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Multi-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
31968 44862 2007 11 PDF Available
Title
Multi-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli
Abstract

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 stoichiometric modeling. All combinations of single (rpoS, appY, yjiD, ycgW, and yhbL), double (yjiD-ycgW) and triple (yjiD-ycgW-yhbL) overexpressions with four gene deletion backgrounds, including single (ΔgdhA, or ΔaceE), double (ΔgdhAΔaceE), and triple (ΔgdhAΔaceEΔfdhF) knockouts, were constructed and evaluated for lycopene production. Investigation of the metabolic landscape spanned by these 40 strains identified the best-engineered strain (T5P-dxs, T5P-idi, rrnBP-yjiD-ycgW, ΔgdhΔaceEΔfdhF, pACLYC), which accumulated 16,000 ppm (16 mg/g cell) of lycopene within 24 h in a batch shake flask with 5 g/L of glucose in M9 minimal medium.

Keywords
Gene targeting; Lycopene; Combinational search
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Multi-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 9, Issue 4, July 2007, Pages 337–347
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us