# Metabolic Engineering _Escherichia coli_ for the Production of Lycopene
## 5 Optimization of Auxiliary Carbon Source and Fermentation Modes  
Besides the optimization of regulatory networks in a metabolic pathway, the supply of an appropriate carbon source is also an essential factor for improving synthetic efficiency, thereby reducing the production costs at the industrial level. In the biosynthesis of lycopene, glycerol or glucose are usually utilized as the primary carbon source [36; 43; 45; 47; 54; 62]. Some researchers explored the effect of an auxiliary carbon source on lycopene production in engineered _E. coli._ When glycerol was used as the primary carbon source, the synergistic effect of the auxiliary carbon sources, glucose, and \(\updownarrow\)-arabinose was related to the endogenous metabolism in _E. coli_ and the stimulation on the exogenous MVA pathway [43]. When glucose, fructose, glycerol, or arabinose were supplied as an auxiliary carbon source, respectively, to the LB medium for lycopene production, 6 g/L fructose exhibited the highest lycopene yield [12]. Citrate was verified to be a positive auxiliary carbon source for enhancing lycopene production, indicating that the citrate pathway might be responsible for accumulating more isoprenoid in engineered _E. coli_[36]. Similarly, in another study, the MVA lower pathway was introduced into _E. coli_, in which glycerol was supplied as the carbon source with addition of mevalonate, and Tween 80 was added to prevent clump formation, resulting in a significant increase in lycopene production [54]. Moreover, an engineered _E. coli_ strain introduced with the fatty acid transport system was capable of utilizing free fatty acids or waste cooking oil to produce lycopene, with the highest yield of 94 mg/g [44].  
Optimization of the fermentation process is a traditional and direct strategy for improving lycopene production. Traditional fermentation that includes shake-flask and fed-batch fermentations is usually applied (Table 1). Besides this, various fermentation optimization strategies have been conducted. For instance, high cell density fermentation was performed in two lycopene-producing _E. coli_ strains to explore the effect of fermentation parameters on lycopene production. Results demonstrated that high oxygen levels and pH values were critical for increasing the lycopene yield. The importance of oxygen,  
Figure 4: Summary of the strategies for engineering the regulatory networks to enhance lycopene production in _E. coli._ (1) Acetyl phosphate (ACP), as an indicator of glucose flux, was set as a signal of the two-component regulon Ntr, to regulate the expression of _idi_ and _pps_ for lycopene biosynthesis. (2) Transcriptional engineering on the global regulator cAMP receptor protein (CRP) was conducted by using error-prone PCR and site-directed mutagenesis, to subtly balancing the whole metabolic pathway networks for improving the lycopene yield. (3) RpoS regulates the transcription of genes induced at the stationary phase and energy metabolism. (4) CrI regulated the expression of _csgBA_ for curli surface fiber formation to promote the accumulation of lycopene in cells. (5) Genomic integration of related genes made the expression stable and controlled the expression intensity by gene copy number. (6) The AppY transcriptional regulator was relative to anaerobic energy metabolism.  
growth rate, and glutamate flux on lycopene production thus indicated the potential of stoichiometric analysis in optimizing the fermentation strategy [45]. In another study, considering the increased carotenoid yield and productivity at 25 \({}^{\circ}\)C in contrast to those at 37 \({}^{\circ}\)C [35; 63], a temperature-shift culture method (37\(\rightarrow\)25 \({}^{\circ}\)C) was adopted to further augment lycopene production [62]. Optimizing the culture conditions of the recombinant _E. coli_ 99 DH cultivated under exposure to light in 2 YT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl), supplemented with glycine addition, exhibited an increased lycopene production rate by 76% [46].