# Caroenoid Biosynthesis in _Fusarium_
## 3 Genes and Enzymes of Caroenoid Metabolism in _Fusarium_  
Some of the enzymes responsible for early steps of the terpenoid pathway were first identified in _F. fujikuroi_ thanks to the similarity with formerly known enzymes. The typical cloning protocol consisted in the identification of conserved protein segments, the design of degenerate primers from such sequences to clone the corresponding internal gene segment by PCR, and the use of the amplified segment as a probe to clone the whole gene through the screening of a genomic library. Such methodology led to clone the genes for HMG-CoA reductase [24], producing mevalonate, and the prenyl transferases responsible for the synthesis of FPP [25] and its conversion to GGPP [26]. The mutants of these genes are expected to be non-viable because of their participation in the synthesis of essential terpenids, and therefore their functions were inferred from their close relatedness with orthologs in other organisms. _F. fujikuroi_ contains a second GGPP synthase located in the cluster for the synthesis of gibberellins, growth-inducing plant hormones of the terpenoid family. This second GGPP synthase gene is presumably specific for the synthesis of these secondary metabolites, as it indicates its coregulation with most of the genes from the gibberellin cluster [27]. The first gene specifically involved in the synthesis of carotenoids known in _Fusarium_ was _carB_[28], identified by a PCR strategy similar to the one mentioned above through its sequence similarity to the _N. crassa_ ortholog _al-1_[29]. The function of the gene was confirmed by complementation of a phytoene-accumulating albino mutant and by generation of the same phenotype by _carB_ targeted mutation. The CarB enzyme is responsible for the five desaturation steps of the NX biosynthetic pathway, as it indicated the identification of a _carB_ mutant allele specifically affected in the fifth desaturation [30]. This was in accordance with former observations with _al-1_ of _N. crassa_: its expression in _Escherichia coli_ or in vitro studies with purified AL-1 enzyme demonstrated its capacity to carry out the five desaturation steps for NX biosynthesis in this species [31]. Closely linked to _carB_ it was found the gene _carRA_ (Figure 1), orthologous of phytoene synthase _al-2_ of _N. crassa_[32] and interpreted as the phytoene synthase of _Fusarium_, a conclusion consistent with the albino phenotype of their mutants [33]. In both species, a carboxylic domain with similarity to caroten cyclases precedes the phytoene synthase domain. The cyclase domain has a high conservation with that of the orthologous gene _crTB_ of the yeast _Xanthophyllomyces dendrohous_, where the cyclase activity was biochemically demonstrated [34]. No mutants specifically affected in the cyclase activity have been described in_Fusarium_, but the mutants of the cyclase domain of the _al-2_ gene of _N. crassa_ were shown to be defective in the cyclization step [35; 36]. Taken together, the available data strongly suggest the same function for the amino domain of the CarRA protein of _F. fujikuroi_. The reactions from torulene to NX include a carotenoid cleavage step, typically achieved by a family of enzymes known as carotenoid oxygenases [37]. A gene for an enzyme of this family, called _carX_, was found to be linked and transcribed divergently from the gene _carRA_ (Figure 1). However, its targeted mutation did not impede the synthesis of NX [38], and the study of its enzymatic activity revealed that it cleaved \(\beta\)-carotene symmetrically to produce retinal [39]. Interestingly, the _carX_/_carRA_/_carB_ cluster is linked to a rhodopsin gene, called _carO_ because of its regulatory connections with the rest of the genes of the cluster (see next sections).