# Metabolic Engineering _Escherichia coli_ for the Production of Lycopene
## 4 Engineering Regulatory Networks to Enhance Lycopene Production  
As mentioned above, most studies on lycopene production were focused on the overexpression of the key rate-limiting enzymes, and the elimination or inactivation of the competitive branch pathway. The regulation of metabolic pathways could be used to reprogram the metabolic genes to improve the yield of the target products by eliminating metabolic imbalance. The phosphorylated response regulator NRI (\(glnG\) product) included in the two-component system Ntr could activate transcription from the \(glnAp2\) promoter by binding to its cognate binding sites on the DNA. Further, NRI itself is capable of sensing the level of acetyl phosphate (ACP), an indicator of glucose flux. Therefore, an artificially engineered \(glnAp2\) promoter containing NRI binding sites and the core \(glnAp2\) promoter was constructed to serve as a control valve that controls gene expression according to the ACP level. Then, this control valve was adopted to regulate the expression of IDI and Pps, which have been identified to control the flux to the final product, and the balance between pyruvate and G3P. The introduction of this regulatory circuit resulted in the full utilization of the excess carbon flux for the enhancement of lycopene production and bypassing the toxic product, acetate [41]. Moreover, transcriptional engineering of the global regulator cAMP receptor protein (CRP) was conducted using an error-prone PCR and site-directed mutagenesis to subtly balance the whole metabolic pathway networks to improve lycopene yield. The mutant strain, MT-1, with the engineered CRP encoded by a mutant gene (mcrp26) showed a higher lycopene production (18.49 mg/g DCW) compared with the original strain. Besides, the differential expression of the global genes between the MT-1 mutant and wild type was also explored, in which the genes of \(p\
ot{k}A\), \(f\!u\!A\), and \(is\!pG\) involved in the lycopene biosynthetic pathway were up-regulated. Thus, it helped reveal the possible mechanism for the improvement in lycopene production caused by the engineered CRP. As explored, in mcrp26, residue D8 (Asp) had been mutated into V (Val), which belongs to the N-proximal cAMP-binding domain, altering the cAMP-binding capacity. This influenced the CAP-dependent promoters, which raised the differential expression of the above genes related to lycopene production [42]. In addition, the modulation of global regulatory proteins RpoS (Sigma S factor), AppY (transcription activator for genes related to anaerobic energy metabolism), and Crl (transcriptional regulator of \(csgBA\) for curli surface fiber formation) also enhanced lycopene production by regulating the expression of lycopene synthesis enzymes and energy metabolism operons [59], or the hydrophobic interaction between curli fiber molecules and lycopene [29; 48; 60]. Another study was performed to disclose the mechanisms involved in the differences in the lycopene production and MEP pathway flux of six \(E\). \(coli\) host strains through systems analysis including genetic complementation, quantitative sequential windowed acquisition of all theoretical fragment ions (SWATH) proteomics, and biochemical analysis. It revealed that RpoS could help accumulate lycopene by decreasing oxidative stress in the growth stationary phase, which reduced the degradation of lycopene to its colorless oxidation--and cleavage products [61]. These strategies for engineering the regulatory networks to enhance lycopene production in \(E\). \(coli\) have been summarized in Figure 4. Thus, the engineered regulators controlling the gene expression gave rise to a significant potential for the regulatory design of metabolic pathways in \(E\). \(coli\) for the production of lycopene and other biochemicals. Although the biochemistry properties of the MVA pathway have been comprehensively revealed and extensively utilized for the industrial production of isoprenoids, few applications of the regulation of the MVA pathway have been explored in lycopene biosynthesis.