## 4 Discussion
ISPD catalyzes the conversion of 2-C-methyl-D-erythnitol 4-phosphate to 4-(cytidine-5'-diphosphate)-2-C-methyl-D-erythnitol. A new enzyme, ISPD, was discovered in _E. coli_[32]. These genes _ygbP_ and _ygbB_ corresponding to ISPD may be involved in this step of transformation [33]. ISPE catalyses transform 4-(cytidine-5'-diphosphate)-2-C-methyl-D-erythnitol into 2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythnitol. A new enzyme, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythnitol kinase (ISPE), was discovered in _E. coli_[34]. Earlier experiments, overexpressing the gene _ychB_ corresponding to ISPE in _E. coli_ and combining with isotopic labeling, found the catalytic activity of ISPE. The predicted protein sequence similarity between YchB and tomato cDNA pTOM41 was 30%, and this phenomenon was related to the transformation of chloroplast to chromosome [35]. ISPF catalyzes the conversion of 2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythnitol to 2-C-methyl-D-erythnitol 2,4-cyclodiphosphate. A new enzyme, ISPF, was discovered in _E. coli_[36]. Earlier experiments, overexpressing the gene _ygbB_ corresponding to ISPF in _E. coli_ and combining with isotopic labeling, found that the catalytic activity of ISPF [37]. GcpE catalyses transform 2-C-methyl-D-erythnitol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-butenyl-4-diphosphate. Combining database information and heterologous expression of _gcpE_ and _petF_ genes indicated that PetF could transfer electrons to GcpE [38]. Experimental results indicate that GcpE is a ferredoxin-dependent enzyme with [4 Fe-4S] cluster [39]. In bacteria, GcpE relies on NADPH/flavodoxin/flavodoxin reductase as a reductive shuttle system for electron transfer [40]. ISPH catalyzes the conversion of 1-hydroxy-2-methyl-2-butenyl-4-diphosphate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMPP). Expression of the ispH (_lytB_) gene in _E. coli_ converts 1-hydroxy-2-methyl-2-butenyl-4-diphosphate to IPP and DMPP [41]. Further studies reveal that the enzyme possesses molecular oxygen-sensitive [4 Fe-4S] clusters [42]. Overexpression of the _ispH_ gene in _E. coli_ using the isc operon resulted in at least a 200-fold increase in the catalytic activity of the purified protein ISPH [43]. FDPS catalyses transform DMPP or IPP into geranyl diphosphate (GPP), and also catalyses transform IPP into farnesyl diphosphate (FPP). Previous studies have obtained isoprenyltransferase from _Microoccus lysodekitcus_, which catalyzes the synthesis of all-trans GPP from FPP and IPP [44]. FPP synthase, obtained from _Bacillus subtilis_, catalyzes the conversion of IPP and DMPP or GPP to all-trans GPP. The metal ions Mg\({}^{2+}\) or Mn\({}^{2+}\) are crucial to the catalytic activity, but Mn\({}^{2+}\) is less effective [45]. Geranylgeranyl diphosphate synthase is the most important to the initiation of the carotenoid synthesis pathway, producing the only product geranylgeranyl diphosphate (GGPP). This study has not annotated synthase (EC: 2.5.1.29) in the existing database. We predict that there are new synthases with lower homology in the genome of _Sphingomonas morindae_ sp. NBD5 and _Sphingopyxis_ sp. USTB-05. One molecule of FPP condenses with one molecule IPP to form GGPP under the catalysis of CrtE in _Erwinia uredovora_ and _Agrobacteriumium aurantiacum_[46]. The GGPP synthase genes were screened from the genomic DNA libraries of archaebacterial _Sulfolobus acidocalarius_ and _Streptomyces acidocalarius_. Heterologous expression showed that DMPP or GPP were used as substrates producing GGPP [46]. GGPP synthase (EC: 2.5.1.29) was purified from _Methanobacterium thermormicicum_ SF-4. It is a dimeric protein consisting of two identical subunits and is stable upon treatment at 65 \({}^{\circ}\)C for 30 min [47]. GGPP synthase from _Erwinia uredovora_ is overexpressed in _E. coli_, reaction rates and Km values indicate that GPP and FPP are allyl substrates for GGPP synthase, but not DMPP [48].