# Metabolic Engineering _Escherichia coli_ for the Production of Lycopene
## 3 Optimization of Other Metabolic Pathways to Enhance Lycopene Production
By exploring the potential reasons, it was found that _gdhA_ deletion could increase the availability of NADPH, the knockout of _aceE_ would presumably improve carbon flux to pyruvate and formate, and further deletion of _fdhF_ might redirect the formate flux back to pyruvate, resulting in the increase of lycopene production [39]. Furthermore, transposon mutagenesis was utilized to identify combinatorial genetic targets for deletion to increase either the cofactor or precursor supply, resulting in the enhancement of lycopene yield. All the validated deletions were directly or indirectly related to cofactor production or metabolic flux [38]. Based on transposon mutagenesis and screening, the \(\Delta hnr\) (aspartokinase/homoserine dehydrogenase) \(\Delta yliE\) (di-GMP phosphodiesteraseerase) mutant was obtained that significantly improved lycopene production. The Hnr protein could function as RpoS degradation, which is important for carotenoid production in _E. coli_. Moreover, the absence of the hypothetical protein YliE could positively influence lycopene production only in the \(\Delta hnr\) background [40]. Subsequently, a two-dimensional gene target search of systematic and combinatorial approaches was developed to identify the overexpressiontargets and the knockout targets, respectively. More than 40 engineered strains were constructed, and the corresponding lycopene production was detected, combining overexpression with the knockout of the target genes with the highest lycopene yield being 16 mg/g DCW. These mutants involved kinds of genes referring to the synthetic pathway and regulation, and the lycopene of the most mutant strains has been limited by regulatory or metabolic barriers [48]. The adoption of a multi-dimensional search could thus help explore extensive mutant phenotypes. As a result, effective tools and strategies are necessary for identifying potential genes related to product yield in metabolic engineering at a global level. Moreover, the approach used to tune the genetic control of a single gene and modify multiple genes simultaneously also has been developed, including a functional promoter library [57], global transcription machinery engineering [58], engineered global regulators [42], and a functional RBS library [27]. The improvement of these tools and strategies would be beneficial for the optimization of the metabolic landscapes and the construction of effectively engineered strains. Besides the modulation of the metabolite flux, a specific strategy such as membrane engineering plays a role in improving the lycopene yield. As shown in Figure 3, when Almgs (a membrane-bending protein) and two proteins related to the membrane-synthesis pathway, Plsb (glycerol-3-phosphate acyltransferase) and Plsc (1-acylglycerol-3-phosphate-acyltransferase), were overexpressed, the cells produced sufficient intracellular membrane vesicles and thus provided more space, and the amount of the membrane component, glycerophospholipids, was increased. Finally, 36.4 mg/g DCW of lycopene was accumulated in the cell membranes [49]. This novel membrane engineering strategy could be further explored for the synthesis of a wide range of hydrophobic products. In addition, the precursors of lycopene, IPP and DMAPP, were biosynthesized via the MEP or MVA pathway, during which they need NADPH and ATP [19]. Consequently, another critical engineering strategy for enhancing lycopene production is to provide enough NADPH and ATP to the entire metabolic pathway. In another study, the expression of genes encoding a-ketoglutate dehydrogenase, succinate dehydrogenase, and transaldolase B was modulated to increase NADPH and ATP, resulting in a significant increase in the lycopene yield [27]. Figure 3: Diagram of membrane engineering strategy to increase the lycopene production in _E. coli_. Almgs, membrane-bending protein; Plsb, glycerol-3-phosphateacyltransferase; Plsc, 1-acylglycerol-3-phosphate-acyltransferase.