## 5 Biosynthetic Pathway Engineering for Novel C\({}_{30}\) Carotenoid Generation  
Both natural and engineered extended pathways have been identified and developed for the synthesis of C\({}_{30}\) carotenoids [99]. Metabolic engineering and directed evolution allow the development of new pathways for carotenoid production in noncarotenogenic microorganisms through the amalgamation of carotenoid genes from various sources. The enzymes expressed from these genes or gene clusters work in tandem to produce the desired compound. Additionally, the promiscuity of these carotenoid enzymes is based on end-group recognition rather than on the whole carotenoid structure (Figure 5) [100]. The genes involved in the synthesis of the C\({}_{30}\) carotenoid skeleton are dehydrosolane synthase (_crtM_) and dehydrosolane desaturase (_crtN_) [14]. However, several novel C\({}_{30}\) carotenoids have been created through combinatorial biosynthesis using enzymes from different microorganisms. For instance, C\({}_{40}\) carotenoids were modified to produce cyclic C\({}_{30}\) diapocarotonoids using lycopene cyclase, CrtY, which catalyzes the reaction on the \(\psi\)-end groups [101]. Various carotenoid structures can be obtained by manipulating biosynthetic pathways. [PERSON] et al. [36] synthesized the acyclic, monocyclic, and bicyclic forms of C\({}_{30}\) carotenoids from _E. coli_ that had undergone directed evolution and a combination of biosynthetic processes. The extension of the diapolycopene pathway led to the formation of diaponeurosporene and the novel cyclic carotenoid, diapotorulene [99; 58]. Following comparisons with other lycopene cyclase producers, [PERSON] et al. [36] selected C\({}_{40}\) CrtY from _Brevbacterium linents_ (CrtYBL) to catalyze the cyclization of a saturated end of 4,4'-diaponeurosporene and produce the structurally novel monocyclic 4,4'-diapotorulene. CrtN evolution was used to optimize the 4,4'-diaponeurosporene or 4,4'-diapo-\(\zeta\)-carotene biosynthesis pathways for cyclization to produce cyclic C\({}_{30}\) carotenoids not found in nature. Modifications to the enzymes and pathways also led to the formation of other structurally novel compounds. These compounds include bicyclic C\({}_{30}\) 4,4'-diapo-\(\beta\)-carotene; monocyclic C\({}_{30}\) carotenoids harboring modified \(\beta\)-ionone rings, 7-hydroxy-4,4'-diapotorulene and 8-keto-4,4'-diapotorulene; monocyclic C\({}_{30}\) carotenoids with modified acyclic ends, 4,4'-diapotorulen-4'-al and 4,4'-diapotorulen-4'-oic acid; and bicyclic compounds, 4,4'-diapo-\(\beta\)-crytoxanthin, 4,4'-diapozeaxanthin,4,4'-diapoechinenone, and 4,4'-diapo-\(\beta\)-cryptoxanthin glucoside [36]. [PERSON] et al. [37] identified a novel gene (_crtNb_) from _Methylomonas_, and its homologue in Staphylococcus, which converts 4,4\({}^{\prime}\)-diapolcycpene to 4,4\({}^{\prime}\)-diapolcycpene aldehyde, Additionally, they identified an aldehyde dehydrogenase gene (_ald_) involved in the oxidation of 4,4\({}^{\prime}\)-diapolcycpene aldehyde to 4,4\({}^{\prime}\)-diapolcycpene acid. New acyclic carotenoids, diapolcycpenedic acid xylosylesters A-C, and the C\({}_{30}\) aglycone, methyl 5-glucosyl-5,6-dihydro-apo-4,4\({}^{\prime}\)-lycopanoate, were produced by _Rubritalea squalenifica_ and _P. marititins_ strains. The diapolcycpenedic acids xylosylesters A, B, and C had 2-acyl-D-xylose in their structures and were derivatives of the aglycone diapolcycopenedioc acid [61]. In another study on pathway manipulation, diaponeurosporene was formed when CrtN introduced four double bonds into dehydrogenualene to produce a completely conjugated diapolcycpene in recombinant _E. coli_, substituting the three-step desaturation [17]. [PERSON] et al. [58] extended the acyclic C\({}_{30}\) diaponeurosporene pathway using the C\({}_{40}\) carotenoid enzymes spheroidene monooxygenase and lycopene cyclase to produce new oxygenated acyclic products and the novel cyclic C\({}_{30}\) compound, diapotorulene.