## 3 Technology Developments for Production of Carotenoids
### Pathway Engineering for Production of Carotenoids  
Carotenoid biosynthesis emerges from the central isoprenoid pathway, either the MEP pathway or the MVA pathway, existing in all organisms [19, 22]. The expression of carotenogenic genes can yield carotenoids of interest in a heterologous organism [16, 73, 74, 75]. The early attempts led to the production of lycopene, \(\beta\)-carotene, and astaxanthin in _Saccharomyces cerevisiae_ and _Candida utilis_ by the expression of carotenoid enzymes from _Pantoea ananatis_[74, 76]. _Corynebacterium glutamicum_ is a native producer of decaprenoxanthin and its glucosides, and it has been engineered to synthesize C50 carotenoids C.P.450 and sarcinaxanthin [77]. To date, there have been many exemplary illuminations to achieve high carotenoid titers from non-native producers. Carotenogenic enzymes from different sources exhibit different capacities in carotenoid biosynthesis. A two-fold higher lycopene production is obtained in _E. coli_ by the expression of carotenoid enzymes from _P. agglomerans_ (27 mg/L) than from _P. ananatis_ (12 mg/L) [78]. Metabolic engineering approaches allow the assembly of genes from different organisms for production purposes or for building new carotenoids [32, 79, 80]. \(\beta\)-Carotene production has been improved by hybrid expression of carotenoid genes from _P. agglomeras_ and _P. ananatis_ in _E. coli_[81]. In another example, expression of \(\beta\)-end ketolase from _Agrobacteriumium aurantiacum_ extends the zeaxanthin \(\beta\)-d-diglucoside pathway from _P. ananatis_, and synthesizes novel astaxanthin \(\beta\)-d-diglucoside and adonixanthin \(\beta\)-d-diglucoside [29]. Generally, a sufficient precursor supply is a prerequisite for high-yield production of carotenoids. Overexpression of the rate-limiting enzymes 1-deoxy-d-xylulose-5-phosphate synthase and reductoisomerase led to a 3.6-fold increase in lycopene production in _E. coli_ when compared with the native MEP pathway for IPP and DMAPP supply [71]. Overexpression of the rate-limiting enzyme 3-hdroxy-3-methyl-glutaryl-coenzyme A (HNG-CoA) reductase of the MVA pathway from _Xanthophyllomyces dendorrho_ significantly increased \(\beta\)-carotene production in _S. cerevisiae_[82]. A great effort in metabolic engineering of the central carotenoid building block pathway is the introduction of a hybrid MVA pathway of _Streptococcus pneumonia_ and _Enterococcus faecalis_ into _E. coli_, which enables the recombinant host to produce 465 mg/L of \(\beta\)-carotene [83]. With more available genetic tools, microbial organisms such as _Pseudomonas putida_ and _Bacillus subtilis_ have also been developed as platform hosts for carotenoid production [84, 85]. Carotenoids synthesis involves multiple enzymes [2]. The expression level of all the components of a multigene circuit should be orchestrated to optimize metabolic flux and to gain a high yield (Figure 5B) [86]. A random approach is screening of the best orchestra from numerous combinatorial assemblies of required genes and control elements. BioBrick(tm) paradigm is capable of rapidly assembling a biosynthetic pathway in a variety of gene orders from different promoters in plasmids with different copy numbers [87]. It is possible to build a hybrid carotenoid pathway wherein each enzyme possesses a right turnover number, however, BioBrick(tm) assembly is still not in a high throughput to create vast combinatorial expression constructs for the best combination of carotenoid genes. Recently, several advanced assembly methods using homologous recombination, such as sequence and ligation-independent cloning (SLIC), Gibson DNA assembly and reiterative recombination, have been applied to construct multigene circuits [88, 89, 90]. These advances promise to randomize all genetic components, including genes, promoters, ribosome binding sites, and other control modules to build a large number of individual genetic circuits for screening purposes.