# Metabolic Engineering _Escherichia coli_ for the Production of Lycopene
## 2 Metabolic Engineering of Two Major Pathways in _E. coli_ for Lycopene Production
### Metabolic Engineering of the Endogenous MEP Pathway in E. coli  
In the early development stages of lycopene production using engineered _E. coli_, the utilization and modification of the endogenous MEP pathway were extensively investigated. Generally, the overexpression of the key enzymes of the MEP pathway is important in lycopene production, especially the major rate-limiting enzymes (DXS, DXR, and IDI) [19]. When the genes _dxs_ and _dxr_ were overexpressed solely or jointly on different expression vectors using three different promoters and _E. coli_ host strains, the highest lycopene yield (22 mg/L) was reached with the arabinose-inducible promoter on a medium-copy plasmid pBAD24 in the _E. coli_ XL1-Blue strain [31]. \begin{table}
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**Major Methods** & **Optimization Strategies** & **Yield/Titer** & **Culture Conditions** & **References** \\ \hline Overexpression of rate-limiting enzymes & Comparison of _crtEBI_ genes from different strains & 59 mg/L & - & [15] \\ Knockout of _znf_; overexpression of _ali_, & & & \\ _dxs_ and _spD_, _spF_ & 7.55 mg/g DCW & Shake-flask fermentation & [28] \\ Overexpression of _crtE_, _crtE_, _crtI_, _ipi_, _dxs_ & 5.2 mg/g DCW & Shake-flask fermentation & [29] \\ Overexpression of _dxs_, _dxr_ & 22 mg/L & Shake-flask fermentation & [31] \\ Overexpression of _dxs_ & 1.33 mg/g DCW & Shake-flask fermentation & [32] \\ The co-expression of _apyr_, _crl_, and _rpos_ & 4.7 mg/g DCW & [29] \\ with _dxs_ & 45 mg/g DCW & Shake-flask fermentation & [33] \\ Directed evolution & Directed evolution of _GCP_ synthase & 45 mg/g DCW & Shake-flask fermentation & [34] \\ Directed co-evolution of _dxs_, _dxr_ and _ali_ & 0.65 mg/L & - & [34] \\ Whole pathway engineering & Expression of the MVA pathway & 4.28 mg/L & Shake-flask fermentation & [35] \\ Type 2 IDI; heterologous MVA pathway & 198 mg/g DCW & Shake-flask fermentation & [36] \\ Heterologous expression of the MVA & - & Shake-flask fermentation & [37] \\ Removal of competing pathways & _AghlA_, _AacE_, _AgylC_ (_gpmB_), _AfhF_ & 18 mg/g DCW & Batch shake-flask cultivations & [38] \\ Pathway balancing & Combination of gene knockout and overexpression & 2.5 mg/g DCW & - & [20] \\ Genome-wide stoichiometric flux balance & 6.6 mg/g DCW & Shake-flask fermentation & [39] \\ analysis; genes knockout & Gene knockout (_Amr_, _Amr_, _AliE_) & - & Shake-flask fermentation & [40] \\ Regulatory engineering & Ntr regulation, stimulated by excess glycolytic flux through sensing of ACP & 0.16 mg/L/h & Shake-flask fermentation & [41] \\ Engineering of the cAMP receptor protein (CRP) & 18.49 mg/g DCW & Batch fermentation & [42] \\ Optimization of carbon sources & Auxiliary carbon source optimization & 1050 mg/L & Baffled flask fermentation & [12] \\ Supplementing auxiliary carbon sources & 40 mg/L/h & Fed-batch culture & [43] \\ Fermentation with fatty acids or waste & 94 mg/g DCW & Fed-batch fermentation & [44] \\ Optimization of fermentation & High cell density fermentation & 220 mg/L & Batch fermentation & [45] \\ Different types of plasmid expression: optimization of fermentation conditions & 67 mg/g DCW & Shake-flask fermentation & [46] \\ Targeted engineering & Targeted engineering: targeted proteomic and intermediate analysis & 1.23 g/L & Fed-batch fermentation & [47] \\ Two-dimensional search for gene targets & 16 mg/g DCW & Shake-flask fermentation & [48] \\ Cofactor engineering & Modulating supply of NADPH and ATP; overexpression of _dxs_, _ili_ and _crt_ gene & 50.6 mg/g DCW & Fed-batch fermentation & [27] \\ Membrane engineering & Membrane engineering: overexpression of _plb_, _plac_ and _dgka_ & 36.4 mg/g DCW & Shake-flask fermentation & [49] \\ Genome engineering & Synthesis genes were integrated into chromosome & 33.43 mg/g DCW & Shake-flask fermentation & [50] \\ Large-scale programming used to optimize the MEP pathway & 9 mg/g DCW & - & [51] \\ A new combinatorial multi-gene pathway assembly scheme & 448 mg/g DCW & - & [52] \\ \hline \end{tabular}
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