# Metabolic Engineering _Escherichia coli_ for the Production of Lycopene
## 3 Optimization of Other Metabolic Pathways to Enhance Lycopene Production  
In metabolic engineering, inhibition or even knockout of the competitive pathway of intermediate products, and elimination of potential bottlenecks in the upstream pathways not only reduces the generation of by-products but also increases the yield of the target products. Meanwhile, the enhancement of some metabolic pathways is also adopted to modulate the metabolite flux in the synthesis of the target products by supplying more precursors or intermediates. Thus, a rational design strategy of metabolic pathways is of great potential to increase the yield of the target products. As per the description of the MEP pathway, the initial precursors, G3P and pyruvate, are condensed into DXP in equal amounts. The unbalance between the two precursors can reduce the synthetic efficiency, making it necessary to maintain the balance by modulating the metabolic pathways based on a rational design strategy. As is known, the conversion from phosphoenolpyruvate to pyruvate [55], an essential step in the transformation of G3P to pyruvate, is an irreversible reaction [20] inhibiting the inter-conversion between G3P and pyruvate. Consequently, a new circuit bypassing this irreversible step was reconstructed. This was achieved by deleting pyruvate kinases-I and -II (Pyk-I and-II) to cut off the direct conversion of PEP to pyruvate, and overexpressing Ppc and Pck to introduce the bypass pathway between PEP and pyruvate via the oxaloacetate and TCA cycle (Figure 2). Meanwhile, the PEP synthase (Pps) converting pyruvate to PEP was also overexpressed. Thus, lycopene production was significantly increased with the rational reconstruction of the metabolic pathways from G3P to pyruvate [20]. Similarly, the inactivation of the competing pathways at acetyl-CoA and pyruvate nodes was applied to divert more carbon flux to the precursor IPP, including the deletion of the acetate and lactate production pathways. As a result, the engineered strain with the elimination of the acetate pathway accumulated more lycopene than the control strain [35]. Generally, metabolic engineering of pathways was mainly referred to the manipulation of genes directly connected with the product-synthesizing pathway. At the same time, indirectly related genes could also influence the synthesis of target products. For example, the deletion of the _zwf_ gene encoding glucose-6-phosphate dehydrogenase (G6 PD) (Figure 2), which controls the entry of carbon into the pentose phosphate pathway, resulted in the increased carbon fluxes in the Embden-Meyerhof-Parnas (EMP) pathway involving G3P and pyruvate, and indirectly led to an increase in lycopene production [28]. Identification of the genes directly or indirectly related to the production of the target products in metabolic engineering is of utmost importance. The elimination or overexpression of these genes can affect metabolic progress, either by redistributing the metabolic precursors or rewiring regulatory networks. Previous research on metabolically engineering _E. coli_ for lycopene production employed an artificial phenotypic screening system to identify the genes affecting lycopene formation. As a result, multiple genes, including two unknown genes _elb1_ and _elb2_, were identified that might be involved in the early reactions in lycopene synthesis. In more detail, the regulator encoded by _elb2_ could regulate the biosynthesis of ubiquinone, including early steps of isoprenoid biosynthesis [56]. [PERSON] et al. performed a genome-wide stoichiometric flux balance analysis to explore potential genes impacting the whole network properties and cellular phenotype. Consequently, seven single and multiple stoichiometric gene deletion mutants were obtained with increased lycopene production compared with the parental strain. Mainly, the triple knockout mutant of _gdhA/hceE/fdHr_ (encoding NADP-specific glutamate dehydrogenase, pyruvate dehydrogenase, and formate dehydrogenase, respectively) exhibited a nearly 40% increase in lycopene yield (Figure 2).