## 3 Technology Developments for Production of Carotenoids
### Genome Engineering for Strain Development  
For the most efficient carotenoid production, the biological system of the host organism also needs to be optimized, by, for example, redirecting cellular carbon flux to the carotenoid synthesis pathway. The _de novo_ synthesis of carotenoids is initiated from acetyl-coA by the MVA pathway or glycolytic metabolites pyruvate and glyceraldehyde-3-phosphate (G3P) by the MEP pathway. The direct efforts are focused on the modification of associated genes to these pathways. Deletion of pyruvate kinases PykFA can balance the availability of pyruvate and G3P for the MEP pathway, and increase lycopene production by 2.8-fold in _E. coli_[103]. The deletion of glucose-6-phosphoatte (G6P) dehydrogenase Zwf, which branches G6P to pentose phosphate pathway results in an increase by 30% in lycopene production [104]. Deletion of carbohydrate phosphotransferase system yields a seven-fold increase in lycopene production in another study [105]. Replacement of native promoters of the rate-limiting genes of the MEP pathway with the T5 promoter has been carried out for enhancement of the targeted pathway flux, which results in a 4.5-fold increase in \(\beta\)-carotene production [106]. A heterologous pathway is not just an independent entity. It communicates with the native cellular metabolism and is therefore governed by the global regulation of the host organisms. Adaptive laboratory evolution is a traditional route for strain engineering to achieve desirable industrially relevant phenotypes. Owing to the antioxidant properties of carotenoids, adaptive evolution has been successfully applied to an engineered _S. cerevisiae_ with periodic hydrogen peroxide shocking, resulting in a three-fold increase of \(\beta\)-carotene production. Subsequent transcriptome analysis indicates that some genes related with lipid biosynthesis and MVA pathways are up-regulated in the adopted strains [107]. It also suggests that carotenoid production can be improved by modifications (knock-out or overexpression) of distant genes, which are responsible for the overall regulation of the metabolic network or the physiological fitness of the host (Figure 5C). In a genome-wide screening of yeast deletion collection, 24 deletions exhibit significant higher carotenoid levels than the wild type. The triple deletion of _ROX1_, _YJL064W_, and _YJL062W_ shows an almost four-fold increase in total carotenoid production [108]. Gene deletions of _hnr_, _yjifP_, and _yjiD_ related to the improvement of lycopene production have been identified from a global transposon _E. coli_ mutant library [109]. Other gene deletions such as _gdhA_, _cyoA_, _ppc_, _gpmA_, _gpmB_, _eno_, _glyA_, aceE, _talB_, and _fdhF_ have been _in silico_ identified using a stoichiometric model [110]. The triple mutation of _gdhA_, _aceE_ and _fdhF_ was validated to increase lycopene production by nearly 40% in _E. coli_ over the engineered parental strain. A similar set of gene deletions _dhA_, _cyoA_, _gpmA_, _gpmB_, _icdA_, and _eno_ have been also _in silico_ identified using different metabolic network models [111]. Overexpression of some genes encoding global regulatory proteins AppY, Crl, RpoS, and ElbAB, oxidoreductasees TorC, YdgK, and YeiA, and hypothetical proteins YedR and YhDL, result in a significant increase in lycopene production in _E. coli_[112]. With a profound understanding of the landscape of genome manipulation, all these knocked-out and overexpressed alleles have been combined and optimized to generate high-fitness host strains for lycopene production [113, 114]. ATP and NADPH are also important cofactors for the production of carotenoids. Using engineering ATP synthesis, pentose phosphate and TCA modules, recent work has shown the highest \(\beta\)-carotene production of 2.1 g/L by a fed-batch fermentation process in _E. coli_[115]. The advances in synthetic biology greatly boost genome manipulation on a large scale.