Systems biology represents a powerful method to describe and manipulate phenotypes of interest by incorporating biological information from various levels of cellular organization. Such an approach is illustrated from a library of both rationally-directed and combinatorial gene knockout strains of E. coli containing the pAC-LYC plasmid which have been shown to produce elevated levels of the representative isoprenoid lycopene. Global genomic and proteomic expression changes associated with increased lycopene production of mutant E. coli constructs were discovered using whole-genome E. coli DNA microarrays and a novel LC-MS technique, respectively.

Following validation of the DNA microarray method by ANOVA analysis, transcriptional profiling was applied to five high lycopene-producing knockout mutants. One observation was greater than 4 times more differential expression for strains with a deletion in hnr, an important regulator of σ_s factor, as compared to mutants with metabolic targets. Additionally, the conservation of expression trends across a majority of distantly-related mutants for specific genes was seen. Within a specific mutant family of ΔgdhA, ΔgdhA ΔaceE, and ΔgdhA ΔaceE Δ_Pyjid strains, hisH of the histidine biosynthetic pathway was down-regulated more than 10-fold while a gene encoding for a critical component of ATP synthase, atpE, was up-regulated more than 3-fold.

To supplement the genomic data, proteomic expression changes between the strains were analyzed using a novel LC-MS technique for simultaneous identification and label-free quantitation of proteins. The combined database search results from the LC-MS analysis of eight samples provided greater than 500 protein identifications. While a majority of proteins showed little expression change in the mutants relative to the pre-engineered strain, some key proteins were up- or down-regulated by nearly 10-fold. In particular, WrbA, a NADPH quinone oxidoreductase which reduces the quinone pool, was observed up-regulated over 5-fold while MdoG, a periplasmic protein which may affect the membrane storage capacity for lycopene, was down-regulated over 2-fold.

A simultaneous examination of both genomic and proteomic data revealed that the TCA cycle appears generally down-regulated while the PEP-glyoxylate cycle appears generally up-regulated across the various mutants. This is interesting due to the effect of the PEP-glyoxylate cycle upon cellular NADPH/NADH balance since lycopene biosynthesis requires large amounts of NADPH. Accordingly, a repressor of the glyoxylate pathway, IclR, and a membrane-bound pyridine nucleotide transhydrogenase, PntB, were targeted as potentially important to lycopene production.

Current work is progressing to overexpress and delete the hisH, wrbA, iclR, and pntB genes and determine resulting effects upon lycopene production.