Jin-2007-Multi-dimensional ge.pdfVIP

ARTICLE IN PRESS1096-7176/$ - se doi:10.1016/j.ym Correspond chusetts Institu Fax: +617 253 E-mail addrMetabolic Engineering 9 (2007) 337–347 /locate/ymbenMulti-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli Yong-Su Jina,b, Gregory Stephanopoulosa, aDepartment of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA bDepartment of Food Science and Biotechnology, Sungkyunkwan University, Suwon, Republic of Korea Received 17 October 2006; received in revised form 2 March 2007; accepted 11 March 2007 Available online 12 April 2007Abstract Identification of multiple gene targets that exhibit different modes of action toward a desired phenotype is a crucial step in strain improvement. Target identification methods based on traceable genetic perturbations and stoichiometric modeling have been employed before for the mining of putative overexpression and knock-out targets. Most search methods are sequential and, as such, quite limited in the space they can explore. In this study, we investigate a multi-dimensional search approach whereby unknown interactions of gene targets identified by different search methods are assessed by employing orthogonal search strategies. To this end, we combined knock- out and overexpression gene targets, identified through systematic and combinatorial approaches, respectively, in order to improve lycopene production in Escherichia coli. Specifically, we first identified multiple overexpression targets by screening genomic libraries of E. coli in a sequential-iterative manner. Targets so identified confirmed previously amplified genes in the non-mevalonate pathway (dxs and idi) and some regulatory genes (rpoS and appY). Additionally, this method revealed novel gene targets (yjiD, ycgW, yhbL, purDH, and yggT). A two-dimensional search was subsequently undertaken, whereby the selected overexpression targets were combined with the knock-out targets predicted by stoichiometr