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Jin-2007-Multi-dimensional ge
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
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