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

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 (delta gdhA, or delta aceE), double (delta gdhA delta aceE), and triple (delta gdhA delta aceE delta fdhF) knockouts, were constructed and evaluated for lycopene production. Investigation of the metabolic landscape spanned by these 40 strains identified the best-engineered strain (T5(P)-dxs, T5(P)-idi, rrnB(P)-yjiD-ycgW, delta gdh delta aceE delta fdhF, 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.