jun Hyoung Lee , Sang Hee Lee , Mi Sun Kim2, Michael D. Koob3 and Sun Chang Kim1, 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 2Biomass Team, Korea Institute of Energy Research, Daejeon, Korea and 3Department of Laboratory Medicine and Pathology, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA Received November 12, 2007; Revised June 19, 2008; Accepted June 27, 2008 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 pheno- typic 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 pro- teins, with distinct specificities, fused to an E. coli cyclic AMP receptor protein (CRP). By randomly assembling 40 different types of ZFs, we have con- structed more than 6.4 104 ATFs that consist of 3 ZF DNA-binding domains and a CRP effector domain. Using these ATFs, we induced various phe- notypic changes in E. coli and selected for indus- trially important traits, such as resistance to heat shock, osmotic pressure and cold shock. Genes associated with the heat-shock resistance pheno- type were then characterized. These results and the general applicability of this platform clearly indi- cate that novel ATFs are powerful tools for the phe- notypic engineering of microorganisms and can facilitate microbial functional genomic studies. INTRODUCTION Historically, many unique cellular traits of microorganisms have been identiﬁed and modiﬁed, not only for the beneﬁt of human life, but also for industrial applications. To improve the characteristics of useful microorganisms, sev- eral approaches have been explored to expedite the screen- ing for new phenotypes of interest. Because of its simplicity and convenience, random mutagenesis has been widely used for the selection of desired phenotypes (1,2). However, mutagenesis approaches rely on labor-intensive work and luck. Therefore, more rational approaches have been employed for the manipulation of genes relevant to a spe- ciﬁc function (3–5). Most rational approaches involve either the deletion or overexpression of a single gene or sequential multigenic modiﬁcations (4,5). However, the engineering of desired phenotypes in organisms often requires the complete reprogramming of innate gene exp- ression circuits, a process that necessitates multigenic tran- scriptional coordination. Even with the most sophisticated computational methods for physiological pathway analy- sis, complete identiﬁcation of the precise genes involved in achieving a desired phenotype is almost impossible. There- fore, to permit multiple simultaneous gene expression changes, researchers recently developed methods for engi- neering of the global transcription machinery (6,7). Although this methodology permits changes in the expres- sion of many genes that allow organisms to access novel cellular phenotypes, its use has been restricted either to a speciﬁc genotype or to housekeeping genes that are gener- ally expressed and thought to be involved in routine cellular metabolism (7). Here, we have developed novel artiﬁcial transcription factors (ATFs) to completely reprogram innate gene expression circuits in Escherichia coli and thus to elicit broad perturbations in the E. coli transcriptome. Zinc ﬁngers (ZFs) are well-characterized and highly spe- ciﬁc DNA-binding domains found in a wide variety of transcriptional regulatory proteins (8,9). Because of their diversity and modular structure, ZF domains have pro- vided an attractive framework with which to construct diverse, novel ATFs (10–17). The cyclic AMP receptor protein (CRP) is a global transcription factor that regulates gene expression at >200 diﬀerent promoters in E. coli (18–21). CRP activates transcription by interacting with RNA polymerase through CRP’s functionally independent transcriptional *To whom correspondence should be addressed. Tel: +82 42 869 2619; Fax: +82 42 869 2610; Email: sunkim@kaist.ac.kr 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.